Chemisorption of CO2 by chitosan oligosaccharide/DMSO: organic carbamato–carbonato bond formation

Abdussalam K. Qaroush *a, Khaleel I. Assaf b, Sanaa K. Bardaweel c, Ala'a Al-Khateeb d, Fatima Alsoubani d, Esraa Al-Ramahi d, Mahmoud Masri e, Thomas Brück e, Carsten Troll f, Bernhard Rieger f and Ala'a F. Eftaiha *d
aDepartment of Chemistry, Faculty of Science, The University of Jordan, Amman 11942, Jordan. E-mail: a.qaroush@ju.edu.jo
bDepartment of Life Sciences and Chemistry, Jacobs University Bremen, Campus Ring 1, 28759 Bremen, Germany
cDepartment of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan
dDepartment of Chemistry, the Hashemite University, P.O. Box 150459, Zarqa 13115, Jordan. E-mail: alaa.eftaiha@hu.edu.jo
eProfessorship of Industrial Biocatalysis, Department of Chemistry, Technische Universität München, Lichtenbergstraße 4, 85748 Garching, Germany
fWACKER-Lehrstuhl für Makromolekulare Chemie, Technische Universität München, Lichtenbergstraße 4, 85747 Garching bei München, Germany

Received 19th June 2017 , Accepted 15th August 2017

First published on 15th August 2017


A newly formed bond of organic carbamato–carbonato emerged upon bubbling CO2 in a low molecular weight chitosan hydrochloride oligosaccharide CS·HCl/DMSO binary mixture. The aforementioned bond was detected and confirmed using attenuated total reflectance-Fourier transform Infrared (ATR-FTIR) spectroscopy, with two prominent peaks at 1551 cm−1 and 1709 cm−1 corresponding to ionic organic alkylcarbonate (RCO3) and carbamate (RNH–CO2 NH3+–R), respectively. 1H–, 13C–, and 1H–15N heteronuclear single quantum coherence (HSQC) NMR experiments were also employed. According to 13C NMR, two newly emerged peaks at 157.4 ppm and 161.5 ppm attributed for the carbonyl carbon within the sequestered species RCO3 and RNH–CO2 NH3+–R, respectively. Upon CO2 bubbling, cross peaks obtained from 1H–15N HSQC at 84.7 and 6.8 ppm correlated to the ammonium counterpart chemical shift bound to the proton resonances. Volumetric uptake of CO2 was measured using an ATR-FTIR autoclave equipped with a silicon probe. The equilibrium sorption capacity was 0.6 and 0.2 bars through the formation of RCO3 and RNH–CO2 NH3+–R, respectively. Moreover, physisorption by the dried DMSO contributed to additional 0.4 bars. Density functional theory (DFT) calculations supported the occurrence of the suggested dual mechanisms and confirmed the formation of carbonate at C-6 of the glucosamine co-monomer. Moreover, CS·HCl/DMSO showed a slight impact on cell proliferation after 48 hours; this was a clear evidence of its non-toxicity. The biodegradation test revealed that a degradation of about 80% of CS·HCl/DMSO was achieved after 33 days; these results indicated that this method is suitable for green industry. CS·HCl/DMSO showed modest activities against Staphylococcus aureus and Escherichia coli. In addition, CS·HCl/DMSO demonstrated a significant antifungal activity against Aspergillus flavus in comparison with Fluconazole.


1. Introduction

One of the efficient approaches for climate change mitigation is carbon capture and sequestration (CCS). It is considered as an important technology to minimize CO2 emission from flue gas in order to adhere to emission regulations imposed by several governmental agencies in leading industrial countries, viz., China, USA, and countries in the EU. To overcome problems associated with the mature technology of monoethanolamine (MEA) wet scrubbing agent, ‘Green sorbents for carbon dioxide (CO2) capturing’ is a new addition in sustainable chemistry that was introduced very recently by our research group,1–3 although others have also reported significant contributions.4–10

These reports are focused on the synthesis/use of eco-friendly materials, viz., cellulose11,12 and chitin4–7 bio-feedstocks, metal organic frameworks (MOFs)8–10 and synthetic oligomers.1 Moreover, developing less hazardous materials in terms of energy efficiency through the use of benign solvents for sustainable purposes is a highly demanding task.13

Polymers have attracted great interest as CO2 sorbents due to their mechanical and thermal stability, ease of modification and casting into membranes,14 and their use in engineering porous/non-porous solids,15 liquids, and solution forms.16 Solid polymeric sorbents require a sorption temperature as low as 0 °C due to physical adsorption of CO2 unless impregnated with pendant groups.17 The latter point is of major concern due to the leaching of the functionalized material(s). Such unsolved obstacles will persist unless chemisorption with both built-in functional group(s) and fast sorption kinetics are brought into the solution without any crosslinking, decomposition, or evaporative losses that are characteristic of solvents.18

Apart from synthetic polymers, bio-feedstocks such as chitin (the second most naturally-occurring polymer after cellulose) and its derivatives (primarily chitosan) are strong candidates that might serve as green sorbents (chemical structure is shown in Scheme 1).2–6 From an economical point of view, the production of 100 billion tons per annum19 of chitin was estimated to have a revenue of 63 billion US dollars in 201520 in the global market. For example, chitosan is used in wastewater treatment and plasters in the Mt and kt scale per year, respectively. The growing market of chitosan industry will pave the way towards worldwide awareness into a prosperous bio-economy that forms a strong pillar for a more sustainable future.


image file: c7gc01830d-s1.tif
Scheme 1 Chemical structure of chitin (n > m) and its deacetylated form, chitosan (m > n). The numbers designate the carbon atoms across the constitutional repeating units.

One inherent limitation associated with the use of these motifs is the lack of solubility that hinders their processability. The use of solvent mixtures,21 ionic liquids (ILs)22 or even using oligomers2,3 might serve as a proper choice to understand the chemistry of macromolecules in solution. The use of ILs is problematic, as shown by Tom Welton.23 Their high viscosity, high cost, extreme care needed during purification and potential environmental hazards to aquatic and terrestrial ecosystems24 are considered as major disadvantages. In this context, oligomeric sorbents provide a useful platform to act as an attractive tool that combines the parent properties with enhanced processability features by maintaining the unexploited supramolecular interactions that are better resolved.

The chemistry of CO2 capturing takes place throughout by chemisorption and/or physisorption. The former is attained via two well-known approaches: carbonates (whether inorganic or organic) and carbamates (RNH–CO2 NH3+–R), which follow the 1[thin space (1/6-em)]:[thin space (1/6-em)]1 and 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanisms, respectively. The latter has a significant drawback due to the use of sacrificial bases together with its instability in aqueous solutions when forming bicarbonate (HCO3)/carbonate (CO32−) depending on the pH of the respective solution.25 In addition, inorganic carbonates require high temperature for regeneration in the post-combustion capture, viz. 100–150 °C, which leads to additional energy expenditure for the industrial sector. Therefore, organic ionic alkylcarbonates (RCO3) are considered to be a promising approach in terms of regeneration temperature, stability in aqueous media, and reducing additional expenditure associated with highly expensive additives.26,27

In this respect, following the concept of switchable solvents proposed by the Jessop group,14,15 several literature reports demonstrated that the usage of superbases is necessary to activate the alcohols, which results in the formation of ionic organic carbonate species upon CO2 bubbling.28–31 Similarly, Sir J. Stoddart8,32–34 and co-workers reported that hydroxyl groups located at the rim of γ-cyclodextrin-rubidium based MOFs are capable of reacting with CO2 reversibly to form a metal-stabilized RCO3. The chemisorption of CO2 was confirmed by high-resolution solid-state 13C nuclear magnetic resonance (13C NMR) that indicated the emergence of a peak at 158 ppm upon CO2 bubbling. Jessop's breakthrough combined with Stoddart's piece of artistic work led to the solubilization of naturally occurring polymers.28 In 2016, a combination of polymer scission2,3 and acetate effect35 resulted in task-specific oligomers for CO2 capturing. Our research group reported the formation of RCO3 through “supramolecular chemisorption” by reacting chitin-acetate oligomers dissolved in dimethyl sulfoxide (DMSO) at ambient CO2 conditions without the use of further additives,2,3 such as activators or stabilizers (superbases26 and metals,8 respectively). In terms of sustainability and greenness, the use of DMSO coincides with the guidelines set by the father of green chemistry Paul Anastas.13 DMSO is not only considered as a non-volatile organic compound due to its high boiling point,36 but also a natural product that exists in beverages, fruits and vegetables at the micromolar level.37–39 Furthermore, DMSO is also used as a penetration enhancer in topical pharmaceutical formulations.40 Together with its use as a polar aprotic solvent with extra merits such as ease of purification and low costs of production compared to ILs, DMSO is a potential greener candidate compared to polar aprotic solvents such as N-methyl-2-pyrrolidone (NMP), N,N-dimethylformamide (DMF), and N,N-dimethylacetamide (DMAc) as reviewed by J. H. Clark.41

Biodegradability is one of the key characteristic features in the field of green chemistry. To fulfill this need, the International Organization for Standardization (ISO) has issued nine standards42 for biodegradation testing in aqueous, compost, disintegration, soil or anaerobic based media.43 ISO 14855-2:2007/Cor.:2009 or BS EN ISO/DIS 14855-2:2009, have been widely reported for many polymers like poly(lactic acid)43,44 and poly(butylene succinate).45 Based on this method, the degradation was conducted within closed bioreactors using a controlled composite to simulate an accelerated environmental decomposition. The reaction vessels were aerated with CO2-free air. The degree of biodegradation is a percentage of the evolved CO2 (captured with an alkaline trap) to the theoretical evolved CO2.

Herein, a sustainable oligochitosan hydrochloride (CS·HCl) dissolved in DMSO (CS·HCl/DMSO) as a green sorbent for CO2 capturing is exploited. A qualitative and quantitative determination of CO2-chemisorbed species is determined through the formation of novel organic carbonato–carbamato motifs following the 1[thin space (1/6-em)]:[thin space (1/6-em)]1 and 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanisms, respectively. The titled species are well characterized and analyzed via1H-, 13C-, and 15N-NMR spectroscopy, ex situ and in situ attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, as well as density functional theory (DFT). This approach gives a straightforward approach towards the use of sustainable green sorbents for CO2 capturing adapting a benign-by-design approach. To further explore the impact of using DMSO on the greenness/toxicity of the applied binary system, ISO/DIS 14855-2:2007 is chosen to evaluate the compatibility of the described CO2 capturing system using the concept of green chemistry. In addition, the anti-bacterial, as well as anti-fungal activities are further tested for the CS·HCl/DMSO green sorbent.

2. Results and discussion

The main objective of this study was to show the possibility of obtaining a double sequestered species of CO2 using a commercially available, sustainable, and renewable material, namely chitosan hydrochloride (CS·HCl) oligosaccharide. As reported elsewhere, CO2 capture is achieved either by carbonate (organic and inorganic) or carbamate (RNH–CO2 NH3+–R) formation. RCO3 is thermodynamically more stable than RNH–CO2 NH3+–R,3 which is less energy demanding to regenerate compared to inorganic carbonates;25 hence, we anticipate that organic carbonato–carbamato sorbents will show a prominent collaborative performance. In order to enhance the likelihood of RNH–CO2 NH3+–R formation throughout the bio-renewables we used earlier, viz. chitin oligosaccharide,2,3 we focused our attention towards oligochitosan that can be produced from chitin by hydrolysis to increase the number of amine groups per sorbent. Herein, the degree of deacetylation (DDA) of the used chitosan oligomer was ca. 95% as verified by both proton nuclear magnetic resonance (1H NMR) spectra and elemental analysis (EA) (see ESI, Fig. S1 and Table S1).

2.1. Spectroscopic investigations

The absorption spectra of CS·HCl/NaOH pellet/DMSO before and after bubbling CO2 measured by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy (A) and the absorption profiles of CO2 by neat DMSO (B), CS·HCl/DMSO (C), DMSO/NaOH pellet (D), and CS·HCl/NaOH pellet/DMSO (E) obtained using an in situ ATR-FTIR autoclave (4.0 bars, 25 °C, 10 mL dry DMSO) are shown in Fig. 1. In these experiments, the water content of DMSO utilized was 6.1 ppm as measured using a Karl-Fischer titrator.
image file: c7gc01830d-f1.tif
Fig. 1 A. ATR-FTIR Spectra of CS·HCl/NaOH/DMSO before (red) and after bubbling (black) with CO2. Absorption profiles of physisorbed carbon dioxide (blue), organic carbonate (green) and carbamate (gray) as a function of time of B. Neat DMSO. C. CS·HCl/DMSO. D. DMSO/NaOH pellet. E. CS·HCl/NaOH pellet/DMSO, obtained from in situ ATR-FTIR.

CS·HCl/DMSO solution absorbed 0.6 bar CO2 through the formation of organic carbonate via supramolecular chemisorption2,3 (1551 cm−1, Fig. 1A). In this context, the pH of CS·HCl aqueous solution was ca. 5.6, which implies that the material is fully protonated; therefore, NaOH was added to deprotonate the ammonium group in order to be prone to the nucleophilic attack of CO2. The absorption profile of neat DMSO indicated that the absorption capacity was 0.4 bar. NaOH pellets were used due to its ease of filtration, which did not interfere with the overall effect on sorption capacity because of the elimination of water needed to make the side reaction to form sodium bicarbonate. CS·HCl/NaOH/DMSO solution absorbed 0.8 bar of CO2 through the formation of organic carbonate together with an extra contribution of the RNH–CO2 NH3+–R ion (1709 cm−1, Fig. 1A) (0.2 bar of CO2 absorbed as RNH–CO2 NH3+–R upon correcting the DMSO and CS·HCl values from the overall drop in pressure). The sorption capacity of 10% w/v solution through chemisorption was 1.60 mmol CO2 per g of sorbent. Taking into account an additional 0.81 mmol CO2 due to physisorption, the total CO2 absorbed was 2.41 mmol. In comparison with the sorption capacity of MEA aqueous solution (30% in water) reported in the literature, it absorbed 1.59 mmol CO2 per g of sorbent,46 which makes our material a potential candidate to compete with other commercially available compounds in the market.

As inferred from the sorption profiles, two presumed mechanisms (chemisorption and physiosorption, vide supra) were involved during the capturing process. The chemisorption takes place through two independent pathways. The first one is the formation of RCO3 through supramolecular chemisorption via a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 reaction mechanism, where DMSO activates the primary hydroxyl group of the amino pyranose ring towards nucleophilic attack.2,3 The organic carbonate adduct is stabilized through non-bonding interactions along the oligomer backbone. The second pathway results in the formation of RNH–CO2 NH3+–R following a 1[thin space (1/6-em)]:[thin space (1/6-em)]2 reaction mechanism, through the nucleophilic attack of CO2 by the amine's nitrogen.47 The latter does not proceed unless sodium hydroxide is used to activate the glucopyranose amine groups towards nucleophilic attack through deprotonation. It is noteworthy that the faster kinetics of carbamate formation compared to organic carbonate is explained by the higher nucleophilicity of nitrogen compared to oxygen. Chemisorption was the principal competitor to the physisorption process (blue trace, Fig. 1) as demonstrated in both C and E where the kinetic profiles showed a slower sorption for the latter. Scheme 2 summarizes the two suggested chemisorption mechanisms of CO2 capture by CS·HCl dissolved in DMSO; path A and B represent the formation of the RCO3 and RNH–CO2 NH3+–R via the 1[thin space (1/6-em)]:[thin space (1/6-em)]1 and 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanisms, respectively.


image file: c7gc01830d-s2.tif
Scheme 2 Schematic diagram illustrating the proposed chemisorption mechanisms for CO2 capturing upon CS·HCl dissolution in DMSO. Ionic organic alkylcarbonate (RCO3) (Path A) represents a 1[thin space (1/6-em)]:[thin space (1/6-em)]1 mechanism. Ionic carbamates (RNH–CO2 NH3+–R) (Path B) show a 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanism.

Nuclear magnetic resonance (NMR) was used to confirm the formation of carbonato–carbamato sequestered species in the CS·HCl/NaOH/DMSO solution. 13C NMR (Fig. 2A) showed the formation of two new peaks arising at 157.4 and 161.5 ppm, which correspond to the quaternary carbon of RCO3 and RNH–CO2 NH3+–R ions, respectively. Further, 1H NMR (Fig. 2B) shows noticeable changes at two different chemical shift regions. First, a new peak evolved at 6.81 ppm, which corresponds to the ammonium counterpart of the carbamate ion.3 Second, the methylene group neighboring the hydroxyl at C-6 was down-fielded to 4.75 ppm due to the inductive effect of the organic carbonate.


image file: c7gc01830d-f2.tif
Fig. 2 NMR spectra of CS·HCl/NaOH/DMSO-d6 before (red) and after (black) bubbling CO2. A. 13C NMR. B. 1H NMR. C. & D. 1H–15N HSQC spectra.

In addition, 1H–15N heteronuclear single quantum coherence (HSQC) NMR (Fig. 2C and D) spectra provided useful information about the formed RNH–CO2 NH3+–R. Cross peaks at 84.7 and 6.8 ppm (correlated to the ammonium counterpart chemical shift, vide supra) were recorded to the nitrogen-bound proton resonances upon bubbling CO2. The same observation was seen in the monomeric unit of CS·HCl, viz. glucosamine hydrochloride (Gln·HCl, Fig. S2, ESI). In 13C NMR, it is noteworthy that doubling of peaks associated with the monomer was due to the 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanism; the same observation was seen for task-specific ionic liquids as shown by Bates et al.48 Doubling of peaks was not clearly observed in CS·HCl/NaOH/DMSO filtered solution (pellet-free) due to the presumed macromolecular effect (Fig. S2, ESI). Structural simplicity of a monomeric unit cannot be extended to the oligomeric/polymeric form. This is further reflected over other physical properties such as solubility, viscosity, and crystallinity.

The reversible binding character of CO2 was achieved by sonicating the DMSO solution at 55 ± 3 °C and confirmed by diminishing the chemical shifts of the sequestered species obtained by 13C NMR spectroscopy (at ca. 157 and 161 ppm).

2.2. Computational investigation

In order to attain an in-depth understanding of the interaction between CS and CO2, gas phase density functional theory (DFT) calculations were performed using a Gln trimer as a model compound; the optimized structure is shown in Fig. 3. The proton affinity (PA) values for the ionizable amine and hydroxyl groups of the optimized structure were calculated and are shown in Table 1.
image file: c7gc01830d-f3.tif
Fig. 3 The optimized structure of Gln trimer, as a model compound.
Table 1 Calculated proton affinitiesa (PA) of the amino and the hydroxyl group at the central Gln unit in the trimer; the structure is shown in Fig. 3
Possible protonation active sites PA (kcal mol−1)
a PA values were calculated using the Gaussian 09 software (B3LYP/6-31+G* level of theory) as the negative of the enthalpy change (ΔH) of the gas phase reaction, A(g) + H+(g) → AH+(g). Under standard conditions, the value of the enthalpy of the gas-phase proton was taken as 1.48 kcal mol−1.52
Gln-Gln(NH2)-Gln + H+ → Gln-Gln(NH3+)-Gln 293.4
Gln-Gln(NH2)(O-6)-Gln + H+ → Gln-Gln(NH2)(OH-6)-Gln 329.4
Gln-Gln(NH2)(O-3)-Gln + H+ → Gln-Gln(NH2)(OH-3)-Gln 331.2


Although the gas phase is considered as the “ultimate” nonpolar environment,49 solvents play an important role when exploring the acidity/basicity of different compounds. The gas phase calculations provide the intrinsic PA values rather than relative reactivity, which could be exploited to understand the influence of solvation and intermolecular forces on reactivity.50,51 The higher the PA values, the stronger is the base and the weaker the corresponding conjugated acid in the gas phase. Results revealed that the deprotonation of the ammonium (–NH3+) group is the easiest compared to the other possible ionizable sites, followed by the hydroxyl groups at C-6 and C-3, respectively. This implies that the –OH group at C-6 is the second most reactive site towards bases and thus, its alkoxide (RO) ion counterpart is the most susceptible to form RCO3 upon reacting with CO2 even in the absence of supramolecular stabilization as in the corresponding oligomer (vide supra).

Besides the effect of aforementioned electronic parameter on the difference in the acidity between the primary (1°) hydroxyl group at C-6 and its secondary (2°) counterpart at C-3, the solvation effect might also contribute to higher PA values. It seems that DMSO molecules can easily solvate, and hence stabilize the unhindered RO anion at C-6 compared to C-3 once formed. This is consistent with the NMR data obtained for Gln·HCl/NaOH/DMSO-d6 upon bubbling CO2 (Fig. S2 A, ESI), where 13C NMR spectra indicated the emergence of two peaks at 157.7 and 159.2 ppm, corresponding to formation of RCO3 and RNH–CO2 NH3+–R, respectively. The complexity of the 13C NMR of Gln·HCl due to the different organic carbonate/carbamate assemblies, which resulted in peak doubling (vide supra), was better resolved by using glucose as an analogous chemical structure (Fig. S3, ESI). The 13C-NMR of glucose/NaOH/DMSO indicated that the chemical shift of C-6 was down-fielded from 61.7 ppm to a set of split peaks ranging from (63.4 to 64.8) ppm due to the RCO3 formation and presumably the inter- and intramolecular interactions after bubbling CO2. Other hydroxyl groups from C-1 to C-4 were not shifted in the spectrum due to their lower reactivity (2° alcohols).

To fully understand the stability of two possible CO2 adducts (if present), the optimized structures of RCO3 (either at C-6 or C-3) and RNH–CO2 NH3+–R (at C-2) at the central unit of the trimer are shown in Fig. 4. The calculated relative energy values showed that the formation of carbamate and organic carbonate at C-3 (Fig. 4A) is less favorable by 15 kcal mol−1 compared to that formed at C-6 (Fig. 4B), as a result of the strong repulsion between the carbonate at C-3 and the carbamate. This is verified by 13C NMR experiments (no C-3 carbonate was observed, vide supra). The optimized structures (Fig. S4, ESI) and the calculated relative energy values for the hypothetically formed single CO2 species, i.e., either carbonate or carbamate, are presented in Table S2, ESI.


image file: c7gc01830d-f4.tif
Fig. 4 DFT-optimized structures of carbamato-together with organic carbonato-adducts formed at A. C-3 and B. C-6 of the central unit of the glucosamine trimer.

2.3. Biodegradation study

The biodegradation of the chitosan oligosaccharide in a controlled compost was measured in a 1 L stirred tank bioreactor accompanied with the CO2 unit (washing, drying and capturing). Fig. 5 shows the biodegradation diagram as a function of incubation time maintained at 58 °C. Within the first 10 days, results showed that less than 10% of CS·HCl was degraded as seen from the minor differences in the amount of evolved CO2 from the blank and sample vessels. On day 11, the degradation appeared to be more intense until it reached about 80% after 33 days. The high degree of degradation in the relatively short time (according to the ISO protocol, see section 4.1.4) clearly indicated that CS·HCl is well-suited as sustainable and degradable in the presence of DMSO. It also fortified the usage of this solvent and formed a clear-cut evidence that it has no influence on the biodegradation of CS, which encountered concerns presented recently by Clark and coworkers41 on the guidelines of selecting green solvents. This finding could be of great importance for both the academic and industrial sectors.
image file: c7gc01830d-f5.tif
Fig. 5 Biodegradation evaluation method by gravimetric measurement of CO2 evolved via a laboratory-scale test using DASGIP® benchtop bioreactor in a controlled compost at 58 °C following the standard protocol, ISO/DIS 14855-2:2007/Cor.:2009.

2.4. Cytotoxicity results

Fig. 6 presents the percentage cell proliferation determined as (Absorbance of experimental well/Absorbance of negative control well) × 100 as a function of concentration. Statistical analysis was performed by applying the Student's t-test using SPSS 10.0 statistical software package (SPSSFW, SPSS Inc., Chicago, IL. USA). A p-value <0.05 was considered statistically significant. Each experiment represented the average of a series of three replicates. Notably, cell viability was not significantly changed after exposure of human fibroblasts to CS·HCl using the MTS assay. Cell growth of fibroblast cell cultures was evaluated using the MTS assay after 24 h and 48 h exposure periods. As demonstrated in Fig. 6, there is no statistically significant difference between the cell proliferation rates in wells treated with CS·HCl/DMSO at the concentration ranges used relative to control cells (untreated well) after both exposure periods, indicating a reasonable safety margin of CS·HCl/DMSO.
image file: c7gc01830d-f6.tif
Fig. 6 Percentage of cell proliferation after 24 and 48 hours exposure periods.

As shown in Table 2, upon comparison with the antibacterial activity of Norfloxacin, CS·HCl/DMSO exhibited pronounced activities against Staphylococcus aureus and Escherichia coli. In addition, CS·HCl/DMSO demonstrated a significant antifungal activity against Aspergillus flavus relative to Fluconazole.

Table 2 Antimicrobial activity measured by a zone of inhibition (mm). Results represent the means of three independent readings
Microorganism CS·HCl/DMSO Control
Staphylococcus aureus 9 ± 0.7 14 ± 0.9
Escherichia coli 12 ± 0.5 18 ± 1.0
Aspergillus flavus 13 ± 0.9 25 ± 1.0


3. Conclusions

Briefly, a sustainable oligochitosan hydrochloride dissolved in DMSO as a green sorbent for CO2 capturing is reported. Two different mechanisms are described: the formation of organic carbonate through the 1[thin space (1/6-em)]:[thin space (1/6-em)]1 mechanism in the absence of NaOH, and the formation of novel organic carbonato–carbamato species following a 1[thin space (1/6-em)]:[thin space (1/6-em)]2 mechanism. NMR and ATR-FTIR support the formation of carbonato–carbamato species in the CS·HCl/NaOH/DMSO solution. Absorption profiles indicate that the carbamate is formed faster than carbonate in NaOH/DMSO solution. Further, DFT calculations confirm the formation of the carbonate at the C-6 position together with carbamate formation. Further testing showed that CS·HCl/DMSO had a minor effect (non-toxicity) on cell proliferation after two days of the exposure. Moreover, it was shown to be a biodegradable binary mixture. Also, the CS·HCl/DMSO binary sorbent showed both antibacterial and antifungal characteristics with good activities. This confirms that DMSO usage within the sorbent system is eco-friendly and it does not have a negative impact on CS·HCl as a non-toxic/biodegradable green sorbent for CO2 capture. Explicitly, this makes the application of CS·HCl/DMSO to be more suited towards academic and industrial interests upon implementation, which will open new horizons in sustainable green technology. Furthermore, greater focus should be directed towards the dissolution of high molecular weight bio-feedstocks that are able to sequester CO2 as organic ionic alkycarbonates and/or carbamates.

4. Experimental

4.1. Materials and methods

4.1.1. Chemicals. Unless otherwise stated, all chemicals were used without further purification. Low molecular weight chitosan hydrochloride (CS·HCl) was prepared by G.T.C. Bio Corporation (1.5 kDa), Qingdao, China. For experimental manipulations, CS·HCl was dried overnight in a Schlenk tube at 50 °C (oil bath, in vacuuo). CO2 (99.95%, Food Grade) was purchased from Advanced Technical Gases Co. (Amman, Jordan). Glucosamine hydrochloride (Gln·HCl), DMSO-d6, D2O, sodium hydroxide (NaOH) and hydrochloric acid (HCl, 37%) were purchased from Sigma-Aldrich and DMSO was purchased from TEDIA.
4.1.2. Instruments. Solution 1H, 13C, and 1H–15N heteronuclear single quantum coherence (HSQC -nuclear magnetic resonance (NMR)) spectra were collected at room temperature using (AVANCE-III 400 MHz (1H: 400.13 MHz, 13C: 100.61 MHz, 15N: 40.560 MHz)) FT-NMR NanoBay spectrometer (Bruker, Switzerland) in either DMSO-d6 or D2O. Elemental Analysis (EA) was performed using a EURO EA 3000 instrument (Euro Vector, Italy). In situ attenuated total reflectance-Fourier transform infrared (ATR-FTIR) measurements were carried out using an MMIR45 m RB04-50 (Mettler-Toledo, Switzerland) with an MCT Detector, with a silicon windows probe connected via a pressure vessel. Sampling was done from 3500 to 650 cm−1 at 8 wavenumber resolution; scan option: 64; gain: 1×. Ex situ ATR-FTIR spectra were recorded on a Bruker Vertex 70-FT-IR spectrometer at room temperature coupled with a Vertex Pt-ATR accessory. Water content was measured using a Karl-Fischer titrator (TZ 1753 with Diaphragma, KF1100, TitroLine KF). pH measurements were obtained via an RL 150-Russel pH meter.
4.1.3. Computational method. Calculations were performed within Gaussian 09.53 The full optimization was performed using the DFT method (B3LYP/6-31+G*). Minima were characterized by the absence of imaginary frequencies.
4.1.4. Biodegradability study. The biodegradability test was conducted in accordance with the standard operating procedure of ISO/DIS 14855-2:2007/Cor.:2009. The controlled composite was collected from a soil of chicken farms in Munich-Germany. The incubation was continued at 58 °C in 1 L stirred tank glass bioreactors (DASGIP® Benchtop Bioreactors, Eppendorf, Germany). Bioreactors were connected to a designed unit for effective CO2 washing, drying and capture. The evolved CO2 was measured gravimetrically using an electronic balance (Sartorius, Germany) with a display reading down to ±1 mg. According to of ISO14855-2:2007, the polymer should reach a 75% degradation ratio after 45 days to be considered as a biodegradable polymer.
4.1.5. Cytotoxicity study. Human skin fibroblasts were maintained in a phenol red-free culture medium DMEM/F12 (Dulbecco's modified essential medium/Ham's 12 nutrient mixture, Gibco), supplemented with 5% (v/v) fetal calf serum (JS Bioscience, Australia), and 1% (v/v) antibiotic (2 mM L-glutamine, 100 U mL−1 penicillin and 0.1 mg mL−1 streptomycin; Gibco). Cultured cells were incubated at 37 °C in a humidified 5% CO2 incubator. The confluent cell layers were enzymatically removed, using Trypsin/EDTA (Gibco, USA), and resuspended in the culture medium. Cell viability was assessed by vital staining with trypan blue (0.4% (w/v); Sigma, USA), and cell number was determined using a light microscope.

In vitro evaluation of the cytotoxicity was performed using the Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation (MTS) assay to determine the number of viable cells in the culture (Promega, 2005). CS·HCl/DMSO was added to the culture media at concentrations of 2.5, 5, 10, 20, and 40% (w/v DMSO) and incubated at 37 °C with 5% CO2. Two sets of exposure times were carried out. These included 24 h and 48 h exposure periods. At the end of each exposure time, an MTS mixture (20 μL per well) was added. The absorbance of the formazan product was read at 492 nm using a microplate enzyme-linked immunoassay (ELISA) reader. Each experiment was repeated on three independent occasions. Two internal controls were used for each experiment; a negative control consisting of cells only without any treatment; and a positive control consisting of cells treated with vincristine, a microtubulin polymerization inhibitor.

4.1.6. Anti-microbial activity assays. Antibacterial activity of CS·HCl/DMSO was studied against Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 29425. Antifungal activity of CS·HCl/DMSO was examined against mycelial fungi (Aspergillus flavus). Overnight cultures of microorganisms were freshly prepared for each assay.

The agar diffusion method was used to assess the antimicrobial activities of CS·HCl/DMSO, with a minor modification. Briefly, Trypticase Soy agar (Difco) medium was aseptically inoculated with the bacterial or fungal suspension of the microorganism under examination. Wells were drilled and filled with 25 μL of CS·HCl/DMSO. Bacterial plates were incubated at 37 °C for 24 h whereas fungal plates were incubated at 25 °C for 48 h. After completion of the incubation period, the inhibition zones were observed and measured in mm. A positive control, Norfloxacin 1 mg mL−1 for bacteria and Fluconazole 1 mg mL−1 for fungi, and a negative control of the vehicle (DMSO) were employed. Each assay was repeated in triplicate.

4.1.7. Experimental procedure.
4.1.7.1. NMR. In an NMR tube, 30 mg of the substrate was dissolved in 0.5 mL DMSO-d6. Upon dissolution, CO2 was bubbled into the NMR tube via a long cannula for 20 minutes. CO2 Saturation was ensured using NMR spectroscopy.
4.1.7.2. In situ ATR-FTIR. Formation of carbonato-terminated oligosaccharide

The addition of 1.0 g CS·HCl together with 10 mL dry DMSO (6.1 ppm H2O, Karl-Fischer titrator) in a Schlenk flask was performed, and was sonicated till it completely dissolved. This solution was transferred into the IR autoclave, with parameters set at 25 °C, 4 bar CO2, and then the reaction was run for 4 hours (intervals of 15 seconds, initial and end pressure are to be reported). Initial pressure: 4.0 bar, final pressure: 3.0 bar, organic alkyl carbonate contribution through sorption was 0.6 bar.

Formation of carbonato–carbamato-terminated oligosaccharide

One gram of CS·HCl together was added to 10 mL dry DMSO (6.1 ppm H2O, Karl-Fischer titrator) in a Schlenk flask and sonicated till it completely dissolved. Further, 1.0 g of sodium hydroxide pellets was added and stirred for a minute and the solution was filtered in order to prevent any side reactions with the base used. Upon completion, the constituents were transferred into the IR autoclave using the same previous parameters (vide supra). Initial pressure: 4.2 bar, final pressure: 2.8 bar; carbamates contribution through sorption process: 0.2 bar; organic alkyl carbonate contribution through sorption was 0.6 bar.

For correction purposes, 10 mL DMSO was added into the IR autoclave; the solvent contribution was 0.4 bar as a result of physisorption (13C NMR peak identified at 124 ppm, together with ATR-FTIR peak centered at 2337 cm−1).

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

AFE acknowledges the Deanship of Scientific Research at the Hashemite University. Marina Reiter (TUM, Germany) is acknowledged for performing the volumetric uptake measurements using the in situ ATR-FTIR autoclaves. Thanks to Mr Basem R. Nassrallah (HU, Jordan) for performing the EA experiments.

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Footnote

Electronic supplementary information (ESI) available. See DOI: 10.1039/c7gc01830d

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