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1-Ethyl-3-methylimidazolium diethylphosphate based extraction of Bioplastic “Polyhydroxyalkanoates” from bacteria: Green and Sustainable Approach Sonam Dubey, Pankaj Bharmoria, Praveen Singh Gehlot, Vinod Agrawal, Arvind Kumar, and Sandhya Mishra ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03096 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 30, 2017

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ACS Sustainable Chemistry & Engineering

1-Ethyl-3-methylimidazolium

diethylphosphate

based

extraction

of

Bioplastic “Polyhydroxyalkanoates” from bacteria: Green and Sustainable Approach Sonam Dubey,£ Pankaj Bharmoria,£┴ Praveen Singh Gehlot,£┴ Vinod Agrawal,† Arvind Kumar,£┴* Sandhya Mishra£┴* £Salt

& Marine Chemicals Division, CSIR-Central Salt and Marine Chemicals Research

Institute, Bhavnagar 364002, India ┴Academy

†Analytical

of Scientific and Innovative Research (AcSIR), Bhavnagar 364002, India Division and Centralized Instrument Facility, CSIR-Central Salt and Marine

Chemicals Research Institute, Bhavnagar 364002, India Authors: [email protected]; [email protected]; [email protected]; [email protected] *Corresponding authors: [email protected]; [email protected]; Tel: +91-2782567039;

Fax:

+91-278-2567562.;

[email protected];

5801/3805x6160; Fax: +91 278 256 6970/7562.

1 ACS Paragon Plus Environment

Tel.:

+91

278

256

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract An eco-friendly approach towards downstream processing of bacterial biomass for extraction of an intracellular potential bio-plastic material polyhydroxyalkanoates replacing the chlorinated organic solvents is reported using ionic liquid (IL); 1-Ethyl-3-methylimidazolium diethylphosphate ([C2mim][(C2)2OPO3]) as an extractant. [C2mim][(C2)2OPO3] dissolved wet bacterial biomass of Halomonas hydrothermalis (MTCC accession no.5445; NCBI Genbank accession no. GU938192), with ease on account of its high hydrogen bond basicity (β = 1.07). The recovered polymer with a yield of 60% ± 2 % was characterized using 1H-NMR, FT-IR and TGA techniques and confirmed to be polyhydroxybutyrate (PHB). The properties of PHB were found to be in close proximity to the standard PHB. The [C2mim][(C2)2OPO3] was recovered with 60% yield and retention of chemical structure after two consecutive dissolution cycles, which minimized the cost of developed process. The recovered PHB was used to prepare a bioplastic film, which showed good thermal and mechanical stability, as characterized from FT-IR, DSC, TGA and DMA techniques. Keywords: Ionic liquid, Polyhydroxybutyrate, Extraction, Recyclability, NMR, TGA, FTIR, DSC.


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Introduction Polythene is a non-biodegradable plastic which is routinely used worldwide, because of its flexible mechanical properties, despite having adverse effects on biosphere. Therefore, an alternative biodegradable plastic with flexible mechanical properties is in high demand as a replacement material to polythene. Polyhydroxyalkanoates (PHAs) on account of their mechanical flexibility and biodegradable nature are emerging as potential alternative bioplastics. PHAs, are polyesters produced by prokaryotes to store carbon and energy. 1 Apart from bacteria, an increasing number of Archae like; Haloferax mediterranei has also been reported as a potential producer of PHA in hypotonic medium.2,3 Therefore, prokaryotes can be used to produce PHAs at bulk scale considering their natural abundance. However, PHAs production from bacteria is beyond economic feasibility owing to its cost of production as well as the usage of hazardous chemicals during the extraction process. This is because of the fact that extraction process requires the use of excess organic solvents to break the cell wall and separate the polymer accumulated inside the cells. Koller et. al.4 have reviewed various processes of PHA production from bacteria which includes; solvent method (chloroform, cyclic carbonates, fusel alcohol, lactic acid ester method and acetone under reflux), digestion of non-PHAs cell material (enzymatic digestion, alkaline digestion, hypochlorite digestion), mechanical disintegration of PHA-rich cells, high-pressure homogenization, cell disruption in hypotonic medium, bead mills, dissolved air floatation and ultrasound, extraction using supercritical fluids, or a combination of these methods.5 Other important reviews regarding PHAs recovery are reported by Jacquel6 and Madkour7 et al. who have reviewed pretreatment of cell prior to disruption and purification methods and analytical methods of PHAs6 and different processes that result in the release of PHA from the cells, with regard to the suitability at large scale in the industry.7 Among these processes the solvent method in 3 ACS Paragon Plus Environment


particular uses chlorinated solvents such as sodium hypochlorite, chloroform, etc.8 Such solvent based extraction methods are very harmful for the environment and therefore, need to be replaced with more environmental friendly process. In an improvement to solvent recovery method Koller et al.9 recovered PHA from microbial biomass using acetone as antisolvent at high temperature and pressure with 98.4% purity. The acetone was recovered by cooling down the solution, which could be reused thus, signifying greenness of the process. Dimethyl carbonate and switchable anionic surfactants have also been reported as an alternative green solvent to overcome the limitations of chlorinated solvents for the recovery (85 to 95%) of PHAs from Cupravidus necator.10 In a recent development Arikawa et al.11 have reported the extraction of PHA from culture broth using sodium dodecylsulfate (SDS)sonication treatment (with 96% purity) as simple and rapid method. Seeking further simplification of the solvent extraction process herein we have used Ionic liquids (IL) as an extracting solvent of PHA from bacterial biomass. Ionic liquids (ILs) bearing few green properties (low vapour pressure, recyclability and sometimes biocompatible) have emerged as an alternative solvent to harmful Volatile Organic Compounds (VOCs) in the last decade for various processes.12-14 ILs are organic ionic salts which melt below 100 oC and exhibit properties having fair contribution of organic liquids and polar compounds. 14 These properties have instilled special solvating nature in ILs, leading to solubilization of a plethora of compounds including the tough biopolymers like polysaccharides 15-21 and proteins22-27 at high temperature. Among polysaccharides, cellulose being the most abundant naturally occurring biomass has been explored extensively for dissolution in neat ILs. 15-21 In a recent paper Scurto et al.20 reported the solubility of cellulose up to 19.8% at 100oC in 1-ethyl-3methylimidazolium diethylphosphate ([C2mim][(C2)2OPO3]) and explained the solubility of cellulose on the basis of hydrogen bond basicity of diethylphosphate anion [(C 2)2OPO3] (β = 1.07)28. High solubility was accounted to high β = 1.07 of [(C2)2OPO3] anion compared to


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chloride anion (β = 0.87,28 solubility = 10% at 90oC17) in 1-butyl-3-methylimidazolium chloride ([C4mim][Cl]) and acetate anion ( β = 0.95,28 solubility = 16%29) in 1-ethyl-3methylimidazolium acetate ([C2mim][OAc]). A molecular dynamic carried out by Rabideau et al.30 for solubility of cellulose in ([C2mim][(C2)2OPO3]) showed that one [(C2)2OPO3] anion can interact two hydroxyl groups of cellulose, which further supports its ability to dissolve biopolymers having OH functionality. Moreover, cationic imidazolium part of ([C2mim][(C2)2OPO3]) has been shown to interact with negatively charged phospholipid membrane via strong electrostatic interaction to rupture the bacterial membrane.31 Therefore, inspired by the high biomass solubilization ability of [C 2mim][(C2)2OPO3] due to hydrogen bond basicity and potential role of cation to rupture the bacterial cell wall, we investigated it’s utility as solvent to extract another polymer, PHA (which is in fact a bio-polyester)” from bacterial biomass. It is to be noted that PHA has been extracted earlier from plant source using ILs, comprising of ammonium, imidazolium, pyrazolium, oxazolum, quinolinium, isoquinolinium cations and carboxylates, sulphate, and sulfosuccinates anions. 32 However, on account of its high hydrogen bond basicity, [(C2)2OPO3] could be a powerful extracting solvent to collect PHA from biomass, hence, we have made an attempt to utilize the IL; [C2mim][(C2)2OPO3] for PHA extraction from bacterial biomass, and further investigate the greenness of ILs on account of its recyclability scale for a number of cycles.

Materials and Methods Materials 1-Ethyl-3-methylimidazolium diethyl phosphate, [C2mim][(C2)2OPO3] with purity >98% was purchased from Sigma Aldrich. Methanol HPLC grade (99.5%) was purchased from SRL, India. Halomonas hydrothermalis MTCC 5445 with NCBI Genbank accession no. GU938192 isolated from Aadri Veraval, Gujarat (20°57.584’ N 70°16.659’ E)33 has been used in current study was maintained in Zobell marine agar (ZMA) slants at 4 oC until use. 5 ACS Paragon Plus Environment


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The bacterial biomass of Halomonas hydrothermalis was obtained by growing the culture as per the reported procedure in our earlier reports. 34-35 The bacterial culture was initially inoculated in Zobell marine broth (ZMB) for fresh inoculum preparation kept overnight at 37 oC

and 120 rpm in an Orbitek shaker. Next day, 10% of inoculum was transferred aseptically

to the sterile production media for PHA production consisting of 2 % crude glycerol from Jatropha biodiesel waste residue as carbon source and 10% Jatropha deoiled cake hydrolysate as nitrogen source. The detailed composition of the crude glycerol and hydrolysate is : crude glycerol contains 95% glycerol; metal impurities (mg/l) calcium, 8.189; cadmium, 0.002; cobalt, 0.002; chromium, 0.023; copper, 0.046; iron, 0.554; potassium, 2163; magnesium, 3.552; manganese, 0.040; molybdenum, 0.004; sodium, 38.24; nickel, 0.022; lead, 0.152; zinc, 0.131; trace amount of methanol; moisture content 0.30%; and free fatty acids 0.13%. The carbon content and bound nitrogen in the hydrolysate was 2.31% (w/v) and 0.48% (w/v) respectively.34-35 Total working volume for PHA production broth was kept as 100 mL and further incubated for 96 hrs. at 37 oC and 120 rpm. Harvesting of the culture was carried out after 96 hrs. of incubation by centrifuging at 10,000 rpm for 10 minutes. The wet bacterial biomass was collected and used for further studies. Similarly, dried bacterial biomass was obtained by additional drying of bacterial pellets in an oven overnight at 60 oC. The PHA content of the bacterial biomass has been found to be 74% as also reported in our earlier report.30 Methods Nuclear

magnetic

resonance

(NMR):

The

1H

NMR

spectrum

of 1-Ethyl-3-

methylimidazolium diethyl phosphate and recovered ionic liquid were recorded in deuterated DMSO using a Brüker 200 MHz spectrometer. Prior to measurement IL was dried in vacuum oven. The extracted polymer was dissolved in deuterated chloroform for 1H NMR analysis at 500 MHz and compared with standard PHB from Sigma Aldrich. 6 ACS Paragon Plus Environment

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Fourier transform infrared spectroscopy (FT-IR): The extracted polymer was mixed with KBr and FTIR spectrum was recorded with PerkinElmer spectrum GX FTIR spectrometer in 4000 – 400 cm-1 range. Differential scanning calorimetry (DSC): The glass transition and melting temperature of the recovered polymer were observed from DSC. The extracted polymer sample was run in the temperature range of -20 to 500 °C with the heating rate of 10 °C min-1 in a Differential scanning calorimeter (DSC) using DSC 204 F1 Phoenix instrument with Netzsch software. Thermo gravimetric analysis (TGA): The thermal degradation temperature of the PHB was measured from TGA. The extracted polymer sample was subjected to Thermo gravimetric analysis (TGA) with help of TG–DTA system in TG 209 F1 instrument. The analysis were performed over a temperature range from room temperature to 500 °C at a heating rate of 10 °C

min−1 under nitrogen atmosphere.

Gel permeation chromatography (GPC) analysis: The Column used was 2 PL Gel Mixed D (300 mm x 7 mm) with Guard column in series. Mobile Phase CHCl3 stabilised with 1% Ethanol with Flow rate of 1 ml/min and polystyrene was used as calibration standard. Temperature was maintained at 30 °C. Dynamic mechanical analysis (DMA): The mechanical strength of the film was measured from DMA using a NETZSCH DMA instrument. Scans were run from -50 to 300 oC at 5 oC/min

and tension mode of 1 Hz.

Experimental Dissolution and Extraction For dissolution in [C2mim][(C2)2OPO3], we used both wet and dried Halomonas hydrothermalis biomass. In a typical experiment 0.5 g of both wet and dried biomass were taken in a 15 ml glass vial, followed by the addition of 5 g of [C2mim][(C2)2OPO3] and 7 ACS Paragon Plus Environment


tightening of the vial with Teflon coated caps. To further avoid any contamination of water during dissolution, the caps were covered tightly with parafilm followed by additional wrapping with aluminium foil. Initially the dissolution was carried out at room temperature; however, with no substantial dissolution we gradually increased the temperature and observed dissolution temperature to be around 60 oC. Therefore, dissolution was carried out at 60 oC. The samples were incubated at 60 oC for 24 hrs. The complete dissolution was observed from clear orange colour solution (Figure 1b). After complete dissolution of the biomass, methanol as an anti-solvent was added to recover [C2mim][(C2)2OPO3]. White precipitates of PHAs were observed with the addition of methanol to this solution, which can be clearly observed in Figure 1c.

Figure 1. Dissolution of bacterial biomass in [C2mim][(C2)2OPO3] and consequent extraction of PHA and recovery of [C2mim][(C2)2OPO3] using methanol as anti-solvent. The Halomonas hydrothermalis cell is composed of peptidoglycan wall and PHA granules in the cytoplasm. Peptidoglycan is a combination of sugars (N-acetylglucosmine and Nacetylmuramic acid) which are interlinked by peptide cross chains. The PHA granule in the cytoplasm is surrounded by phospholipid layer and granule attached protein.37 Intramolecular H-bonding in peptidoglycan provides toughness to bacterial cell wall, making it hard to break. However, unlike organic solvents8 [C2mim][(C2)2OPO3] breaks up the bacterial cell wall easily and removes the PHAs in a single pot without any discharge into the environment. The [(C2)2OPO3] anion on account of its high β = 1.07,20 interacts with 8 ACS Paragon Plus Environment

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peptidoglycan via intermolecular H-bonding, thus breaking the existing intra chain Hbonding leading to cell wall lysis.29 Moreover, the imidazolium cation interacted with negatively charged phospholipid membrane via electrostatic interactions to dissolve it.30 The possible mechanism of [C2mim][(C2)2OPO3] interaction with peptidoglycan via H-bonding is depicted in Figure 2.

Figure 2. Interaction mechanism of [C2mim][(C2)2OPO3] with peptidoglycan via hydrogen bonding along with the depiction of biomass dissolved. The [C2mim][(C2)2OPO3] along with organic impurities of the biomass (phospholipid and fragmented protein) went into the methanol. The methanol solution was then decanted and excess methanol was added to extract the biopolymer and to remove the IL completely. The methanol solution containing IL and bacterial impurities were then mixed with small amount of charcoal. The solution was stirred for 2 hrs. to remove organic impurities and then filtered. The filtrate was mixed again with small amount of charcoal, stirred for 1hr. and filtered again. The filtrate was subjected to distillation of methanol and any moisture content at 70 oC and zero pressure for 3 hrs. A clear orange coloured liquid (Figure 1d) was obtained at the bottom of round bottom flask which should be [C 2mim][(C2)2OPO3]. The liquid was collected in a glass vial and stored in a desiccator containing P 2O5 as desiccant. The 1H-NMR analysis (Figure S1b, Supporting Information) of the liquid showed that it is [C2mim][(C2)2OPO3]. When compared with the spectra of original IL (Figure S1a,



Supporting Information) obtained from Sigma Aldrich, the spectra of recovered IL was found to be same. 7g of [C2mim][(C2)2OPO3] was collected after extraction, compared to initially used 10 g, hence showing 70% recovery. Out of the 7g of recovered IL, 5 g was again used to solubilize 0.5 g of wet bacterial biomass which showed similar solubilizing ability and dissolved the biomass completely. Similar procedure was followed to recover the IL after PHAs precipitation. The recovered IL with 60% recovery, was again found to be [C2mim][(C2)2OPO3] from NMR analysis (Figure S1c Supporting Information). It is to be noted that no chemical degradation of the structure of IL occurred even after using for 2 cycles. Moreover, the methanol used for extraction could be used again after distillation, thus indicating economy of the process. Characterization of the recovered biopolymer The extracted white precipitates were dried at 70 oC to ensure complete solvent removal. The yield of PHAs extracted was found to be 60%± 2%. The dried precipitates were characterized using 1H-NMR, FT-IR, GPC and TGA techniques. The comparative 1H-NMR spectra of recovered biopolymer and commercial PHB (Sigma Aldrich) is presented in Figure 3.


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C

B A

Figure 3. Comparative 1H-NMR spectra of PHB (A) Sigma Aldrich (B) extracted from dried and (C) extracted from wet biomass using [C2mim][(C2)2OPO3]. The 1H-NMR spectra of PHB showed doublet at δ = 1.28 and 1.27 ppm due to methyl group (1) coupled to one proton (3), doublet of quadruplet at δ = 2.58 and 2.49 ppm, attributed to the methylene group (2) adjacent to an asymmetric carbon, multiplet at δ = 5.25 ppm is characteristic due to methine group (3). The recovered biomass showed all chemical shift values and peak pattern as found in Sigma Aldrich PHB. Additional peaks observed in the range δ = 3.5 to 4.7 can be attributed to residual presence of ionic liquid or small residues of fragmented protein or amino acids remaining in the biomass.38 The microstructure of recovered polymer was further confirmed from FT-IR spectra (Figure 4A-C).



Figure 4. Comparative FT-IR spectra of PHB (A) Sigma Aldrich (B) extracted from dried and (C) extracted from wet biomass using [C2mim][(C2)2OPO3]. The FT-IR spectra of PHB (Sigma Aldrich) showed characteristic peaks, ν-OH str. at 3436 cm-1, ν-CH2 and ν-CH3 str. at 2880 to 2977 cm-1, ν-C=O str.at 1724 cm-1 and ν-C-O str. at 1000-1300 cm-1. The recovered biomass (both dried and wet) showed all characteristic peaks, corresponding to that observed for PHB (Sigma Aldrich). Hence, it was confirmed that the recovered biomass is in fact PHB. Peaks corresponding P-O or P=O vibration of phosphate group of IL (Figure S2 Supporting information) in the region of 700 to 1250 cm -1 could not observed in the recovered biomass. Thus, showing elimination of IL. Additional peak observed in the region of 1550 to 1650 cm-1 can be accounted to the N-H bending vibrations and NH3+ deformation peaks of amino acid impurities.39 Thus, both 1H-NMR and FT-IR spectra indicated slight impurities of fragmented proteins or amino acids in the recovered biomass. The molecular weight of recovered PHB was measured from GPC analysis and found to be Mw = 163.88 k Da with polydispersity index of 1.96. The Mw for the recovered biomass has been found to be less than the one recovered from sodium hypochlorite extraction method (Mw = 432 kDa).30 Therefore, it seems that IL extraction leads to fragmentation of PHB into


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small Mw compounds. On a positive note this method is suitable for the extraction of small molecular weight PHB. Thermal stability of the recovered PHB in comparison with commercial PHB was monitored from thermo gravimetric analysis (TGA) and is presented in Figure 5.

105

0

90

-7

75

-14

60

-21

45

-28

30

DTG%

TG %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


-35 Sigma Aldrich DRY WET

15 0

-42 -49

60

120

180

240

o

300

360

420

T/ C

Figure 5. Comparative TGA profiles of PHB of Sigma Aldrich, extracted from dried and extracted from wet biomass using [C2mim][(C2)2OPO3]. The thermal degradation temperature of recovered PHB from dried and wet biomass was found to be 281 oC and 288 oC respectively, which is higher than 264 oC observed for commercial PHB. The rise in degradation temperature for observed biomass can be accounted to slight strengthening of intra molecular bonding upon interaction with IL or amino acid impurities. The degradation temperature was comparable to that extracted through 4% sodium hypochlorite extraction process from both dried and wet biomass. To assess the presence of cell debris in the extracted samples we carried out elemental analysis (Table 1).



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Table 1. Comparative CHNS analysis of PHB extracted from biomass and obtained from Sigma.

Sample

Elemental analysis

PHA

%C

%H

%N

%S

Standard PHB (Sigma Aldrich)

55.48

9.053

0.00

0.00

Recovered PHB from wet biomass

54.57

7.527

0.95

0.00

The content of C, H, N, S in the sample is an indirect indicator of the impurities present in the extracted polymer. The extracted samples analysed were found to have nitrogen content of 0.95%. The nitrogen content indicates the presence of amino acid impurities in the recovered biomass as observed from 1H-NMR and FT-IR spectra. This % age of N corresponds to 14% amino acid impurity, thus percentage purity of PHB in the recovered biomass is 86%. Preparation of polymeric bio-plastic from recovered biomass To utilize the recovered polymer for the preparation of bio-plastic film we adopted solvent casting method. The film was prepared from the PHB recovered from wet biomass. The recovered PHB was dissolved in chloroform and poured into a watch glass. The solvent was allowed to evaporate under room temperature and a film was obtained after evaporation. The digital images of the film formed are provided in Figure 6. As can be seen the film is showing good transparency. When compared to PHB film prepared from Chloroform or acetic acid as reported in literature the transparency of the prepared film is in par with the reported ones.40


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Figure 6. (A), (B) and (c) Digital images of the bioplastic film prepared from PHB recovered from wet biomass in different views. The prepared film was characterized by using FT-IR, DSC, TGA, and DMA. The FT-IR spectrum with 2-D images of the bio-plastic film is presented in Figure 7.

(B)

(A)

ν-C-O, 1274 cm-1

ν-C=O, 1727 cm-1

(C)

(D)



Figure 7. FT-IR spectra of the bio-plastic film prepared from recovered PHB of wet biomass; (A) 2-D FT-IR spectral image of film (B) FT-IR spectra of the film showing region between 2500-1000 cm-1 (C) 2-D images of film indicating C=O str. region at 1727 cm -1 (D) 2-D images of film indicating C-O str. region at 1274 cm-1 The FT-IR spectra and spectral images of the film showed characteristic functional group peaks corresponding to PHB. The ν-C=O str. due to carbonyl was observed at 1727 cm-1 whereas the ν-C-O str. due to glycosidic linkage of PHB was observed at 1274 cm -1 (Figure 6). Similar peaks were observed for the PHB recovered from chloroform from the same source of bacteria using chloroform.30 It is to be noted that film did show the peak corresponding to amino acid impurities with very low intensity, however, bulk of the spectra corresponds to PHB. It is possible that amino acids impurity might be present in bonded situation with the PHB. The thermal stability of the prepared film was observed from DSC and TGA profiles (Figure 8). It is to be noted that in the melting temperature range, thermal motions in the film increases which ultimately lead to melting of the polymer chains under thermal stress.

(A)

100

0

Tm= 175.56 C

-0.3

-1

-1

Cp = 2.38 J.g .K

TG %

-1

o

-0.6

-0.9

80

-6

60

-12

40

-18

20

-24

0

-30

(B)

-1.2 0

60

o

120

60

180

120 180 240 300 360 420 480 o

T/ C

T/ C

Figure 8. Thermal analysis profiles of the prepared bioplastic film (A) DSC (B) TGA The DSC thermogram of the prepared film (Figure 7A) showed good thermal stability with melting temperature of 175.16oC. The melting temperature is comparable to the literature 16 ACS Paragon Plus Environment

DTG %

0.0

Exo

DSC / mW.mg

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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value of commercial PHB film casted using acetone (T m = 180 oC) 36 which shows that amino acids impurity does not affect the film structure to a greater extent. The film melted with endothermic enthalpy changes with the heat capacity (ΔC p) at Tm of 2.38 J.g-1.K-1. Further analysis of the thermal stability of film was carried out using TGA (Figure 7B). The thermal degradation temperature of the film was found to be 235.15 oC which is less than the one reported in literature (295 oC).36 The difference can be accounted to the film casting procedure, otherwise the degradation temperature observed for the recovered PHB is 288 oC which close to the literature value. Therefore, high melting and degradation temperatures observed for the prepared film indicated that it possesses a good thermal stability. The mechanical strength of the film was monitored from storage modulus (E´) vs temperature profile obtained via dynamic mechanical analysis (DMA), as presented in Figure 9.

1.2 1.0

E' / G.Pa

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.8 0.6 0.4 0.2 0.0 0

50

100

150

200

250

300

o

T/ C

Figure 9. Dynamic mechanical analysis (DMA) presented as storage modulus (E´) vs temperature plot. Storage modulus (E´) is defined as the stress in phase with the strain in a sinusoidal shearing deformation divided by the strain. High magnitude of E´, is an indicative of higher mechanical strength of the film. The E´ = 0.86 G.Pa at room temperature indicates good strength of the prepared film.40 The strength however decreased with the increase in 17 ACS Paragon Plus Environment


temperature which can be accounted to the increase in thermal motion of molecules leading to uncoiling of different layers. The E´ became constant at around 138 oC which indicates the arrival of melting temperature. It is to be noted that melting temperature observed was a bit higher (175 oC) in DSC experiments which can be accounted to the sensitivity of different instruments. Upon comparison to PHB film prepared from chloroform or acetic acid, these films have also been reported to undergo change in their elastic modulus at around 140 oC, which is not much dissimilar change in storage modulus temperature of our film (138 oC). Over all schematic of the entire process is depicted in Scheme 1.

Scheme 1. Schematic showing extraction of PHB from bacterial biomass using IL; 1-Ethyl-3methylimidazolium diethylphosphate in a downstream process and consequent preparation of bioplastic film of PHB and recovery of IL.


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Conclusion We have developed an eco-friendly and cost-effective process for the extraction of polyhydroxybutyrate (a potential bio-degradable plastic material) from marine bacteria (both dried and wet biomass) of Halomonas hydrothermalis. The extractant used is a low vapour pressure IL; 1-Ethyl-3-methylimidazolium diethylphosphate. The IL solubilized the bacterial biomass with ease on account of its higher hydrogen bond basicity (β= 1.07) which assisted it in forming new hydrogen bond and breaking the existing one in bacterial cell membrane, leading to solubilization. PHB was precipitated from the solution using methanol with the recovery of IL. The % age purity of the PHB was found to be 86% from CHNS analysis. The IL was used for two consecutive cycles to solubilize the biomass and could be used further since, it did not lose its structural integrity during dissolution thus, minimizing its high cost in the process. Moreover, a plastic film was prepared from the PHB recovered from wet biomass. The prepared film showed good structural integrity with thermal and mechanical stability hence, it can be utilized as potential bio-plastic. We need to further optimize the entire process to use it at industrial scale with possible 100% efficiency.

Author Information Corresponding Authors *Tel.: +91-278-2567760. E-mail: [email protected] (S.M.). *E-mail: [email protected] (A.K.). ORCID Sandhya Mishra: 0000-0002-2412-4927 Arvind Kumar: 0000-0001-9236-532X



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Acknowledgements The manuscript has been assigned the PRIS number PRIS 062/2017. Authors would like to acknowledge analytical discipline CSIR-CSMCRI for the help in characterization of the extracted samples. SD would like acknowledge CSC 0203 for financial support and MKBU for PhD registration. PSG acknowledges UGC SRF for financial support and AcSIR for PhD registration.

References 1. Doi, Y.; Steinbüchel, A. Biopolymers. 2002. Weinheim, Germany: Wiley-VCH. 2. Rodriguez-Valera, F.; Lillo, J. A. G. Halobacteria as producers of polyhydroxyalkanoates FEMS Microbiol. Rev., 1992, 103, 181–186. DOI: 10.1016/0378-1097(92)90308-B 3. Escalona, A. M.; Varela, F. R.; Gomis, A. M. US Patent 5536419, 1996. 4. Koller, M.; Niebelschütz, H.; Braunegg, G. Strategies for recovery and purification of poly[(R)-3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass. Eng. Life Sci. 2013, 13, 549–562. DOI: 10.1002/elsc.201300021 5. Quines, L. K. M; Schmidt, M; Zanfonato, K; Schmidell, W; Aragão, G. M. F. Química Nova., 2015, 38, 1207-1218. DOI: 10.5935/0100-4042.20150129 6. Jacquel, N.; Lo, C.-W.; Wei, Y.-H.; Wu, H.-S.; Wang, S. S. Isolation and purification of bacterial poly(3-hydroxyalkanoates). Biochem. Eng. J. 2008, 39, 15–27. DOI: 10.1016/j.bej.2007.11.029 7. Madkour, M. H.; Heinrich, D.; Alghamdi, M. A.; Shabbaj, I. I.; Steinbüchel, A. PHA recovery

from

biomass.

Biomacromolecules,

2013,

14,

2963–2972.

DOI:

10.1021/bm4010244 8. Noda, I., and Schechtman, L. A. U.S. Patent No. 5,942,597. 1999. Washington, DC: U.S. Patent and Trademark Office. 20 ACS Paragon Plus Environment

Page 21 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


9. Koller, M.; Bona, R.; Chiellini, E.; Braunegg, G. Extraction of short-chain-length poly[(R)-hydroxyalkanoates] (scl-PHA) by the ‘‘anti-solvent’’ acetone under elevated temperature and pressure. Biotechnol Lett. 2013, 35, 1023–1028. DOI: 10.1007/s10529013-1185-7 10. Samorì, C.; Basaglia, M.; Casella, S.; Favaro, L.; Galletti, P.; Giorgini, L.; Marchi, D.; Mazzocchetti, L.; Torri, C.; Tagliavini. E. Dimethyl carbonate and switchable anionic surfactants: two effective tools for the extraction of polyhydroxyalkanoates from microbial biomass. Green Chem., 2015, 17, 1047-1056. DOI: 10.1039/C4GC01821D 11. Arikawa, H.; Sato, S.; Fujiki, T.; Matsumoto, K. Simple and rapid method for isolation and quantitation of polyhydroxyalkanoate by SDS-sonication treatment. J Biosci Bioeng. 2017, 124, 250-254. DOI: 10.1016/j.jbiosc.2017.03.003 12. Niedermeyer, H.; Hallett, J. P.; Villar-Garcia, I. J.; Hunt, P. A.; Welton, T. Mixtures of ionic liquids. Chem. Soc., Rev. 2012. 41, 7780-7802. DOI: 10.1039/C2CS35177C 13. Bharmoria, P.; Damarla, K.; Trivedi, T. J.; Malek, N. I.; & Kumar, A. A reciprocal binary mixture of protic/aprotic ionic liquids as a deep eutectic solvent: physicochemical behaviour and application towards agarose processing. RSC Adv. 2015. 5, 99245-99252. DOI: 10.1039/C5RA22329F 14. Rogers, R. D., Seddon, K. R., Volkov, S. NATO Science Series, Kluwer, Dordrecht, 2002. 15. Brandt, A.; Gräsvik, J.; Hallett, J. P.; & Welton, T. Deconstruction of lignocellulosic biomass

with

ionic

liquids.

Green

Chem.

2013.

15(3),

550-583.

DOI:

10.1039/C2GC36364J 16. Fukaya, Y.; Hayashi, K.; Wada, M.; Ohno, H. Cellulose dissolution with polar ionic liquids under mild conditions: required factors for anions. Green Chem. 2008, 10, 44-46. DOI: 10.1039/B713289A



Page 22 of 25

17. Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. Dissolution of cellulose with ionic liquids. J. Am. Chem. Soc. 2002. 124, 4974-4975. DOI: 10.1021/ja025790m 18. Zhu, S.; Wu, Y.; Chen, Q.; Yu, Z.; Wang, C.; Jin, S.; Dinga, Y.; Wu, G. Dissolution of cellulose with ionic liquids and its application: a mini-review. Green Chem. 2006. 8, 325327. DOI: 10.1039/B601395C 19. Trivedi, T. J.; Kumar, A. Efficient extraction of agarose from red algae using ionic liquids. Green and Sustainable Chem. 2014. 4, 190-201. DOI: 10.4236/gsc.2014.44025 20. Minnick, D. L.; Flores, R. A.; De Stefano, M. R.; Scurto, A. M. Cellulose solubility in ionic liquid mixtures: temperature, cosolvent, and antisolvent effects. J. Phys. Chem. B. 2016, 120, 7906-7919. DOI: 10.1021/acs.jpcb.6b04309 21. Ohno, H.; Fukaya, Y. Task specific ionic liquids for cellulose technology. Chem. Lett. 2008, 38, 2-7. DOI: 10.1246/cl.2009.2 22. Bihari, M.; Russell, T. P.; & Hoagland, D. A. Dissolution and dissolved state of cytochrome c in a neat, hydrophilic ionic liquid. Biomacromolecules. 2010, 11, 29442948. DOI: 10.1021/bm100735z 23. Tamura,

K.;

Nakamura,

N.;

Ohno,

H.

Cytochrome

c

dissolved

in

1‐allyl‐3‐methylimidazolium chloride type ionic liquid undergoes a quasi‐reversible redox reaction up to 140 °C. Biotech. Bioeng. 2012, 109, 729-735. DOI: 10.1002/bit.24357 24. Idris, A.; Vijayaraghavan, R.; Patti, A. F.; Mac Farlane, D. R. Distillable protic ionic liquids for keratin dissolution and recovery. ACS Sustain. Chem. Eng. 2014, 2, 18881894. DOI: 10.1021/sc500229a 25. Tomlinson, S. R.; Kehr, C. W.; Lopez, M. S.; Schlup, J. R.; Anthony, J. L. Solubility of the corn protein zein in imidazolium-based ionic liquids. Ind. Eng. Chem. Res. 2014, 53, 2293-2298. DOI: 10.1021/ie403659x


Page 23 of 25 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


26. Bharmoria, P.; Kumar, A. Unusually high thermal stability and peroxidase activity of cytochrome c in ionic liquid colloidal formulation. Chem. Commun. 2016, 52, 497-500. DOI: 10.1039/C5CC05722A 27. Strassburg, S.; Bermudez, H.; Hoagland, D. Lysozyme solubility and conformation in neat ionic liquids and their mixtures with water. Biomacromolecules. 2016, 17, 22332239. DOI: 10.1021/acs.biomac.6b00468 28. Zhang, S.; Qi, X.; Ma, X.; Lu, L.; Deng, Y. Hydroxyl ionic liquids: the differentiating effect of hydroxyl on polarity due to ionic hydrogen bonds between hydroxyl and anions. J. Phys. Chem. B. 2010, 114, 3912-3920. DOI: 10.1021/jp911430t 29. Zavrel M.; Bross D.; Funke M.; Büchs J.; Spiess A. C. High-throughput screening for ionic liquids dissolving (ligno-) cellulose. Bioresour. Technol. 2009, 100, 2580-2587. DOI: 10.1016/j.biortech.2008.11.052 30. Rabideau, B. D.; Ismail, A. E. Mechanisms of hydrogen bond formation between ionic liquids and cellulose and the influence of water content. Phys. Chem. Chem. Phys. 2015, 17, 5767-5775. DOI: 10.1039/C4CP04060K 31. Birkner C.; von der Eltz, H.; Soukopova, M. Reagents for lysis of bacterial cells, US Patent 2016, No. US20160264925 A1. 32. S. E. Hecht, R. L. Niehoff, K. Narasimhan, C.W. Neal, P. A. Forshey, D. V. Phan, A. D. M. Brooker, K. H. Combs, EP1874996 A2, 2008, PCT/US2006/015152. 33. Bharadwaj V.; Shrivastav, A.; Dubey, S.; Ghosh, T.; Paliwal, C.; Maurya, R.; & Mishra, S. Draft genome sequence of Halomonas hydrothermalis MTCC 5445, isolated from the west

coast

of

India.

Genome

Announc,

2015.

3,

e01419-14.

DOI:

10.1128/genomeA.01419-14 34. Shrivastav, A.; Mishra, S. K.; Pancha, I.; Jain, D.; Bhattacharya, S.; Patel, S.; Mishra, S. Biodegradability studies of polyhydroxyalkanoate (PHA) film produced by a marine



Page 24 of 25

bacteria using Jatropha biodiesel byproduct as a substrate. World J Microbiol Biotechnol. 2011, 27, 1531-1541. DOI: 10.1007/s11274-010-0605-2 35. Bera, A.; Dubey, S.; Bhayani, K.; Mondal, D.; Mishra, S.; Ghosh, P. K. Microbial synthesis of polyhydroxyalkanoate using seaweed-derived crude levulinic acid as conutrient.

Int.

J.

Biol.

Macromolec.

2015,

72,

487-494.

DOI:

10.1016/j.ijbiomac.2014.08.037 36. Ghosh, P.K.; Mishra, S.; Gandhi, M.R.; Upadhyay, S.C.; Paul, P.; Anand, P.S.; Popat, K. M.; Shrivastav, A.; Mishra, S. K.; Ondhiya, N.; Maru, R. D.; Gangadharan, D.; Brahmbhatt, H.; Boricha, V.P.; Chaudhary, D. R.; Rewari, B. Integrated process for the production of Jatropha methyl ester and by-products, European Patent, 2014, 2, 475, 754. 37. Kunasundari, B.; Sudesh, K. Isolation and recovery of microbial polyhydroxyalkanoates. eExpress Polym. Lett. 2011, 5, 620-634. DOI: 10.3144/expresspolymlett.2011.60 38. Sindhu, R.; Ammu, B.; Binod, P.; Deepthi, S. K.; Ramachandran, K. B.; Soccol, C. R.; Pandey, A. Production and characterization of poly-3-hydroxybutyrate from crude glycerol by Bacillus sphaericus NII 0838 and improving its thermal properties by blending with other polymers. Braz Arch Biol and Technol. 2011, 54, 783-794. DOI: 10.1590/S1516-89132011000400019 39. Leifer, A.; Lippincot, E. R. The infrared spectra of some amino acids. J. Am. Chem. Soc., 1957, 79, 5098-5101. DOI: 10.1021/ja01576a006 40. Anbukarasu, P.; Sauvageau D.; Elias, A. Tuning the properties of polyhydroxybutyrate films using acetic acid via solvent casting. Sci. rep. 2015, 5:17884, 1-14. DOI: 10.1038/srep17884 41. Herring, H. Lockheed Martin Engineering & Sciences, Hampton, Virginia. 2003. NASNCR-2 12422.


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Green approach for extracting polyhydroxyalkanoates using ionic liquids.


Polyhydroxyalkanoates - ACS Publications - American Chemical Society

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