Mechanochemical Amorphization of Î±-Chitin and Conversion into

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Mechanochemical amorphization of #-chitin and conversion into oligomers of N-acetyl-D-glucosamine George Margoutidis, Valerie Parsons, Christina Sheila Bottaro, Ning Yan, and Francesca Maria Kerton ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b02870 • Publication Date (Web): 22 Dec 2017 Downloaded from http://pubs.acs.org on December 26, 2017

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ACS Sustainable Chemistry & Engineering

Mechanochemical amorphization of α-chitin and conversion into oligomers of N-acetyl-D-glucosamine George Margoutidis,a Valerie H. Parsons,a Christina S. Bottaro,a Ning Yan,b Francesca M. Kerton*a a

Department of Chemistry, Memorial University of Newfoundland, St. John’s, NL, A1B 3X7,

Canada. b

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4

Engineering Drive 4, 117585, Singapore. * Corresponding author. E-mail: [email protected]

Phone: +1-709-8648089

Fax: +1-709-

8643702 Supporting Information Available. Abstract Mechanochemical treatment offers great potential for environmentally sustainable processing of chitin within the context of biomass valorization. Using powder X-ray diffraction, we show that crystallinity can be reduced by 50% in 2 h in a controlled way using a ball mill. We correlate this crystallinity reduction with a decrease in interchain hydrogen bonding using infrared spectroscopy as a structural probe. Furthermore, our quantitative interpretation of the spectra reveal a decrease in glycosidic linkage content and retention of N-acetyl groups. The addition of a natural clay, kaolinite, in the ball mill leads to a significant increase in the solubility of the milled materials (up to 75.8% water soluble products in 6 h cf. 35.0% without kaolinite). The products of this process were characterized as oligomers of N-acetyl-Dglucosamine (chitin oligomers) with degrees of polymerization (DP) between 1 and 5 using a new quantitative matrix-assisted laser desorption-ionization (MALDI-ToF) mass spectrometric method. These data were complemented by a colorimetric assay of reducing ends and size-exclusion chromatography (SEC). N-acetyl-D-glucosamine (the monomer) and N,N’-diacetylchitobiose (the dimer) were obtained in yields of 5.1 wt.% and 3.9 wt.% respectively within 6 h, which is comparable with yields of glucose and cellobiose from cellulose ball milling. Keywords: Biomass, Food waste valorization, Ball milling, Separation-free analysis, Chitin, N-acetylchito-oligomers, Biopolymer, Catalysis

1 ACS Paragon Plus Environment

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Introduction The biorefinery concept has already started complementing fossil fuels whilst using just a few of the infinite biosynthesized functionalities available.1 Within this new way of thinking, one that balances profitability and environmental burden,2 food supply chain waste is seen as renewable on a human timescale and suitable for decentralized production of fuels and chemicals.3 Focusing on the challenges originating from inedible parts of seafood,4 our groups have highlighted processes for a range of products including lipids, pigments, minerals, and biopolymers (proteins and chitin).5, 6 Chitin makes up 20-30 wt.% of the shells of crustaceans which account for at least 50% of the fish harvested.7, 8 Global production of decapods (shrimps, crabs and lobsters) is over 13 million tonnes.9 Hence, tonnes of chitin are produced annually in the world and currently discarded. This by-product stream could satisfy demands for many substances including biologically-active materials,10,

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nanomaterials and fibres,12,

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and nitrogen-

containing platform molecules.14 Various methods for carbohydrate valorization have been explored to date. High-temperature processes can lead to unselective reactions of polysaccharides and production of humins (via monosaccharide dehydration).15 Enzymatic conversions are selective,16,

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however, they often need some sort of pre-

treatment step to enhance productivity (e.g. dissolution in an ionic liquid).18 Furthermore there is a trend towards the use of cocktails of enzymes in order to efficiently hydrolyze carbohydrates,18, 19 and concerns have been raised regarding process scale-up at the present time due to the high capital and operating costs, and the maturity of the biotechnologies used (e.g. global availability, stability and cost of biocatalysts). Hence, alternative methods that generate high energy micro-environments such as microwaves, ultrasound and ball-milling are promising and complementary tools for the conversion of biomass.20 In particular, the localized pressures and temperatures developed in mechanochemical processes21 offers an effective method to induce reactivity in organic materials including cellulose and chitin. This approach overcomes the need for solvent use and can disrupt the crystalline structure of polysaccharides by weakening the intermolecular hydrogen bonding network.22-24 In the context of the current work, reductions in crystallinity are thought to be essential for the conversion of α-chitin (which is the most abundant polymorph of chitin). Among other weak forces, hydrogen bonding is also responsible for the layered structure of the mineral kaolinite.25 The breakage of those bonds by mechanical treatment leads to an increase of its surface area which in turn led to remarkable catalytic activity in the hydrolysis of cellulose.26 After 3 h of milling, up to 84% of cellulose could be transformed to water-soluble material. Based on the aforementioned method, Meine et al. managed to fully convert cellulose into soluble products in 2 h after impregnating with acid and milling with modified parameters.27 Following on this momentum, a recent study has revealed a


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decrease in energy consumption with upscaling of biomass ball-milling processes.28 In this paper, we present a study of kaolinite’s mechanocatalytic potential towards α-chitin depolymerization as well as an investigation of the effect of ball milling parameters on the crystallinity, hydrogen bonding and solubility of α-chitin.

We also present a quantitative, separation-free analytical method for the quantitation of

carbohydrates using MALDI-ToF mass spectrometry.

Experimental Section Details of instrumentation and analytical studies are provided in the Supporting Information Mechanochemical treatment of α-chitin Milling experiments were conducted using a SPEX SamplePrep 8000M mixer/mill. A hardened 440C stainless steel vial with a volume of 65 mL was charged with either 2.00 g α-chitin, or 1.00 g α-chitin with 1.00 g kaolinite. Stainless steel balls were used with diameters of half inch (0.5ʺ) and quarter inch (0.25ʺ). Milling times reported herein refer to the time the vial was shaken at 1080 cycles per minute. The temperature of the outer surface of the vial was monitored with a thermocouple and did not exceed 65 °C in any case. This is in accordance with previous studies using the same mixer/mill (steel equipment) that reports plateaus for maximum temperatures throughout milling times dependent on the packing degree of the vial.

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In representative experiments, the vial containing α-chitin and/or kaolinite together

or separately was weighed at room temperature before and after milling and no appreciable mass loss was realised within the range of the standard deviation of the balance. Maximum soluble products of α-chitin were obtained when a 1:1 w/w mixture of α-chitin:kaolinite was milled with 2  0.5ʺ and 68-70  0.25ʺ balls for 6 h. Gravimetric analysis to determine the mass of soluble products (sample solubility %) 250 mg of milled sample was vortex mixed in 7.5 mL of either distilled water or 0.1 M acetic acid (pH 2.9) for one minute. A compact solid was achieved via centrifugation at 5000 rpm for 30 min and soluble products were separated with a pipet. The residue was dried overnight (70 °C) and weighed. Solubility for the samples from milling α-chitin alone was calculated by subtracting the mass of the undissolved residue from 250 mg (the sample taken from the mill), and reported as a weight percentage. Solubility for the samples from milling α-chitin with kaolinite was calculated by subtracting the mass of the undissolved residue from 250 mg and reported as a weight percentage of half of the weight of the milled sample, as kaolinite accounts for 50% w/w of the original sample. Each sample was analysed in triplicate. Solutions of the soluble material were noted to increase in colour as milling-time increased (Figure S1). This is likely due to Maillard-type reactions that occur between the amine groups and the reducing sugars formed


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to yield a range of intensely-pigmented substances (from small molecules like pyrazines to polymeric ones like melanoidins).

Results and Discussion Effect of milling on α-chitin’s crystallinity and origins of resulting changes Analogous with cellulose,30-32 one of the major challenges for α-chitin conversion is its robust crystallinity with extensive inter- and intramolecular hydrogen bonds. Mechanochemical systems that can reduce the crystallinity of α-chitin should prove advantageous in its transformation. We started by milling chitin with 2  0.5ʺ balls and produced a set of eight samples for which milling time increased in 15 min intervals (maximum 120 min). We repeated by milling with 16  0.25ʺ balls to see if ball size had any effect on the process. The vial’s degree of packing was approximately 3% in both cases. X-ray diffraction data over time for α-chitin ball milled with 2  0.5ʺ balls is shown in Figure 1. Crystalline reﬂections characteristic for α-chitin were observed at 9.3° (020), 19.2° (110) and 26.2° (130). Each reflection decreased in intensity with increasing milling time. The crystallinity changes for both sets of experiments were calculated using the peak height method and are shown in Figure 2. The peak height method is considered a measure of relative crystallinity and is not used here for estimating the absolute amount of crystalline and non-crystalline material in our samples. However, it shows that ball milling for 120 min reduces the crystallinity of α-chitin from 91% to 51% with the 0.5ʺ balls and to 35% with the 0.25ʺ balls.

During the

course of our ongoing research, other groups have reported that steel and zirconia vial ball milling systems with different motion modes, operating frequencies (rpm) and α-chitin/balls volume ratios, were also able to reduce α-chitin’s crystallinity.23, 24 In the study most similar to ours,24 which aimed to reduce crystallinity in order to increase enzymatic degradation, the crystallinity index (CrI) decreased from 68% (after 10 min) to 40% (after 30 min) but a much higher degree of packing was used in their study. However, in the study by Osada et al., they did not perform experiments to elucidate the molecular origin of the reduction in crystallinity. Elucidating the reasons for crystallinity reductions is essential in order to further optimize and improve methods for processing chitin.



(110)

Intensity (x 103 a.u.)

8 6

(020)

16.0°

(130)

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Figure 1. XRD patterns of untreated (black signal) and milled α-chitin with 2  0.5ʺ balls for 30 (red), 60 (green), 90 (blue) and 120 (pink) min. Crystallinity Index (CrI) %

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100 90 80 70 60 50 40 30 20 0

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Milling time (min)

Figure 2. Crystallinity Index (CrI) % of α-chitin when milled with 2  0.5ʺ (blue) and 16  0.25ʺ (red) balls. We thought the decrease in crystallinity might be correlated with the bifurcated hydrogen bond which is critical to the decrystallization of α-chitin,33 and therefore, we employed FT-IR spectroscopy in an attempt to prove this hypothesis. A direct interpretation of α-chitin’s infrared spectrum offers the chance to investigate the well-known amide I split.34 Indeed, our IR spectra for untreated α-chitin show the amide I band splitting at 1652 and 1621 cm-1 (Figures S2 and S3). The lower frequency vibration can be attributed to the C=O2 group hydrogen bonding with N1-H and O61´-H. [O6 refers to the oxygen of the primary alcohol, superscripts denote the number of chain and the prime symbol (´) refers to the neighboring sugar unit within a chain (Figure S4)]. The vibration at higher frequency can be attributed to the C=O2 group hydrogen bonding with N1-H exclusively. Figure 3(A) shows the ratio between the absorbance intensity at 1621 and 1652 cm-1 versus milling time for the 16  0.25ʺ ball set. A decrease in


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this ratio with no error bar overlap is observed for the first 75 min of ball milling and is indicative of a weakening within the hydrogen bonding network. After 75 min, the decrease is less significant which is consistent with the behaviour of the corresponding crystallinity index as discussed and shown above (Figure 2). In Figure S3, the band at 1635 cm-1, which appears to be growing with increased milling time, corresponds to the bending mode of adsorbed water. The most intense C-H stretching band in the IR spectra (at 2884 cm-1 in recent literature,35 at 2875 cm-1 in our spectra) can be assigned to the methine groups (of the ring carbons) and it can be used as a reference to approximate possible depolymerization and/or deacetylation during ball milling. Figure 3(B) shows the peak ratio for the glycosidic linkage decreasing steadily during 120 min of ball milling (from 1.44 to 1.25) with no error bar overlap between 15, 30, 60 and 120 min – this suggests that depolymerization is occurring. Figure 3(C) shows the peak ratio for the amide II stretch remaining constant (~2.70) for 120 min of ball milling with error bar overlap for all samples – indicating that no deacetylation has occurred during milling. The larger error bars and lower signal intensities for the untreated (non-ball milled) αchitin is due to the flaky texture (bigger particles, Figure S5, SEM data) of our α-chitin which limits contact with the diamond ATR surface. These IR data suggest that the ball milling process reduces crystallinity not just through disruption of intermolecular hydrogen bonding but also because of glycosidic linkages breaking. The results also confirm our expectations that depolymerization is favoured over deacetylation in our steel ball mill system. Moreover, we show that FT-IR spectroscopy can be a useful technique to approximate crystallinity changes, depolymerization and deacetylation during ball milling processes of polysaccharides like chitin/chitosan. Further data described below (e.g. MALDI-ToF mass spectra) support depolymerization occurring and no deacetylation during ball milling of chitin.



Absorbance ratio

(A) 1.13 1.03 0.93 0.83 0.73 0

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Absorbance ratio

(B) 1.49 1.39 1.29 1.19 0

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(C) Absorbance ratio

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2.81 2.71 2.61 2.51 0

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Milling time (min)

Figure 3. IR data (peak ratios) for milled α-chitin over time when milled with 16  0.25ʺ balls, (A) 1621 cm-1 / 1652 cm-1, (B) 1154 cm-1 / 2875 cm-1, (C) 1552 cm-1 / 2875 cm-1. The morphologies of our native and ball milled samples are shown in scanning electron microscopy (SEM) images in Figure S5. These micrographs show that ball milling with 3% packing reduces the size of our α-chitin particles from approximately >500 μm (Figure S5a, b) to

Mechanochemical Amorphization of Î±-Chitin and Conversion into

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