Pulping of Crustacean Waste Using Ionic Liquids: To Extract or Not To

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Pulping of Crustacean Waste using Ionic Liquids: To Extract or Not to Extract? Julia L. Shamshina, Patrick Stephen Barber, Gabriela Gurau, Chris S. Griggs, and Robin D. Rogers ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b01434 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 13, 2016

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ACS Sustainable Chemistry & Engineering

Pulping of Crustacean Waste using Ionic Liquids: To Extract or Not to Extract J. L. Shamshina,[a],[b],[c],† P. S. Barber,[a],†,‡ G. Gurau,[b],[c] C. S. Griggs,[a],§ and R. D. Rogers[a],[c],* [a] Department of Chemistry, The University of Alabama, Tuscaloosa, AL 35487, USA [b] 525 Solutions, Inc., 720 2nd Street, Tuscaloosa, AL 35401 [c] Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal QC H3A 0B8, Canada

†

J. L. Shamshina and P. S. Barber have given equal contributions towards experiments and writing of this manuscript ‡ Current address: Department of Chemistry, Williams College, Williamstown, MA 01267, USA § Current address: U.S. Army Engineer Research and Development Center, 3909 Halls Ferry Rd., Vicksburg, MS 39180, USA * Corresponding Author: E-mail: [email protected]

1 ACS Paragon Plus Environment


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Keywords: chitin • pulping • isolation • ionic liquid • sustainable

Abstract: Ionic Liquids (ILs), such as hydroxyammonium acetate ([NH3OH][OAc]) can reactively demineralize and remove proteins from shrimp shells in an efficient one-pot pulping process, thus allowing the isolation of native chitin with >80% purity and a high degree of acetylation and crystallinity. Compared to a previously reported IL extraction using 1-ethyl-3-methylimidazolium acetate, [C2mim][OAc], these less expensive ILs can achieve comparable chitin yields and purity, at up to ten times the biomass loading, although potentially result in lower molecular weight (MW) chitin. Since the IL is not recovered or recycled, the cost can additionally be further reduced by the sequential addition of hydroxylamine and acetic acid (or vice versa) to conduct the pulping process in situ. Though each methodology results in a comparable yields and purity of chitin material, the varying production costs and process safety issues are still unknown. This work presents a step toward narrowing the choices for chitin isolation technologies that can lead to an economically and environmentally sustainable process replacing the current hazardous, energy consuming, and environmentally unsafe process.


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Introduction Chitin, a linear biopolymer composed of β(1→4) linked 2-acetamido-2-deoxy-β-Dglucose elements,1 is a biocompatible, antimicrobial, and biodegradable polymer which has been utilized for a range of high-end (e.g., medicine, biotechnology) to low-end (e.g., nutrition, food processing) applications, and many reviews are available on the subject.2,3 According to Global Industry Analysis, Inc., the global $63 billion market is projected to exceed 118 thousand metric tons by 2018.4 Chitin is most commonly obtained from crustacean shells, where it exists in a matrix consisting primarily of CaCO3, along with proteins and pigments. The current industrial pulping method relies on a waste intensive two-step process using hydrochloric acid (HCl) at varying concentrations (up to 10% w/v) for 1-3 h5,6 at room temperature for inorganic salt removal, followed by treatment with sodium hydroxide (NaOH) at temperatures up to 160 °C for a few days to removes proteins.7-9 Production of 1 kg of chitin using this method requires 6.3 kg of HCl, 1.8 kg of NaOH, and 1.4 tons of water at elevated temperatures of 100 oC for at least 72 h.3 The use of harsh acid and caustic causes some depolymerization of the product and affects the inherent properties of the chitin, decreasing its molecular weight and degree of acetylation (%DA). The method is considered to be environmentally unsafe due to high emissions associated with the process and large amounts of waste, which is a likely reason why no chitin pulping plants are in operation in North America.10 Furthermore, while feasibility studies have been conducted for pulping plants construction, there are US Environmental Protection Agency (US EPA) restrictions and National Pollutant Discharge Elimination System (NPDES) limits for the conveyance and storage of hazardous chemicals in coastal regions that present a lot of environmental regulation challenges for producing chitin in the US.11 Though other methods for chitin isolation that utilize milder organic acids for removal of mineral salts (e.g. formic acid (HCOOH), acetic acid (HOAc), or their mixtures,12 ethylenediaminetetraacetic acid (EDTA13)) and bases for deproteinization (e.g. Na2CO3, NaHCO3, K2CO3, Ca(OH)2, Na2S, CaHSO3, and Na3PO4)14 have been reported, these methods have never been industrially adopted. In 2010, we proposed a chitin extraction method as an alternative process, using the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]).15,16 The method includes a 3 ACS Paragon Plus Environment


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facile microwave-assisted dissolution of the chitin from the chitinous biomass into the IL, followed by coagulation of chitin by addition of water or another anti-solvent. The advantages of IL-extraction include retention of high molecular weight (MW) and %DA and the opportunity to form fibers, beads, and membranes directly from the extract, as well as increased safety due to the non-volatile IL nature of the IL.15 The main disadvantages include that the method requires large amounts of, currently expensive, IL which must be recycled. While there are several studies in this direction, the process is yet proven useful on an industrial scale.17,18 We thus became interested in finding alternative IL pulping methods (i.e., those that remove the matrix from around the chitin without dissolving it) in which we could design not only basicity into the IL that would remove proteins, but also acidity that would allow for the removal of the inorganic minerals such as CaCO3. In essence, we were looking for a single, inexpensive IL that would replace both the HCl and the NaOH from the industrial process and pulp crustacean shells in a single step. We hoped that such an approach would reduce the harsh chemicals, high emissions, and process waste generated during industrial pulping while providing the benefits that ILs might provide in the design of more environmentally friendly processes.6,8 We thus sought easy to synthesize and low-cost ILs which contain both acidic and basic functionalities reactive enough to remove the organic and inorganic shell matrix and leave the chitin undissolved. In this study we discuss our efforts to chemically pulp chitin from

shrimp

shells

utilizing

three

ILs;

choline

acetate

([Cho][OAc]),

2-

hydroxyethylammonium acetate ([NH3(CH2)2OH][OAc]) and hydroxylammonium acetate ([NH3OH][OAc]). This work compares the pulping of two shrimp shell sources as well as a comparison of the pulped chitin properties to those of the chitin material produce from an extraction with [C2mim][OAc].

Results and Discussion The biomass source is a key variable to maintain in obtaining chitin with certain properties. For all experiments presented here, we utilized two types of chitinous biomass: shrimp shells from a local seafood processor which had been dried at a specialized facility by pressing with a screw press, followed by heating at 160 °C until moisture content became < 5 wt%


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(“processed” shells; see Experimental) and shrimp shells obtained by peeling commercial frozen shrimp (“raw” shells; see Experimental). For comparison, the chitin content in biomass was assessed using the Black and Schwartz method19 (see Experimental) and it was determined that the “processed” biomass contained 22(1)% chitin and the “raw” material contained 31(2)% chitin.

Pulping We first investigated the use of cholinium acetate ([Cho][OAc]) since it has been studied for the pretreatment of lignocellulosic biomass.20-22 The synthesis of [Cho][OAc] was conducted according to published protocol.23 As water content affects the properties of the IL significantly, the synthesized [Cho][OAc] was dried vigorously through sparging with N2 at 40 °C and then stored under high vacuum at 60 °C. The IL, a hygroscopic, light tan solid, was obtained with 86 % yield and a melting point (mp) of 85-87 °C. Combining 2 wt% “processed” shells with [Cho][OAc] and heating the mixture via microwave irradiation to the melting temperature of [Cho][OAc] and then to ~110-115 °C using 2-3 s pulses for total 3 min led to a vigorous reaction with significant bubbling. After 3 min, the solution was centrifuged (while still hot) to remove the insoluble material which was determined to be an inseparable mixture of chitin, calcium carbonate, and biomass residue. The supernatant was decanted into water and let sit overnight, at which point a precipitate was observed. The solid was isolated by filtration, thoroughly washed with water, and dried in air, yielding a light brown solid which was identified as chitin. The chitin yield was found to be 20% and is the percent mass of the isolated chitin from the available chitin in the biomass. These results suggested that [Cho][OAc] was extracting some but not all chitin, and at the same time reactively dissolving the inorganic/protein matrix of the shell. This is consistent with observations on the treatment of lignocellulosic biomass with [Cho][OAc], where hemicellulose and lignin (the matrix) are dissolved but cellulose (the polysaccharaide) is not.22 The low chitin yield of 20% was much less than the near quantitative yields reported for [C2mim][OAc],15 suggesting that there was a chitin solubility limit in [Cho][OAc]. Continuing towards our goal of pulping, rather than extraction, and embedding an acidic feature into, we switched our focus to two ILs containing primary ammonium cations to gain more protic character. Two protic ammonium cations, 2-ethanolammonium [NH3(CH2)2OH]+ 5 ACS Paragon Plus Environment


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(pKa 10.8) and hydroxylammonium [NH3OH]+ (pKa 5.9) were paired with the acetate anion through a simple Brønsted acid-base proton transfer.24-26 The ILs were prepared by drop-wise addition of acetic acid to a methanolic or aqueous solution of the corresponding amine (Scheme 1) and upon completion of the reaction through overnight stirring, the solvents were removed under vacuum, and a crystalline solid formed in each case. The previously reported IL [NH3(CH2)2OH][OAc] appeared as a light tan solid, though it’s been reported as a viscous liquid.26-28 The IL [NH3OH][OAc] appeared as a white crystalline solid, which produces large monolith-type crystals when recrystallized from water.29,30 Both ILs were fully characterized through 1H and 13C nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy, and agreed with the previously reported literature values.26,30 The melting points were determined to be 60 °C and 87 °C for [NH3(CH2)2OH][OAc] and [NH3OH][OAc], respectively.

Scheme 1. Synthetic pathway for hydroxylammonium salts.

26,30

Initially, we began with “processed” shrimp shells as our crustacean source, having previously worked with this material.17,31 Upon heating a mixture of 2 wt% shrimp shells and solid [NH3OH][OAc] in a round bottom flask equipped with a condenser to the first signs of melting, a bubbling was observed. Upon further melting, the reaction began vigorously foaming, and the reaction eventually started to reflux (Figure 1). The reaction mixture was kept at ca. 90 °C until foaming slowed and then completely subsided (after ca 1.5−2 h), after which the reaction was kept at this temperature for another 8 h. After cooling to room temperature, the reaction mixture appeared as a white solid covered with a thick frothy orange/yellow liquid, with a smell of acetic acid. DI water was added to dilute the reaction mixture, and the solid was isolated through centrifugation. After centrifugation, washing with copious quantities of water, and oven drying, a light tan solid was obtained. IR spectroscopy


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confirmed the solid as chitin indicating that the reaction between the shells and the IL was, indeed, a chemical pulping.

Figure 1. Photographs showing the chemical pulping process using [NH3OH][OAc] in progress. To optimize the yield of the process and prevent possible hydrolysis of the polymer caused by continuous reaction between the acid and chitin, which could result in a lower molecular weight (MW) material, we increased the mass loading of “processed” biomass to 10 wt% (yet maintained a molar excess based on CaCO3 content of the shrimp shells) and decreased the reaction time to 2 h. Upon melting, the mixture appeared to vigorously start bubbling and develop a foam similar to the prior reaction. The reaction was removed from heat once the foaming subsided, after approximately 2 h, and allowed to cool to room temperature. Water was added to the mixture, and the solid was separated as above, resulting in 22(1)% of the original shell mass (hereafter referred to as “pulp yield”, Table 1, Figure 2). This value corresponded roughly to the chitin content of the shrimp shells. Further investigation of the processing parameters revealed that the mass loading of shells could be increased to 20, 30, and even 50% without significantly affecting the pulp yields - determined to be 31(1), 35(1) and 31(5) % for aforementioned loads, respectively. Under identical conditions for the 10% mass load, untreated “raw” shells resulted in a 40(1)% pulp yield, as expected due to the higher chitin content of the “raw” shell biomass. Shifting to the significantly less hazardous amine, though more acidic IL, [NH3(CH2)2OH][OAc] was tested with a slightly modified procedure to account for the lower mp of the IL. A mixture of 10 wt% of “processed” shrimp shells and [NH3(CH2)2OH][OAc] 7 ACS Paragon Plus Environment


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was initially heated to ca. 60 °C (the mp of the IL); however, while the mixture reacted and bubbled, it did not react as vigorously, nor did it create as much foam, as in the experiment with [NH3OH][OAc]. Once completely melted, the temperature was increased to 90 °C, and heated for ca. 1.5−2 h. After cooling to room temperature, the reaction mixture appeared as thick foam covering an orange liquid, similarly to the observations made when using [NH3OH][OAc]. After dilution with water, centrifugation, washing, and drying, the pulp yield was 39(1)% (Table 1, Figure 2). With a percentage of almost double the amount of chitin available in the biomass, this yield suggests a lower chitin purity. While an IL can deliver acidic and basic functionality in a single compound and reduce processing steps, we also wanted to determine whether pulping was possible with sequential addition of NH2OH and HOAc (or vice versa), circumventing the need for prior synthesis of the IL and thus potentially reducing the cost. A downside to this approach is the direct use of hazardous 50 wt% aqueous solutions of NH2OH, rather than safer, less volatile IL. To conduct this study, we pulped both “processed” and “raw” shell in two steps. First, treatment with the appropriate amount of 50 wt% aqueous NH2OH (or glacial HOAc), followed by treatment with an equimolar amount of glacial HOAc (or 50 wt% aqueous NH2OH) (see Figure S6 and Experimental for details). In both cases, upon addition of the second reagent, a vigorous exothermic reaction with significant foaming, similar to what we observed when pulping with the IL, took place, even before heating was applied. It is important to note that visually, no reaction was observed until after the addition of the second reagent suggesting the acid/base combination (i.e., in situ formation of the IL) is required to generate the reactivity. The resultant mixtures were kept at room temperature until the foaming ceased (ca. 1.5−2 h) and then heated to 90 °C for 7–8 h. After cooling, centrifugation, washing, and drying the isolated pulp yields were 28−29% (Table 1, Figure 2). These yields were only slightly higher than that obtained using the corresponding IL at a 10% mass loading and were independent of the order of addition. Study of the experimental pulping parameters demonstrated that (similarly to the pulping with IL itself) the reaction time could be reduced to 1.5−2 h. In comparing the two methods (IL or sequential acid-base pulping), it is worth noting that while the reaction of shells with the corresponding IL proceeded through initial melting of the salt, the sequential acid-base pulping was able to proceed at room temperature rather than 8 ACS Paragon Plus Environment

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being heated first. We believe this is due to the IL existing in equilibrium in solution and reacting in a solid-liquid reaction compared to the IL and shrimp shells reacting in a solid-solid mixture that would require heat to convert the salt to the acid and base. Chitin Content and Chitin Yield Chitin Content For all of the pulps obtained, chitin content was assessed by the Black and Schwartz method15 (see Experimental, Table 1, Figure 2). Pulps obtained with [NH3OH][OAc] at 10 wt% biomass load contained 83(1)% and 76(1)% chitin, for “processed” and “raw” shells, respectively, and the pulp obtained using 20 wt% “processed” biomass load contained 78(1)% chitin. These values were comparable to the purity of marketed ‘pure chitin’ (81(8)%) and ‘practical grade’ (PG) chitin (78(9)%), as well as the chitin extracted using [C2mim][OAc] (81(1)%).15 However, the chitin content was compromised with increasing biomass loading, where the 30 and 50 wt% loads yielded pulps of 64(1)% and 30(1)%, respectively. This suggests that additional purification steps would be required for pulps obtained with loads > 20 wt%. The most probable explanation for these observations is an insufficient quantity of the IL to completely react with the shell matrix. When pulping with [NH3(CH2)2OH][OAc], the chitin content was low at 46(1)% and thus we did not continue experiments using this IL. For shrimp shell pulped using sequential addition of NH2OH and HOAc (or vice versa), the chitin purities were only slightly lower than those obtained with the corresponding IL ([NH3OH][OAc]) and was independent of the order of addition (Table 1, Figure 2). Though we could not find literature precedent for use of amines for protein removal in shrimp shells, we believe it's most likely due to the relatively low basicity of NH2OH (pKb = 8.0) compared to that of NaOH (pKb = 0.5). This suggests that it is the formation of the IL that makes the process work, where [OAc]− becomes the base and [NH3OH]+ the acid. The sequential addition of acid then base (or vice versa) would thus form the reactive IL in situ where it could immediately react without the melting step required for the premade IL.



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Table 1. Comparison of pulp and chitin yields, chitin content, and the associated properties. Crude (extraction) or Pulp

Source Trial

IL

Shrimp Shells

Chitin Content (%)

CaCO3 (%)

Crude Yield (%)

Load (%)

Chitin Content (%)

Chitin Yield (%)

Chitin Properties CaCO3 (%)[a]

DA (%)[b]

Visc. (cP)[c]

Relat. Visc.[d]

1

[Cho][OAc]

Processed

22(1)

26(1)[e]

2

5

-

20

-

-

-

-

2

Extraction with

Processed

22(1)

26(1)

2

96(3) [f]

81(1)

77(7)

0

79(5)

134(1)

2.00

3

[C2mim][OAc]

Raw

31(2)

12(1)

2

96(4) [f]

81(1)

77(7)

0

55(5)

80(1)

1.20

4

Processed

22(1)

26(1)

10

22(1)

83(5)

83(5)

0

77(1)

94(1)

1.47

5

Processed

22(1)

26(1)

20

31(1)

78(1)

100(3)[g]

0

68(1)

93(1)

1.47

Processed

22(1)

26(1)

30

35(1)

64(1)

100(5)[g]

0

- [h]

93(3) [i]

1.47

[h]

64(1)

[i]

0.96

82(1)

6

[NH3OH][OAc]

7

Processed

22(1)

26(1)

50

31(5)

30(1)

42(5)

0

-

8

Raw

31(2)

12(1)

10

40(1)

76(1)

96(3)

0

83(1) [h]

90(1)

1.23 [j]

9

[NH3(CH2)2OH][OAc]

Processed

22(1)

26(1)

10

39(1)

46(1)

81(1)

5.4(5)

-

10

HOAc+NH2OH

Processed

22(1)

26(1)

10

29(2)

73(1)

96(1)

0

75(3)

85(3)

1.34 1.27

11

NH2OH+HOAc

Processed

22(1)

26(1)

10

28(2)

72(1)

98(3)

0

80(1)

85(1)

1.27

12

HOAc+NH2OH

Raw

31(2)

26(1)

10

42(1)

76(1)

99(1)

0

64(1)

74(1)

1.11

[a] The CaCO3 content of each sample was determined using the decomposition of CaCO3 at 700 °C by calculating mass loss as CO2. [b] Degree of acetylation, %. [c] Viscosity of 0.5 wt% [C2mim][OAc] solution, cP. [d] Relative viscosity (with respect to pure [C2mim][OAc]). [e] The apparent CaCO3 content is higher in the “processed” biomass as proteins are mostly denatured in the “processed” shells. [f] Chitin obtained by extraction according to ref. [15] with isolated yield of 96 % for both biomass sources. [g] The method provides ca. 5-10% error. [h] %DA was not determined due to low chitin content. [i] Viscosity of samples with low chitin content, indicating that viscosity might not be directly correlated here with molecular weight.


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Chitin Yield Chitin yield was determined from both pulp yield and chitin content (See Experimental for calculations). The amount of chitin obtained from either “processed” or “raw” biomass using extraction (conducted in accordance with reference [15]), depended on microwaving time with the highest, 96% yield, obtained at 6 or 10 minutes microwaving, with both “processed” or “raw” biomass types. The amount of chitin obtained when pulping with [NH3OH][OAc] at 10 or 20% loading, compared to the amount of chitin available in the source, was similar to or higher than that isolated by extraction with [C2mim][OAc] (Table 1, Figure 2). Using [C2mim][OAc] for chitin extraction, yields as much as ca. 77% of the available chitin were achieved, and pulping with [NH3OH][OAc] at 10% loading resulted in isolation of ca. 83% of the chitin from “processed” shells and nearly all of the chitin from “raw” shells (Table 1, Figure 2). While the yields for pulping with [NH3OH][OAc] and extraction are essentially the same, pulping allows for 5 times higher biomass loading (10 wt%) than the extraction does (2 wt%).

Figure 2. Chitin yield (% of available chitin recovered) and chitin content (% chitin in the pulp). The numbers represent the trial numbers in Table 1. Black dots represent pulping trials; red triangles represent extraction trials.



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Overall comparison of chitin yields from “processed” or “raw” shrimp shells pulped directly with the IL indicate very little difference. Both sources give high chitin yields with “raw” biomass perhaps slightly lower yielding than the “processed” source. Pulping of “processed” and “raw” shells through sequential addition of acid and base was, again, independent of the order of addition and resulted in almost quantitative chitin yields for both sources. All results are comparable, if not better, to the chitin resulting from the IL extraction process. In summary, pulping with the IL (or sequential acid-base addition) at a mass loading of 10−20% and reaction times of 1.5−2 h at 90 °C yielded maximized chitin yield and purity. Under these conditions, the chitin produced is effectively separated (in some cases nearly quantitative) and the chitin content is similar to that obtained by IL extraction and equivalent-to-slightly higher than commercially available products.

Characterization While pulp yield and chitin content are critical economic parameters for chitin isolation methods, the properties of the resulting chitin are equally important. We compared the various chitins obtained in this study by characterization of the degree of acetylation (%DA, Table 1), CaCO3 content via thermal gravimetric analyses (TGA) (Table 1 and Figure 3), crystallinity via powder X-ray diffraction data (PXRD) (Figure 4) and viscosity (which correlates with molecular weight, MW15,16, Table 1). The results were also compared to chitin extracted using [C2mim][OAc].15,16 The degree of acetylation (DA) The degree of acetylation (%DA) was measured on the pulped material to determine ratio of chitin (amide functional group) to chitosan (amine functional group), as this ratio has a large impact on the material properties of the final product.32 The %DA for the chitin pulps were determined using acid-base titration,32 however it should be noted that due to the possibility of residual CaCO3 reacting with the acid or proteins reacting with the base, only pulps that contain high amounts of chitin were characterized this way. In brief, excess of HCl was added to the chitin samples, allowing acid react with the available free amines (-NH2). The excess HCl was then titrated with NaOH to the equivalence point and


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from this, the amount of acid consumed in the reaction with the -NH2 on chitin was found, and the %DA was calculated following a method reported in the literature.33 It was found that chitins obtained from [NH3OH][OAc] pulping had similar and relatively high %DA of ≥ 70%, regardless of whether “processed” or “raw” shrimp shells were used. Interestingly, the pulping of “raw” shells leads to slightly lower %DA compared to the use of the “processed” shells, excluding the pulping of 10 wt% “raw” biomass with [NH3OH][OAc] (Trial 7). This might to be due to the ease of accessing the shrimp shell matrix in the “raw” biomass which has not been previously pressed and oven-dried. Other possibility could be a difference in the species of shrimp, which are well-known to contain a wide-range of naturally deacetylated chitins. Overall, the pulping of “processed” and “raw” biomass with IL or the sequential addition of acid and base resulted in highly acetylated chitin. These values are comparable to commercially-produced chitin which typically has a %DA of 60–90%.3,9 The results also indicated that the high DA degree was consistent and reproducible for the same biomass load while the increase of biomass load respectively decreased DA. For the extraction process, the %DA of extracted “raw” chitin seems to be much lower than that of extracted “processed” chitin. This difference was the first noticeable difference between the extraction and pulping processes.

Analysis by thermal gravimetric analyses Further examination of pulped materials was conducted by thermal gravimetric analyses (TGA, Figure 3 – selected, Figure S8 - complete) on at least two independent samples. Analysis of “processed” shrimp shell biomass (aqua trace) shows a three step decomposition, - first decomposition step (ca. 75−150 oC) most likely arising from the decomposition of proteins or lipids, second decomposition step (ca. 250−400 oC) attributed to the decomposition of chitin, and a third step decomposition (starts at ca. 700 °C) is characteristic of CaCO3 decomposition to CaO and CO2.34



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Figure 3. Thermogravimetric analysis curves of extracted “processed” chitin (black), “processed” chitin pulped with [NH3OH][OAc] (blue), “processed” chitin pulped with [NH3(CH2)2OH][OAc] (red), “processed” chitin pulped with HOAc followed by NH2OH (dark red), shrimp shell biomass (aqua). The CaCO3 content of each sample was determined using the decomposition of CaCO3 at 700 °C by calculating mass loss as CO2. Thermal analysis of both “processed” (blue trace) or “raw” chitin, obtained through pulping with [NH3OH][OAc], shows a simpler trace with only one decomposition step correlated to chitin, suggesting a pure material. This profile closely resembles decomposition of chitin extracted

with

[C2mim][OAc]

(black

trace).

However,

the

chitin

pulped

with

[NH3(CH2)2OH][OAc] (red trace) indicated 5.4(5)% CaCO3 in the final product, whereas the material pulped with [NH3OH][OAc] showed none. This confirms our initial suggestion that [NH3(CH2)2OH][OAc] is not acidic enough to react completely with CaCO3 available in biomass. TGA plots of the samples pulped with corresponding sequential acid-base additions (dark red) regardless of the order of addition also demonstrate presence of a single chitin decomposition step. No proteins or CaCO3 were present in the final products, though residual mass attributed to silicates and ash was slightly higher than in the case of pulping with the corresponding IL.


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Analysis by powder X-ray diffraction Analysis by powder X-ray diffraction (PXRD, Figure 4) was conducted only on samples obtained from “processed” biomass and pulped with [NH3OH][OAc], as samples pulped with [NH3(CH2)2OH][OAc] were of insufficient purity and had CaCO3 remaining (Table 1). Samples that were pulped with [NH3OH][OAc] were compared to chitin pulped with sequential acid-base addition. Powder X-ray diffraction patterns were also recorded on the shrimp shell biomass and chitin extracted using [C2mim][OAc].

Figure 4. PXRD analysis of extracted “processed” chitin (black), “processed” chitin pulped with [NH3OH][OAc] (blue), “processed” chitin pulped with [NH3(CH2)2OH][OAc] (red), “processed” chitin pulped with HOAc followed by NH2OH (dark red), shrimp shell biomass (aqua). PXRD analysis of the shrimp shells (aqua trace) compared to the pulped “processed” material from the [NH3OH][OAc] (blue trace) provided additional support of the purification of the chitin through this pulping process. The diffraction pattern of the biomass showed 4 major peaks, 9.4, 11.9, 19.3, and 30 ° 2θ corresponding to α-chitin and CaCO3 (30°).35,36 After pulping, the peaks at 9.4, 11.9, and 19.3 ° increased in intensity, and a peak at 26 ° appeared, while the peak at 30 °, associated with CaCO3 disappeared. The intensity change supports an increase in αchitin concentration via removal of the inorganic CaCO3 phase. The lack of movement and 15 ACS Paragon Plus Environment


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broadening within the peaks correlated to chitin suggests that the chemical pulping of the chitin from the shrimp shell matrix maintains the natural crystallinity of the material throughout the process. By contrast, the IL extraction process through dissolution of the chitin and quick coagulation of the polymer, results in a more amorphous material (shown through the black trace). Diffraction patterns of the samples pulped with sequential acid-base addition are identical to those pulped with the IL directly.

Relative viscosity measurements of chitin-IL solutions Comparing viscosity of similar concentration solutions of polymers can provide evidence supporting molecular weight comparisons (Mark-Houwink equation37). We thus prepared [C2mim][OAc] solutions of the resulting chitin materials and compared the viscosities of pulped and extracted chitins. Because viscosities depend on the purity of the pulp, we focused on comparison of the viscosities of samples with comparable purity (thus samples pulped with 30 and 50 wt% biomass were excluded from this study). For viscosity determination, solutions of the various chitins in [C2mim][OAc] solvent were prepared at 0.5 wt% loading by dissolving approximately 0.015 g chitinous sample in ca. 3 g of [C2mim][OAc]. The mixtures were heated at 90 °C on an oil bath with magnetic stirring until the solutions appeared clear (3-6 h) and the viscosity of the samples was measured at a chosen temperature (35 °C), on three independently loaded samples. The relative viscosities are shown in Table 1 and were calculated using equation 4 (see Experimental). We observed that the viscosity of IL solutions of pulped chitins were lower than chitin extracted with [C2mim][OAc] from the “processed” biomass source. Also, for a given method of chitin isolation, the viscosity of the IL solutions of the chitin obtained from “processed” shells were higher than those determined for the chitin obtained from “raw” biomass. The data suggests that the MW of the chitin obtained through pulping is lower than that of chitin obtained through extraction, and also that the MW of chitin obtained from “raw” biomass is lower than that obtained from the “processed” biomass source. This is consistent with an understanding that the use of [C2mim][OAc] leads to chitin dissolution making deacetylation easier. Additionally, the pulping methodologies (including the current commercial process) treat the shells with an acid and base which are known to lead to chitin hydrolysis if given enough time. When comparing the viscosities of chitin pulping methods, the sequential acid-base sequence yielded only slightly 16 ACS Paragon Plus Environment

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lower viscosities than the values obtained from the [NH3OH][OAc] method and did not depend on the order of addition. The viscosity, similarly to chitin purity and %DA, depended on the initial biomass loading. For example, chitin obtained by pulping 10 and 30 wt% loading yielded identical viscosities, while chitin pulped with 50 wt% loading resulted in a much lower viscosity. In fact, viscosity was lower than viscosity of the solvent itself, suggesting the entanglement density of polymer chains was low, due to possibly high amount of impurities (proteins, salts and lipids) present in the sample.

Conclusions The current industrial pulping method of crustacean shells to produce chitin is costly, polluting, and results in low quality chitin. An alternative IL-extraction method with [C2mim][OAc], while increasing safety and preserving higher chitin quality (higher MW and higher %DA), utilizes a non-biodegradable and currently expensive IL. Here we present another alternative: inexpensive ILs made of highly acidic and basic ions, such as [NH3OH][OAc], can be used to pulp shrimp shells with comparable or even higher chitin yields and purity. Furthermore, the cost of production of [NH3OH][OAc] is relatively low and this inexpensive IL pulping of shrimp shells with comparable or even higher chitin yields than the extraction process could improve the economics of the process. Reducing the cost of making the reactive IL first, can be achieved by the sequential addition of NH2OH and HOAc (or vice versa) to prepare the IL in situ, but here the disadvantage is the direct use of NH2OH. Reactive pulping allows for ten times higher biomass loading (20 wt%) than the extraction method does (2 wt%) without compromising purity (78% and 81% chitin respectively). Altogether these factors presume the favorable economics of the process from a chemicals perspective, but this does not address possible environmental regulations, energy usage, etc. which could factor into a commercial process. We are currently carrying out such an economic analysis on the extraction process using the IL [C2mim][OAc] funded by a Department of Energy (DOE) Small Business Innovative Research (SBIR) Phase II grant and it has become clear to us that it takes an incredible amount of time and expertise to develop a true cost/economic analysis of any process.



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On the other hand, with pulping, the relative viscosity is lower suggesting lower MW and thus the use of IL-extraction method with [C2mim][OAc] might be advantageous in some applications requiring a chitin which is more similar to the native chitin polymer. Here, more work is needed to determine if this is due to hydrolysis from the aggressive chemicals needed to remove the crustacean shell matrix from the chitin and to obtain more accurate determinations of MW. All of these methods might compete in free-market factors and they differ greatly in the economics of chitin production (reagent costs and safety, biomass load, temperature and time (energy), etc.) vs. chitin yield and quality (purity, MW, %DA, etc.). Though there are still many unknowns in comparative bulk chemical costs, plant safety, and water and energy usage, we believe

that

this

new

pulping

method

deserves

further

study.


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Experimental Section Materials Chemicals All reagents were used as obtained. Acetic acid (HOAc), sodium hydroxide (NaOH) and hydrochloric acid (HCl) were purchased from VWR (Radnor, PA, USA); HOAc and NaOH were manufactured by BDH Merck Ltd. (Poole Dorset, UK) and HCl was manufactured by EMD Chemicals Inc. (Gibbstown, NJ, USA). Hydroxylamine and ethanolamine were purchased from Alfa Aesar (Ward Hill, MA). (CAUTION! Hydroxylamine can become explosive if heated and/or concentrated above 50 wt%. Please refer to Material Safety Data Sheet (MSDS) for further details (CAS # 7803-49-8)). Choline hydroxide was purchased as choline hydroxide solution 45 wt% in methanol from Sigma Aldrich (St. Louis, MO, USA). Solvent grade methanol was purchased from VWR (Radnor, PA, USA). It was used as received without additional puriﬁcation. 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) ionic liquid, used for chitin extraction and relative viscosity measurements, was purchased from Iolitec (Ionic Liquids Technologies, Inc., Tuscaloosa, AL). Deionized (DI) water was acquired from an in-house system (Culligan Water Systems, Chattanooga, TN) with a measured resistivity of 17.4 MΩ cm. For the titration, normalized calibrated solutions were used. NaOH, 0.100 Normal (N/10) solution (manufactured by Ricca Chemical Company, Arlington, TX) and HCl, 0.1 N volumetric solution, BAKER ANALYZED® Reagent (manufactured by JTBaker, Center Valley, PA) were purchased from VWR (Radnor, PA, USA) and used as received.

Chitinous Biomass Dried shrimp shells (hereafter indicated as “processed”) were received from the Gulf Coast Agricultural and Seafood Cooperative in Bayou La Batre, AL, where the shrimp shells were dried at a specialized facility by pressing with a screw press to eliminate the majority of the water, followed by heating at up to 160 °C in a fluidized bed dryer until the material had a final moisture content of less than 5 wt%. The dried material was pulverized with a hammer mill to particles 0.635 cm diameter and smaller. This dried, pulverized material was shipped to The University of Alabama. Before each laboratory experiment, the shrimp shells were additionally ground using an electric lab mill (Model M20 S3, Ika®, Wilmington, NC). The ground shells 19 ACS Paragon Plus Environment


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were then separated by particle size using a set of four brass sieves with wire mesh (Ika Labortechnik, Wilmington, NC) decreasing in size (1000 µm, 500 µm, 250 µm, and 125 µm) into a collecting pan. Particle sizing was carried out in small aliquots of ca. 1-2 g, until a sufficient amount of ground shrimp shells with desired particle sizes (

Pulping of Crustacean Waste Using Ionic Liquids: To Extract or Not To

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