Tannery trimming waste based biodegradable bioplastic: Facile synthesis and characterization of properties

Journal Pre-proof Tannery trimming waste based biodegradable bioplastic: Facile synthesis and characterization of properties Vimudha Muralidharan, Michael Selvakumar Arokianathan, Madhan Balaraman, Saravanan Palanivel PII:

S0142-9418(19)31166-3

DOI:

https://doi.org/10.1016/j.polymertesting.2019.106250

Reference:

POTE 106250

To appear in:

Polymer Testing

Received Date: 5 July 2019 Revised Date:

16 November 2019

Accepted Date: 23 November 2019

Please cite this article as: V. Muralidharan, M.S. Arokianathan, M. Balaraman, S. Palanivel, Tannery trimming waste based biodegradable bioplastic: Facile synthesis and characterization of properties, Polymer Testing (2019), doi: https://doi.org/10.1016/j.polymertesting.2019.106250. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Tannery trimming waste based biodegradable bioplastic: Facile synthesis and characterization of properties Vimudha Muralidharana, Michael Selvakumar Arokianathana, Madhan Balaramanb,*, and Saravanan Palanivelc, a

Leather Process Technology Division (LPTD), CSIR- Central Leather Research Institute, Adyar, Chennai, 600 020, India b

Centre for Academic and Research Excellence (CARE), CSIR- Central Leather Research Institute, Adyar, Chennai, 600 020, India

c

Project Planning and Business Development (PPBD), CSIR- Central Leather Research Institute, Adyar, Chennai, 600 020, India

*

Corresponding author information

B. Madhan Principal Scientist, Centre for Academic and Research Excellence CSIR-CLRI, Adyar, Chennai – 600 020 India Email id: [email protected]; [email protected] Contact number: +91-44-2443 7169

1

ABSTRACT

In the present research, the untanned proteinaceous trimming waste from tanneries was used to prepare highly flexible and transparent bioplastic films. Composite bioplastic films were fabricated by blending trimming hydrolysate powder and polyvinyl alcohol using the solution casting method. In addition, a non-toxic and relatively inexpensive bio-crosslinker – citric acid was used as a plasticizer / crosslinking agent. The effects of citric acid concentration on the mechanical properties, thermal stability, transparency and anti-microbial properties of the bioplastic films were investigated. Crosslinking interactions by the citric acid on the constituents of the bioplastic were confirmed using FTIR/ATR. Also, the surface microstructure of the films was studied using SEM. The resultant bioplastic films were smooth, uniform and defect-free. Citric acid used in the bioplastic blend formulation clearly acted as a plasticizer at higher concentrations. The trimming waste-based bioplastic with the citric acid concentration of 40% exhibited an outstanding tensile strength above 20 MPa and extremely high elongation at break value greater than 343%. The bioplastic degraded to an extent of 62% within 70 days under the soil burial test. The transparency of the bioplastics was comparable with the LDPE and PP-like conventional plastics. The anti-microbial properties of the films are the positive aspects brought about by the presence of citric acid interactions. Consequently, trimming based bioplastics may become a future friendly alternative to fossil derived plastics having applicability in packaging, wound healing and other biocompatible applications.

Keywords: Tannery wastes; bioplastic; citric acid; biodegradable; antimicrobial plastic

2

1. Introduction Leather is one of the important sectors globally, which contributes for major economic development of a country. Nonetheless, the process of conversion of raw hides or skins into non-putrescible leather involves usage of large volume of water, chemicals and mechanical processes to remove unwanted components so as to meet the quality standards of leather. The leather manufacture process, in turn, generates a variety of solid wastes and stream of wastewater [1-4]. The leather processing includes unit operations viz. pretanning, tanning, post tanning and finishing. It is reported that about 1000 kg of wet salted hides when processed would yield only 200 kg of leather and about 700 kg of solid wastes are generated during the conversion [5]. The solid wastes generated from the leather industry can be classified into untanned and tanned wastes [6]. The raw trimmings, fleshing wastes (green and limed) and keratin wastes fall into the untanned wastes category. The chrome shaving, chrome buffing dust and leather trimmings make up the tanned wastes of leather industry. The earlier disposal methods of these wastes included incineration, combustion and landfilling. These wastes are primarily protein rich organic components that face the unsafe disposal issues thereby causing environmental pollution. Owing to the resource potential of these wastes, they have been identified as “new raw materials” by many researchers as well as industries [7]. Of all the untanned wastes, the trimming wastes are of prime interest owing to its high collagen content. Masilamani et al., 2016 have stated that about 300,000 ton of raw trimmings waste generated in the leather industry, contains a minimum of 20% w/w collagen [8]. These raw trimming wastes have been used for the production of glue, industrial gelatin, feed and fertiliser [9,10]. On the other hand, about 300 million tons of petroleum based plastic is produced every year globally and approximately 8 million tons of plastic end up in the oceans each year [11]. Biodegradable plastics or bioplastics with characteristics matching up to the conventional

3

ones have been proposed as their alternatives. Turning industrial wastes into useful material for industrial applications will aid in circular economy. Such a benevolent approach can be adopted in case of leather processing industry to tackle the twin issue of accumulating leather solid waste and plastic pollution. Biopolymer blends with collagen and collagen hydrolysates for replacing petroleum based plastics have been studied [12-14]. However, the protein based films have poor mechanical properties and have low water stability owing to its hydrophilic nature. A noteworthy method to overcome this problem is to blend these protein fragments with synthesized polymeric gel networks such as polyvinyl alcohol (PVA). PVA provides multiple properties to the biodegradable plastic blend system which includes film forming capabilities [15], chemical resistance, optical properties and physical properties [16]. PVA has been used to prepare composite films with collagen/gelatin and the characteristics of the films have been elucidated extensively [17,18]. In order to make the protein based bioplastic system more workable and comparable to that of fossil based counterparts, it is necessary to incorporate plasticizer and a crosslinker to the system. Crosslinking of gelatin obtained from the leather waste by chemical crosslinkers such as glutaraldehyde, oxazolidine II, ethylene glycol diglycidyl ether (EGDE) and hexamethyl diisocyanate (HMDC) has revealed that these chemicals reduce the glass transition temperature of the films, imparting flexibility [19]. Glutaraldehyde based crosslinking systems are inexpensive, efficient but unsafe since they are known to cause pollution [20]. Other crosslinking agents such as genipin were proved safe, had low crosslinking efficiency but found to be expensive [21]. Polycarboxylic acids have been proved effective in cross-linking proteins. These polycarboxylic acid are usually inexpensive, can be derived from natural products with large availability, and have low toxicity [22], and thus could be prospective non-toxic alternative cross-linkers. Citric acid with one hydroxyl and three carboxyl group exists widely in citrus fruits which can be a non toxic and inexpensive alternative to the chemical crosslinkers.

4

Citric acid has been used in several studies for crosslinking of polysaccharides [23-25]. As a multicarboxylic acid, an esterification reaction could take place between the carboxyl groups of citric acid and hydroxyl groups of the protein, thus enhancing the water resistibility of the films [26]. As compared to the hydroxyl groups on glycerol (commonly used plasticiser), the carboxyl groups on citric acid can form stronger hydrogen bonds with the hydroxyl groups protein polymer blends, thus improving the interactions between the molecules and thus acting as a plasticiser too [27, 28]. The mechanism of crosslinking and the effect of its concentration on the plant protein based biodegradable plastics have been explored [29]. Crosslinking of protein from animal origins such as casein, keratin and gelatin have also been attempted with the citric acid. Citric acid and a catalyst based crosslinking of casein fibers improved their wet stability and pH stability in the range of 3.0-9.0 [30]. Compression moulded thermoplastics made from chicken feather wherein citric acid and sodium hypophosphite catalyst behaved as crosslinker system, which improved the dry tensile strength of the material thereby making it useable for applications such as packaging, biopolymeric applications, agricultural mulches and biomaterials etc. [31]. The wet and dry crease recovery angle of the citric acid treated wool fabric was found to be increased. The interaction between wool protein and citric acid made the wool fabric antimicrobial in nature [32]. Citric acid acted as a plasticizer yielding higher elongation at break values for wool keratin based bioplastic materials [33]. Wet spun gelatin fibers crosslinked using citric acid was found to have higher crosslinking densities than the heat crosslinked fibers, owing to the reactivity between the amine groups of gelatin and citric acid [34]. Recently, a method of intra-fibrillar crosslinking in electrospun marine collagen fibers by the citric acid (with high temperature annealing) increased the wet stability of the collagen fibers [35]. The present work aimed at utilizing the trimming hydrolysate from tannery solid wastes for the preparation of bioplastic material in the presence of a non-toxic bio-crosslinker. The

5

effect of varying concentrations of citric acid on the mechanical, optical and antimicrobial properties were studied. The degradability of the bioplastic material under soil burial test was also studied.

2. Materials and Methods 2.1 Materials Raw animal skin trimmings were obtained from the pilot tannery at CSIR-CLRI, Chennai, India. Polyvinyl alcohol, which was 99% hydrolysed with an average polymerization degree of 1700-1800, was purchased from Loba Chemie Pvt. Ltd (Maharashtra, India). Citric acid anhydrous was purchased from SRL chemicals (Chennai, India). Microbiological media components such as nutrient broth, agar, and yeast extract were purchased from Himedia Pvt. Ltd (Germany).

2.2 Methods

2.2.1 Pre-processing of raw skin trimmings Raw trimmings were washed, cleaned and rinsed thoroughly with water to remove dirt and salt. After soaking the trimmings in water for 6 h, the trimmings were treated with 5% lime [Ca(OH)2] and 3% Na2S (percentage weight based on total weight of trimmings) for dehairing in a pit for 24 h to accomplish unhairing and fiber opening of the trimming matrix. The limed trimmings were delimed in a sample drum with 2% ammonium chloride salt for 2 h to remove the lime and washed extensively with water until the pH reached 7.0-7.5. The delimed trimmings were then cut into small fragments (size 3 x 3 cm) for extraction of trimming hydrolysate. 2.2.2 Extraction of trimming hydrolysate

6

Trimming hydrolysate was prepared by hydrothermal hydrolysis. The delimed trimmings were mixed with distilled water in a 500 mL beaker at a ratio 1:2 (w/v) and kept for hydrolysis in an autoclave at temperature 121 °C and 15 psi pressure for 60 min, 120 min and 180 min time durations. After hydrolysis, the hydrolyzed mixture was left to cool down at room temperature overnight. The trimming hydrolysate was filtered out using a mesh cloth, separating it from the solid sludge and centrifuged at 10,000 rpm, 10 °C for 15 min. The supernatant was filtered using Whatmann No. 1 filter paper and lyophilized (LyoDel-Delvac, Tray type, Chennai, India) for 18 h, in order to obtain trimming hydrolysate powder (THP). Further characterization of the hydrolysate was done using both filtered supernatant as well as lyophilized sponge.

2.3 Characterization of Trimming Hydrolysate

2.3.1 Degree of hydrolysis (DH %) and Total protein content The degree of hydrolysis of trimming hydrolysate was determined by the percentage of soluble protein in 10 % (w/v) trichloroacetic acid (TCA) in relation to the total protein content of the sample as described by Hoyle and Merrit, (1994) with few modifications [36]. Trimming hydrolysate aliquots of 500 µL were mixed with 500 µL of 20% (w/v) of TCA solution to obtain the soluble and insoluble fractions in 10% TCA. The mixture was incubated for 30 min at room temperature and then centrifuged (Eppendorf Centrifuge 5810R) at 3000g for 10 min. The total protein content of the trimming hydrolysate aliquots was determined by the method of Lowry et al., 1951 [37]. The protein content of the supernatant (soluble protein) from the centrifugation was also determined by the same method. Bovine serum albumin (BSA) was used as a protein standard for Lowry’s protein

7

estimation. The Total protein content of the trimming hydrolysate was expressed in mg/mL. The degree of hydrolysis (DH) was calculated according to the Eq. 1. % =







10% ℎ

× 100

2.3.2 Characterization of THP The elemental composition of THP was determined using Elementar (Vario-Cube). The nitrogen content of the samples was determined by the Dumas method using the elemental analyzer. The THP exhibited gelling capability at 6-10 °C. The bloom/gel strength of the THP sample was determined using Texture analyser (Brookfield Engineering Lab, DVII, USA). The THP of concentration 6.67% (w/v) was prepared by homogenizing the sample in distilled water for an hour at 70 °C and stored at 10 °C for 17 h [38]. The sample was tested for gel strength as against a standard commercial gelatin solution (Nitta Gelatin India Ltd, India). The extent of hydrolysis of trimming wastes was determined by calculating the weight difference between the samples before and after hydrolysis. The percentage extent of hydrolysis is calculated by the Eq. 2 !



=

"

# $ℎ

"

%% $ − ' # $ℎ # $ℎ %% $

ℎ

× 100

The hydrolysate yield of the obtained lyophilized powder samples was calculated based on the weight ratio between the trimming hydrolysate powder (THP) and the weight of delimed trimmings (on dry weight basis). The moisture content of the delimed trimmings were determined by calculating the weight loss ratio between the initial weight and final weight after drying them at 105 °C for 24 h. The delimed trimmings has moisture content

8

ranging from 67 to 70%. The total solid content of the delimed trimming wastes is around 30%. The yield is calculated on dry weight basis, and expressed in percentage by Eq. 3 (

% =

) $ℎ

) $ℎ * $ % %% $ #

$

× 100

2.4 Preparation of Bioplastic Blend formulation Bioplastic films were prepared by solution casting method. THP and PVA were maintained at a constant concentration of 10% w/v in the bioplastic blend formulation and in the equal ratio of 50/50. THP and PVA were dissolved in distilled deionized water separately under stirring and then both were mixed together to form a blend of constant volume 15 mL. In succession, the citric acid was added to the mixture at a concentration ranging from 6% to 80% w/w (based on the total solids in the bioplastic blend) as given in Table S1 (Supplementary file). The bioplastic blend formulation (BBF) was stirred using a mechanical stirrer for 30 min at 50 °C and 350 rpm. The bubbles formed in the mixture was removed by sonication for 5 min and poured on a levelled polystyrene petri dish (90 mm diameter). It was left to evaporation at room temperature for 18 - 24 h. The dried bioplastic films were peeled off and stored in desiccator for at least 48 h prior to all other characterization, according to the ASTM D618-00 method [39].

2.5 Characterization of the Bioplastic films

2.5.1 Mechanical properties of the films The tensile strength and elongation were evaluated for all the bioplastic films with a Universal Testing Machine (Instron 3369/J7257, Controlled by Blue-hill software, version 3). The samples were conditioned at room temperature before sampling for tensile testing. Dumbbell shaped specimens were cut from the samples. The gauge separation was kept 50 9

mm. The crosshead speed was at 25 mm/min. [40]. The tests were carried out at 25 °C temperature and 50% relative humidity. The four samples were taken from each bioplastic film for the tensile testing and the averages with standard deviation were reported.

2.5.2 Thermal analysis of bioplastic films The thermal properties of the films were measured using a Differential Scanning Calorimeter (DSC) (Q200, TA instruments, Waters, Austria). The native trimming hydrolysate poweder (TH), polyvinyl alcohol (PVA) and two control films TH-CA and PVACA were taken for thermal analysis as well. All the samples were kept at room temperature before sampling for DSC. Sample weighing 5 ± 0.3 mg was cut from the film samples and was placed into the sample pan. The reference was an empty pan. Samples were scanned at a heating rate of 10 °C/min from 0 to 300 °C to determine the heat stability of the bioplastic films. The DSC scans were analyzed to determine the changes in melting point of the films.

2.5.3 Spectral characterization of the films (ATR-FTIR) FTIR spectra were recorded with a Fourier transform spectrophotometer (JASCO FT/IR 4200) appended with Attenuated Total Reflectance (ATR) technique. The samples were kept at room temperature before sampling. Film samples (10 mm x 10 mm) were placed on the ATR crystal to cover the crystal surface. The sample was gently squeezed by a screw to promote contact with the crystal. The FTIR spectra of the film samples were recorded in transmittance mode at the 4000-400 cm-1 region at a resolution of 4 cm-1 at room temperature. The angle of incidence for ATR crystal was 45°. The empty crystal was used as a background. The native THP powder was mixed with fine crystals of KBr and a pellet was made for spectral scanning.

10

2.5.4 Surface morphology of the films The microstructural analysis of the films was carried out by using a Scanning Electron Microscopy SEM (Phenom Pro). The film samples were preconditioned in a desiccator and cut into 1 cm x 1 cm size samples mounted onto aluminium stubs using double sided adhesive tape. The samples were sputter coated with a thin layer of gold. The samples were tested at an accelerating voltage of 10 kV. The samples were viewed at magnification levels starting from 500x up to 2500x.

2.5.5 Transparency of the films The transparency of the films was determined by percentage transmittance (%T) at 600 nm using a spectrophotometer (JASCO V660) according to method ASTM D1746-03 [41]. The bioplastic films were cut into 4 cm x 1 cm strips and placed in the glass cuvette. An empty cuvette (air blank) was used as a reference. Transparency (T600) was determined as per Eq. 5, T = log (%T)/δ

eq. 5

,where δ is the film thickness (in mm). Each sample was tested in duplicate and the results are presented as average with standard deviation.

2.5.6 Antibacterial activity by the films The trimming based bioplastic films were tested for in-vitro antimicrobial activity by agar overlay method as per the procedure presented in Kemme and Weiland, 2018 [42]. Briefly, the method combines the agar disc diffusion with a homogenous lawn of microbial cells within a thin layer of agar across the surface of the plate to ensure uniform microbial distribution. Based on the Kirby-Bauer disc susceptibility test, the method is adapted from seeded lawn overlay spot assay and activity was evaluated by determining the zone of growth

11

inhibition. The test microorganisms gram positive Staphylococcus aureus (S. aureus, ATCC 33592), gram negative Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853) and Escherichia coli (E.coli ATCC 25922) were maintained on nutrient agar and stored at 4 °C and refreshed weekly. The basal layer of the agar overlay plate comprised of nutrient agar (2.5% w/v nutrient broth and 1.5% w/v agar) poured on sterilized petri dishes to a depth of 34 mm and was allowed to solidify. The control bioplastic film was prepared with THP, polyvinyl alcohol and glycerol as plasticizer (for film forming), without citric acid crystals. Discs of diameter 1 cm were punched out from the bioplastic films for evaluation. For agar diffusion, each disc was placed onto surface of basal layer and overlaid with 10 mL of seeded soft agar (0.5 % w/v agar and 0.3 % w/v yeast extract) pre-warmed to 42 °C. The soft agar was seeded with 1 mL of 24 h grown liquid inoculum of bacteria (standardized to 106 – 107 cfu/mL). After solidification of the agar, the plates were incubated at 30 °C for 18 h for observing the zone of clearance. The width of the clear zone was determined using the formula: )

ℎ



/ =

0



ℎ

.



%



&

/ 2

−

.



%



3



All the experiments were conducted in triplicates and the data reported are the averages of the readings.

2.5.7 Degree of biodegradation of bioplastic films The degree of degradation for the tannery waste based bioplastic film was determined by soil burial test. The bioplastic film with 40% citric acid (THPCA40) and a control PVA film was selected for the test. The films were cut into 2 x 2 cm2 pieces which were pre-dried in a hot air oven at 80 °C for 4 h and then weight noted as W1. The film was put in a gauze

12

cloth pocket with the labelling for identification purpose. The pockets with films were buried 6 cm from the surface of the soil in a pot with moistened mud (50% moisture) from CSIRCLRI garden. After certain time interval, each film was washed with water and dried at 105 °C to a constant weight W2. The degree of degradation was calculated by the weight loss ratio of the particular sample, as given in the equation. Degree of degradation % =

W1 − W2 × 100 W1

3. Results and Discussion 3.1 Characteristics of trimming hydrolysate The trimming wastes were thermally hydrolysed for 60, 120 and 180 min under the autoclave conditions and were labelled as T1H, T2H and T3H respectively. The degree of hydrolysis, the extent of hydrolysis, hydrolysate yield and nitrogen content are given in Table 1. The supernatant was used for determining the degree of hydrolysis. The degree of hydrolysis was found to increase with time. The total protein content of each of the hydrolysate was found to be between the 24.89 and 34.85 mg/mL (data not shown). All of the hydrolysates were lyophilized to obtain moisture free trimming hydrolysate sponges, sealed in polyethene bag until further use. Most of the proteins that have been isolated from animal tissue contain about 16-18% nitrogen [43]. Gelatin is known to contain about 17% nitrogen [43]. The nitrogen content of the hydrolysates, ranged between 16.48% and 17.16 %, was similar to that of gelatin obtained from meat sources. From Table 1, it is evident that trimming hydrolysates were found to be highly nitrogenous with higher solubility and high water binding capacities.

13

Table 1 Characteristics of Trimming hydrolysate from tannery trimming wastes Sample Code (w:v)

Degree of Hydrolysis (%)

Extent of Hydrolysis (%)

Weight of THP (g)

Yield (%)

Nitrogen Content (%)

T1H (1:2)

23.22 ± 0.68

55.31

18.25

60.83

17.16 ± 0.65

T2H (1:2)

22.03 ± 0.25

78.91

22.24

74.13

16.39 ± 0.24

T3H (1:2)

28.89 ± 0.71

80.48

24.06

80.2

16.48 ± 0.43

Since the hydrolysates had characteristic similarity to the gelatin (based on nitrogen content), the gel strength of trimming hydrolysates was also determined and compared with that of commercial gelatin. The gel strength of hydrolysate was found to be between 25 and 30 bloom (data not shown), which was very low compared to that of high gel strength commercial gelatin (>200). Low gel strength of the hydrolysates indicates their low film forming ability [38]. In order to aid film forming, polymer polyvinyl alcohol PVA was added to the bioplastic blend formulation.

3.2 Characteristics of Bioplastic films One of the hydrolysates with high protein content was chosen to make bioplastic films. The hydrolysate powder from 120 min hydrolysis (T2H) was chosen as THP for the preparation of bioplastic films. All three batches of hydrolysates were equally rich in nitrogen, but the film forming ability of T2H was found to be optimum for bioplastic application, hence it was chosen for further research. When the hydrolysate solution and polyvinyl alcohol was homogenized without citric acid (THPCA0), the sonication of the mixture separated the two polymers with an interfacial layer. This solution when cast onto petri plates, did not form uniform film on drying (broken, discontinuous films as seen in Fig 1a & 1b). Upon addition of citric acid, the film forming ability was improved and the citric

14

acid behaved as a compatibilizer between THP and PVA. The other bioplastic films with citric acid (THPCA06 up to THPCA 80), were found to be uniform, smooth and easy to peel off from the petri plates after drying. From the Fig. 1c and 1d, the uniformly dried, transparent bioplastic films (THPCA40) can be clearly observed, which shows the effect of citric acid.

Fig. 1.

3.2.1 Mechanical properties of the bioplastic films The tensile strength and elongation at break for the bioplastic films are given in Table 2. Mean thickness of 0.35 ± 0.04 mm was obtained from seven different samples. When citric acid concentration in bioplastic blend formulation was 6 and 8%, the tensile strength of the films was found to be 32.08 and 41.62 MPa respectively. Although the films had higher tensile strength, it was brittle and exhibited poor flexibility. The elongation at break values for these films was found to be very low ranging between 6 and 8 %. The films with citric acid concentrations of 10% and 20% also had very low elongation at break values at 5.33% and 8.43% respectively. Nevertheless, the bioplastic films THPCA10 and THPCA20 were physically robust and tough unlike the ones with lower concentrations. This could be due to the initiation of intermolecular interactions between the citric acid and the protein hydrolysate present in the system. When the concentration of citric acid increased further, enhancement in plasticization effect was observed, which is evident from the significant increase in elongation at break values of the bioplastic films. The elongation at break value of THPCA30 was found to be 69.95%, which is a much higher value than the films with lower concentrations. Similar trend of increasing elongation at break values and decreasing tensile strength values with addition of citric acid was observed when films with soybean residue

15

and citric acid were made along with a catalyst [44]. The THPCA40 bioplastic film had elongation at break value of 343.38% with good tensile strength (20.75 MPa). Furthermore, the citric acid concentration was doubled from 40% to 80%, to understand the effects of the excess acid on the bioplastic. Although, the 80% concentration of citric acid was practically too high for any workable application, it provided some insights on the interaction between the components. The maximum elongation at break value of 437.54% was obtained when 80% of citric acid was incorporated in the BBF wherein the tensile strength dropped to 11.15 MPa. The THPCA80 was found to be more flexible but with a tackier surface texture making it more hygroscopic and unsuitable for any practical applications. The low tensile strength and poor texture of that films inhibited further inclusions with increased citric acid concentration for the experimentation.

Table 2 Mechanical strength of Bioplastic films Sample Code THPCA06 THPCA08 THPCA10 THPCA20 THPCA30 THPCA40 THPCA80

Tensile Strength (MPa) 32.08 ± 2.68 41.62 ± 8.01 36.39 ± 7.07 35.34 ± 3.21 22.72 ± 4.30 20.75 ± 1.43 11.15 ± 0.12

Elongation at Break (%) 6.14 ± 1.12 8.25 ± 1.73 5.33 ± 1.47 8.43 ± 2.51 69.95 ± 66.73 343.38 ± 75.40 437.54 ± 42.17

The mechanical properties of the films showed that the citric acid acted majorly as plasticizer in the system. Plasticization effect of citric acid is attributed to increasing molecular mobility, making the polymeric networks less dense, caused due to decreased intermolecular forces [45,46]. Some studies have reported citric acid effects on films only as a crosslinking agent [47-49] while others reported it acts only as a plasticizer [50,51]. The present work reveals that both the effects coexist but in an inconsistent manner. At low

16

concentrations of citric acid, the crosslinking ability was exhibited whereas, at higher concentrations, the plasticization effect was predominant. Similar kind of duality was exhibited when different percentages of citric acid were added to starch and polyvinyl alcohol blends [52]. Citric acid acts as a plasticizer and crosslinker providing the desired properties to the films as required by the application. Citric acid crosslinking in melt extruded amylose system yielded more flexible and cohesive films without external plasticizer (glycerol) [53]. Crosslinking interaction of wool keratin and citric acid without any catalyst and heat curing showed elongation at break values up to 646% but with very low tensile strength (0.28 MPa) [33]. Crosslinking of peanut protein by citric acid increased tensile strength while decreasing the elongation at break values [54]. Similar kind of increased tensile strength was observed when sesame protein was crosslinked by citric acid [55]. The tensile strength of the bioplastic films from trimming wastes was comparable to those of commodity plastic films such as high density polyethylene HDPE (22–23 MPa), low density polyethylene LDPE (19–44 MPa), and polypropylene PP (31–38 MPa) [56]. On the other hand, the values of elongation at break are analogous to some commercial and semicommercial bio-based materials (such as polylactate, wheat starch and corn starch) [57].

3.2.2 Thermal properties Differential Scanning Calorimetry (DSC) is one of the methods to establish the phase transition taking place in the composite films in the presence of heat. The DSC thermograms of the selected bioplastics are presented in Fig. 2a. Normally, a highly crosslinked polymer would be completely amorphous, i.e. DSC thermograms of these type of materials would not present endothermic events [58]. Nevertheless, the thermograms of all the films produced in the present study were typical of partially crystalline materials. The melting peak temperatures (Tm) for all four bioplastic films made from THP appeared between 195 and

17

215 °C. The decrease in crystalline melting temperatures is attributed to the increased plasticizer effect in case of bioplastics [59]. The broad melting peak in case of THPCA40 is indicative of the thermoplasticity induced by the plasticization effect of the citric acid. The DSC thermograms corroborate well with the results from the mechanical characteristics of the THPCA bioplastics, where the citric acid acts as a plasticizer at higher concentration. The glass transition temperature is a representative of the crosslinking interactions. The cross linking will limit the movement of molecular segments, resulting in increasing the Tg. The glass transition temperature increased from 34.5 °C to 79.4 °C when the concentration of the citric acid was increased from 10% to 20%. Further, the Tg was found to increase very slightly with addition of citric acid. At higher concentrations of citric acid, the plasticization effect was more pronounced than the crosslinking interactions. This was evident from the broad melting peak of THPCA40. The thermal pictograms of another system involving interactions between starch and citric acid in the presence of glycerol showed that there exists not much shift in Tm or Tg with increase in concentrations of citric acid, instead only the melting enthalpy decreased as the citric acid increased [52]. The crystalline melting temperature of native porcine skin gelatin film was found to be around 220 °C [60]. In Fig. 2b., the native trimming hydrolysate (TH) exhibits two endothermic melting peaks at 108 °C and 227 °C, respectively. The first peak can be attributed to the evaporation of absorbed moisture by the trimming hydrolysates and the second peak represents the melting temperature of the hydrolysate. On addition of citric acid to the THP, there also occurred two endothermic peaks which is not expected when crosslinking has occurred. The melting peak of the hydrolysate shifts slightly to the lower temperature indicating the plasticization effect of citric acid. The melting point of PVA without and with citric acid is also depicted in Fig. 2b. The peak at 218 °C is assigned to the melting point of PVA and there occur a slight shift in this peak temperature when citric acid

18

is added to the PVA crystals. The addition of citric acid on individual components of the bioplastic blend formulation gives no further insight related to its effect on the final bioplastic films. Fig. 2a. & Fig. 2b.

3.2.3 Spectral characteristics of the bioplastic film The spectra of the native trimming hydrolysate powder and bioplastic film THPCA40 are shown in Fig. 3. The peak position at around, 3296 cm-1 corresponds to the stretching of OH group because of intermolecular and intramolecular hydrogen bondings [61-63]. Citric acid is known for its high capacity to generate hydrogen bonds [64, 65]. Also, the amide A band was assigned to the stretching vibrations of N-H bonds at around at 3310 cm-1 [66]. The peaks at around 2929 cm-1 correspond to C-H stretching. The peak at 1715 cm-1 (marked by an arrow in Fig. 3) in THPCA40 film, indicate the formation of ester linkage causing C=O stretching vibration, suggesting crosslinking interaction between citric acid and hydrolysate [67,68]. The native THP and THPCA40 revealed characteristic peaks at around 1630 cm-1 and 1540 cm-1 indicating the C=O and N-H stretching, respectively [69,70]. Presence of these peaks indicates that there is some remnant citric acid present in the THPCA40 films even after esterification. The unreacted citric acid is found to be beneficial for the films. The intense peak found at 1081 cm-1 from the spectra of bioplastic was assigned to stretching of the C-O functional group [71]. Thus, FTIR spectrograms revealed that the esterification reaction caused by the citric acid in the bioplastic blend enabled crosslinking interactions in the system.

Fig. 3.

3.2.4 Surface morphology of the bioplastic films 19

The surface of the four bioplastic films was characterized for morphology using scanning electron microscope for their morphology. Microstructures can reflect homogeneity and compactness of the films. The visual examination of the films revealed that all of it was continuous films with smooth texture. The micrographs of the films at 500x and 2500x magnification are shown in Fig. 4. At 2500x, the THPCA10 exhibited a cracked surface signaling a brittleness caused by low plasticization effects. Further images of the surfaces of the bioplastic films with increasing citric acid concentration indicate homogenous matrices with clear surfaces. Analogously, the microstructure of the soybean residue films’ surface crosslinked with citric acid became smoother with increase in citric acid concentration [44]. These results validate well with the mechanical properties of the films. Fig. 4.

3.2.5 Transparency of the films Optical properties of films are an important attribute which influences its appearance, marketability, and their suitability for various applications [72]. The transparency values of the bioplastic films are given in Table 3. The transparency of the films was found to be increasing with increased citric acid concentration, thus lowering the opacity of the films. This could be due to an increase in total solids content in the bioplastic blend. The films made with gelatin and CMC showed a similar trend when an increased concentration of xanthan gum was added to the system [73]. The transparency values of the THP bioplastics were comparable with the conventional petroleum based plastics such as LDPE (2%) and polypropylene PP (6%) [74]. The transparency values of films prepared with fish gelatin and citric in the presence of glycerol were found to be in the similar range (0.5 – 6) [75]. Table 3 Transparency values Sample Code

Transparency

20

THPCA10

2.76 ± 0.38

THPCA20

3.27 ± 0.54

THPCA30

4.29 ± 0.81

THPCA40

4.51 ± 0.72

3.3 Antimicrobial activity The antimicrobial activity of THPCA films is shown in Fig. 5. The zone of inhibition (in cm) of the films against the selected test organisms is tabulated in Table 4. Antimicrobial activity of citric acid has been previously reported in the literature [76, 77]. A clear zone of inhibition was observed for all the sample discs of bioplastic against the microorganisms. The width of the clearance zones was observed to increase with increasing citric acid concentration. The widest clear zone of 0.825 cm was observed against S.aureus with THPCA40 film. As distinct from the results, Gram-positive bacteria (S. aureus) are more susceptible to the bioplastic discs than Gram-negative bacteria (E.coli). Nevertheless, the bioplastic films were successful in inhibiting the growth of both the Gram-positive and Gram-negative bacteria. The inhibition of the growth in the plates by the bioplastic discs was sustained for a maximum of 7 days. Table 4 Zone of Inhibition by the bioplastic films Sample Code Control (No Citric Acid) THPCA10 THPCA20 THPCA30 THPCA40

Width of Clear zones of inhibition (cm) against microorganism P. aeruginosa S. aureus E. coli -

-

-

0.06 ± 0.014 0.25 ± 0.014 0.37 ± 0.03 0.6 ± 0.07

0.09 ± 0.012 0.42 ± 0.03 0.67 ± 0.014 0.825 ± 0.17

0.03 ± 0.014 0.22 ± 0.02 0.22 ± 0.014 0.55 ± 0.07

Fig. 5.

21

3.4 Rate of degradation of tannery waste bioplastic To evaluate the applicability of the trimming waste bioplastic in industrial applications, the degradation of films was done by soil burial test. The results of soil degradation are depicted in Fig. 6. The observation of degradation under soil was done for 80 days, where the samples were checked for weight loss at particular intervals of time. Three random strips of dimension 2 x 2 cm2 was cut from the same THPCA40 film and the results are given as mean of the three samples, whereas PVA film was used as a control. The degradation profile was similar for both the PVA and THPCA40 films until the first two weeks of observation. The rate of degradation for THPCA40 film was consistently enhanced through the next 30 days. The overall surface of the films was found to be shrunken. On comparison to the PVA film, the THPCA showed maximum rate of degradation of 62% on the 70th day and there was no further weight loss recorded even after a week. This saturation in degradation rate could be attributed to the difficulty in breaking down the ester linkages by the citric acid. The protein enriched THPCA40 films were observed to more amenable source for the soil bacteria than PVA films. Fig. 6. 4. Conclusion The present work demonstrated a balanced way to utilize the tannery solid waste for preparation of bioplastic films and characterized it for its properties. The trimming hydrolysate containing composite blends were found to be sturdy, transparent and antibacterial in nature. The inclusion of multifunctional polycarboxylic acid led to significant changes in mechanical, morphological and antibacterial properties of the bioplastic films. Increasing citric acid content caused a reduction in tensile strength but enhanced the flexibility, thereby resulting in an increased elongation at break values greater than 400%.

22

However, the higher percentages of citric acid provided acid in excess that resulted in anti microbial activity by the bioplastic films. The esterification reaction with THP caused by the multicarboxylic groups of citric acid is confirmed by the FTIR/ATR technique. The citric acid interaction has given a flexible yet versatile character to the bioplastic that they are mechanically rigid but easily degradable at the same time. These bioplastic films can be used for several high value applications requiring high mechanical performance, and other applications such as packaging.

Acknowledgement One of the authors, Vimudha Muralidharan wishes to thank the Department of Science and Technology, India for providing funds through DST INSPIRE fellowship (Grant No. DST/INSPIRE[IF170352]). Authors are also thankful to the CATERS department (testing facility) of CSIR-CLRI, Chennai, India, for providing the analytical services.

Conflict of interest None.

Data availability The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of an ongoing study.

References [1] A. Bódalo, J. L. Gómez, E. Gómez, A.M. Hidalgo, A. Aleman, Study of the evaporation process of saline waste from the tanning industry. Waste Manage. Res. 25 (2007) 467– 474. DOI: 10.1177%2F0734242X07079869.

23

[2] C. Chen, H. Fan, P. Feng, Development of comprehensive utilization of leather solid waste. Lea. Sci. Eng. 18, (2008) 27–33 [3] C. Collivignarelli, G. Barducci, Waste recovery in the tanning industry. Waste Manage. Res. 2 (1984) 265–278. DOI: 10.1016/0734-242X(84)90031-4. [4] J. Kanagaraj, T. Senthilvelan, R.C. Panda, S. Kavitha, Eco-friendly waste management strategies for greener environment towards sustainable development in leather industry: A comprehensive review. J. Clean. Prod. 89 (2015) 1–17. DOI:10.1016/j.jclepro.2014.11.01 [5] L.F. Cabeza, M.M. Taylor, E.M. Brown, W.N. Marmer, Isolation of protein products from chromium-containing leather waste using two consecutive enzymes and purification of final chromium product: Pilot plant studies. J. Soc. Lea. Technol. Chem. 83 (1999)14– 19 [6] S. Tahiri, M. de la Guardia, Treatment and valorization of leather industry solid wastes: a review. J. Am. Leather Chem. Assoc. 104 (2009) 52-67. [7] J. Hu, Z. Xiao, R. Zhou, W. Deng, M. Wanga, S. Ma, Ecological utilization of leather tannery waste with circular economy model. J. Clean. Prod. 19 (2011) 221-228. DOI: 10.1016/j.jclepro.2010.09.018 [8] D. Masilamani, B. Madhan, G. Shanmugam, S. Palanivel, B. Narayan, Extraction of collagen from raw trimming wastes of tannery: a waste to wealth approach. J. Clean. Prod. 113 (2016) 338-344. DOI: 10.1016/j.jclepro.2015.11.087 [9] L. Dong, Y. Wei, L. Guo-ying, 2008. Extraction of native collagen from limed bovine split wastes through improved pre-treatment methods. J. Chem. Technol. Biotechnol. 83 (2008) 1041-1048. DOI: 10.1002/jctb.1912. [10]

L. Olle, S. Sorolla, C. Casas, A. Bacardit, Developing of a dehydration process for

bovine leather to obtain a new collagenous material. J. Clean. Prod. 51 (2013) 177-183. DOI: 10.1016/j.jclepro.2013.01.044

24

[11]

Plastic Oceans: The facts, 2018. https://plasticoceans.org/the-facts/ (accessed online

on 18th February 2019) [12]

D. Castiello, E. Chiellini, C. Patrizia, S. D’Antone, M. Puccini, M. Salvadori, M.

Seggiani, Polyethylene-Collagen hydrolizate thermoplastic blends: thermal and mechanical properties. J. App. Pol. Sci. 114 (2009) 3827-3834. DOI: 10.1002/app.31000 [13]

F. Langmaier, P. Mokrejs, K. Kolomaznik, M. Mladek, Biodegradable packing

materials from hydrolysates of collagen waste proteins. Waste Manage. 28 (2008) 549556. DOI:10.1016/j.wasman.2007.02.003. [14]

C.K. Liu, N.P. Latona, M.M. Taylor, M.L.A. Ramos, Biobased films prepared from

collagen solutions derived from untanned hides. J. Am. Lea. Chem. Ass. 110 (2015) 2532. [15]

P. Cinelli, E. Chiellini, L.S.H. Gordon, L.E. Chiellini, Characterization of

biodegradable composite films prepared from blends of poly(vinyl alcohol), cornstarch, and lignocellulosic fiber. J. Pol. Environ. 13 (2005) 47–59. DOI: 10.1007/s10924-0041215-6. [16]

Y. Nakayama, M. Takatsuka, T. Matsuda, Surface hydrogelation using photolysis of

dithiocarbamate or xanthate: Hydrogelation, surface fixation, and bioactive substance immobilization. Langmuir. 15 (1999) 1667–1672. DOI: 10.1021/la981169h. [17]

B. Sarti, M. Scandola, Viscoelastic and thermal properties of collagen/poly(vinyl

alcohol) blends. Biomaterials. 16 (1995) 785–792. DOI: 10.1016/0142-9612(95)99641-X [18]

P. Alexy, D. Bakos, S. Hanzelová, L. Kukolíková, J. Kupec, K. Charvátová, E.

Chiellini, P. Cinelli, Poly(vinyl alcohol)–collagen hydrolysate thermoplastic blends: I. Experimental design optimisation and biodegradation behaviour. Pol. Test. 22 (2003) 801–809. DOI: 10.1016/S0142-9418(03)00016-3.

25

[19]

M. Catalina, G.E. Attenburrow, J. Cot, A.D. Covington, A.P.M Antunes, Influence of

crosslinkers and crosslinking method on the properties of gelatin films extracted from leather solid waste. J. App. Pol. Sci. 119 (2011) 2015-2111. DOI: 10.1002/app.32932 [20]

F. Di Stefano, S. Siriruttanapruk, J. McCoach, P. Sherwood Burge, Glutaraldehyde:

an occupational hazard in the hospital setting. Allergy. 54 (1999) 1105-1109. [21]

S. V. Van, P. Dubruel, E. Schacht, Biopolymer based hydrogels as scaffolds for tissue

engineering applications: a review. Biomacromol. 12 (2011) 1387-1408. [22]

C. Menzela, E. Olssonb, T. S. Plivelicc, R. Anderssona, C. Johanssonb, R. Kuktaited,

L. Jarnstromb, K. Koch, Molecular structure of citric acid cross-linked starch films. Carb. Pol. 96 (2013) 270-276. DOI: 10.1016/j.carbpol.2013.03.044 [23]

J. Bonilla, E. Talon, L. Atares, M. Vargas, A. Chiralt, Effect of the incorporation of

antioxidants on physicochemical and antioxidant properties of wheat starch-chitosan films. J. Food Eng. 118 (2013) 271-278. DOI:10.116/j.jfoodeng.2013.04.008. [24]

V. Coma, I. Sebti, P. Pardon, F.H. Pichavant, A. Deschamps, Film properties from

crosslinking of cellulosic derivatives with a polyfunctional carboxylic acid. Carb. Pol. 51 (2003) 265-271. DOI: 10.1016/S0144-8617(02)00191-1. [25]

E. Olsson, M.S. Hedenqvist, C. Johansson, L. Jarnstrom, Influence of citric acid and

curing on moisture sorption, diffusion and permeability of starch films. Carb. Pol. 94 (2013) 765-772. DOI: 10.1016/j.carbpol.2013.02.006. [26]

E. Borredon, D. Bikiaris, J. Prinos, C. Panayiotou, Properties of fatty-acid esters of

starch and their blends with LDPE. J. App. Pol. Sci. 65 (1997) 705–721. [27]

R. Shi, Z. Z. Zhang, Q. Y. Liu, Y. M. Han, L.Q. Zhang, D. Chen, W. Tian,

Characterization of citric acid/glycerol co-plasticized thermoplastic starch prepared by melt blending. Carb. Pol. 69 (2007) 748–755. DOI: 10.1016/j.carbpol.2007.02.010 [28]

Y. Jiugao, W. Ning, X. Ma, The effects of citric acid on the properties of

26

thermoplastic starch plasticized by glycerol. Starch 57 (2005) 494–504. DOI: 10.1002/star.200500423.
 [29]

L. Shen, H. Xu, Y. Yang, Quantitative correlation between crosslinking degrees and

mechanical properties of protein films modified with polycarboxylic acids. Macromol. Mater. Eng. 300 (2015) 1133-1140. DOI: 10.1002/mame.201500145. [30]

Y. Yang, N. Reddy, Properties and potential medical applications of regenerated

casein fibers crosslinked with citric acid. Int. J. Biol. Macromol. 51 (2012) 37-44. DOI: 10.1016/j.ijbiomac.2012.04.027. [31]

N. Reddy, L. Chen, Y. Yang, Biothermoplastics from hydrolyzed and citric acid

crosslinked chicken feathers. Mater. Sci. Eng. C 33 (2013) 1203-1208. DOI: 10.1016/j.msec.2012.12.011. [32]

M. Mohsin, U. Farooq, Z.A. Raza, M. Ahsan, A. Afzal, A. Nazir, Performance

enhancement of wool fabric with environmentally-friendly bio-cross-linker. J. Clean. Prod. 68 (2014)130-134. DOI: 10.1016/j.jclepro.2013.12.083. [33]

Ramirez, D.O.S., Carletto, R.A., Tonetti, C., Giachet, F. T., Vareseno, A., Vineis, C.,

2017. Wool keratin film plasticized by citric acid for food packaging. Food Pack. Shelf Lif. 12, 100-106. DOI: 10.1016/j.fpsl.2017.04.004. [34]

M. Nagura, H. Yokota, M. Ikeura, Y. Gotoh, Y. Okhoshi, Structures and physical

properties of cross-linked gelatin fibers. Pol. Jou. 34 (2002) 761-766. DOI: 10.1295/polymj.34.761. [35]

M.H. Cumming, A.R. Leonard, D.S. LeCorres-Bordes, K. Hofman, Intra-fibrillar

citric acid crosslinking of marine collagen electrospun nanofibers. Int. J. Biol. Macromol. 114 (2018) 874-881. DOI: 10.1016/j.ijbiomac.2018.03.180. [36]

N.T. Hoyle, J.H. Merrit, Quality of fish protein hydrolysates from Herring (Clupea

harengus). J. Food Sci. 59 (1994) 76-79. DOI:10.1111/j.1365-2621.1994.tb06901.x.

27

[37]

O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with the

Folin-phenol reagent. J. Biol. Chem. 193 (1951) 265–275. [38]

D. Masilamani, V. Srinivasan, R.K. Ramachandran, A. Gopinath, B. Madhan, P.

Saravanan, Sustainable packaging materials from tannery trimming solid waste: A new paradigm in wealth from waste approaches. J. Clean. Prod. 164 (2017) 885-891. DOI: 10.1016/j.jclepro.2017.06.200. [39]

ASTM D618-00, 2000. American Society Standard Testing and Materials. Standard

Practice for Conditioning Plastics for Testing; Philadelphia. [40]

ASTM D882-91, 1996. Annual book of ASTM, American Society for Testing and

Materials., Philadelphia, PA [41]

ASTM D1746-03, 2003. American Society Standard Testing and Materials. Standard

Test Method for Transparency of Plastic Sheeting. ASTM; Philadelphia. [42]

M. Kemme, R.H. Weiland, Quantitative assessment of antimicrobial activity of PLGA

films loaded with 4-Hexylresorcinol. J. Func. Biomater. 9 (2018) 1-11. DOI: 10.3390/jfb9010004. [43]

D.B. Jones, Factors for converting percentages of nitrogen in foods and feeds into

percentages of proteins. Circular 183, (1931) United States Department of Agriculture, Washington. D.C. [44]

W. Ma, S. Rokayya, L. Xu, X. Sui, L. Jiang, Y. Li, Physical-Chemical properties of

edible film made from soybean residue and citric acid. J. Chem. (2018) 1-8, DOI: 10.1155/2018/4026831 [45]

Wang, S., Ren, J., Li, W., Sun, R., Liu, S., 2014. Properties of polyvinyl

alcohol/xylan composite films with citric acid. Carb. Pol. 103, 94-99. DOI: 10.1016/j.carbpol.2013.12.030. [46]

H.M.C. Azeredo, C.K. Vrettou, G.K. Moates, N. Wellner, K. Cross, P.H.F. Pereira,

K.W. Waldron, Wheat straw hemicellulose films as affected by citric acid. Food Hyd. 50

28

(2015) 1-6. DOI: 10.1016/j.foodhyd.2015.04.005. [47]

J.B. Olivato, M.V.E. Grossmann, A.P. Bilck, F. Yamashita, Effect of organic acids as

additives on the performance of thermoplastic starch/polyester blown films. Carb. Pol. 90 (2012) 159-164. DOI: 10.1016/j.carbpol.2012.05.009. [48]

N. Reddy, Y. Yang, Citric acid cross-linking of starch films. Food Chem. 118 (2010)

702-711. DOI: 10.1016/j.foodchem.2009.05.050. [49]

H. Xu, L. Shen, L. Xu, Y. Yang, Low-temperature crosslinking of proteins using

non-toxic citric acid in neutral aqueous medium: Mechanism and kinetic study. Ind. Crops Prod. 74 (2015) 234-240. DOI: 10.1016/j.indcrop.2015.05.010 [50]

H. Abdillahi, E. Chabrat, A. Rouilly, L. Rigal, Influence of citric acid on

thermoplastic wheat flour/poly(lactic acid) blends. II. Barrier properties and water vapor sorption

isotherms.

Ind.

Crops

Prod.

50

(2013)

104-111.

DOI:

10.1016/j.indcrop.2013.06.028. [51]

E. Chabrat, H. Abdillahi, A. Rouilly, L. Rigal, Influence of citric acid on

thermoplastic wheat flour/poly(lactic acid) blends. I: Thermal, mechanical and morphological

properties.

Ind.

Crops

Prod.

37

(2012)

238-246.

DOI:

10.1016/j.indcrop.2011.11.034. [52]

R. Shi, J. Bi, Z. Zhang, A. Zhu, D. Chen, X. Zhou, L. Zhang, W. Tian, The effect of

citric acid on structural properties and cytotoxicity of the polyvinyl alcohol/ starch films when

molding at

high

temperature.

Carb.

Pol.

74

(2008) 763-770.

DOI:

10.1016/j.carbpol.2008.04.045. [53]

D. Sagnelli, K. Hooshmand, G.C. Kemmer, J.J.K. Kirkensgaard, K. Mortensen,

C.V.L. Giosafatto, M. Holse, K.H. Hebelstrup, J. Bao, W. Stelte, A.B.

Bjerre, A.

Blennow, Cross-linked amylose bioplastic: a transgenic based compostable plastic alternative. Int. J. Mol. Sci. 18 (2017) 1-12. DOI: 10.3390/ijms18102075.

29

[54]

N. Reddy, Q. Jiang, Y. Yang, Preparation and properties of peanut protein films

crosslinked

with

citric

acid.

Ind.

Crop

Prod.

39

(2012)

26-30.

DOI:

10.1016/j.indcrop.2012.02.004. [55]

L. Sharma, H.K. Sharma, C.S. Saini, Edible films developed from carboxylic acid

cross-linked sesame protein isolate: barrier, mechanical, thermal, crystalline and morphological

properties. J. Food Sci.

Technol. 55

(2018) 532-539. DOI:

10.1007/s13197-017-2962-4. [56]

S. E. M. Selke, J.D. Cutler, R.J. Hernandez, Plastics Packaging: Properties,

Processing, Applications and Regulations, 
2nd ed.; Hanser: Munich, Germany, (2004) p. 203. [57]

K. Petersen, P.V. Nielsen, M.B. Olsen, Physical and mechanical properties of

biobased materials-starch, polylactate and polyhydroxybutyrate. Starch, 53 (2001) 356– 361. [58]

P.J.A. Sobral, A.M. Q.B. Habitante, Phase transitions of pigskin gelatin. Food Hyd.

15 (2001) 377-382. DOI: 10.1016/S0268-005X(01)00060-1. [59]

T.M. Maria, R.A. de Carvalho, P.J.A. Sobral, A. M. Q. B. Habitante, J. Solorza-Feria,

The effect of the degree of hydrolysis of the PVA and the plasticizer concentration on the color, opacity, and thermal and mechanical properties of films based on PVA and gelatin blends. J. Food Eng. 87 (2008)191-199. DOI: 10.1016/j.jfoodeng.2007.11.026. [60]

J.P.D. Garcia, M.F. Hsieh, B. T. Doma, D.C. Peruelo, I.H. Chen, H.M. Lee, Synthesis

of gelatin-γ-polyglutamic acid-based hydrogel for the in vitro controlled release of epigallocatechin gallate (EGCG) from Camellia sinensis. Polymers 6 (2013) 39-58. DOI: 10.3390/polym6010039. [61]

P.G. Seligra, C.M.

Jaramillo, L. Fama, S. Goyanes, Biodegradable and non-

retrogradable eco-films based on starch-glycerol with citric acid as crosslinking agent.

30

Carb. Pol. 138 (2016) 66-74. DOI: 10.1016/j.carbpol.2015.11.041. [62]

A.S. Singha, H. Kapoor, Effects of plasticizer/cross-linker on mechanical and thermal

properties of starch/PVA blends. Iran Pol. J. 23 (2014) 655-662. DOI: 10.1007/s13726014-0260-9. [63]

V.M. Azevedo, M.V. Dias, S.V. Borges, R.V. de B. Fernandes, E.K. Silva, E.A.

Medeiros, N. de F.F. Soares, Optical and structural properties of biodegradable whey protein isolate nanocomposite films for active packaging. Int. J. Food Prop. 20 (2017) 1869-1878. DOI: 10.1080/10942912.2017.1354883. [64]

B. Ghanbarzadeh, H. Almasi, A.A. Entezami, Improving the barrier and mechanical

properties of corn starch-based edible films: Effect of citric acid and carboxymethyl cellulose.

Ind.

Crops

and

Prod.

33

(2011)

229–235.

DOI:

10.1016/j.indcrop.2010.10.016.
 [65]

R. Ortega-Toro, S. Collazo-Bigliardi, P. Talens, A. Chiralt, Influence of citric acid on

the properties and stability of starch-polycaprolactone based films. J. App. Pol. Sci. (2016) 42220. DOI: 10.1002/app.42220. [66]

A. A. Khalil, S.F. Deraz, S.A. Elrahman, G. El-Fawal, Enhancement of mechanical

properties, microstructure, and antimicrobial activities of zein films cross-linked using succinic anhydride, Eugenol and citric acid. Prep. Biochem. Biotech. 45 (2015) 551-567. DOI: 10.1080/10826068.2014.940967. [67]

A. Awadhiya, D. Kumar, V. Verma, Crosslinking of agarose bioplastic using citric

acid. Carb. Pol. 151 (2016) 60-67. DOI: 10.1016/j.carbpol.2016.05.040 [68]

T. G. Dastidar, A.N. Netravali, ‘Green’ crosslinking of native starches with malonic

acid

and

their

properties.

Carb.

Pol.

90

(2012)

1620-1628.

DOI:

10.1016/j.carbpol.2012.07.041. [69]

S. F. Wang, L. Shen, Y.J. Tong, L. Chen, I.Y. Phang, P. Q. Lim, T.X. Liu,

31

Biopolymer

Chitosan/

Characterization.

Polym.

Montmorillonite Degrad.

Nanocomposites:

Stab.

90

(2005)

Preparation 123–

131.

and DOI:

10.1016/j.polymdegradstab.2005.03.001. 
 [70]

B. S. Chiou, H. Jafri, T. Cao, G. H. Robertson, K.S. Gregorski, S.H. Imam, G.M.

Glenn, W.J. Orts, Modification of wheat gluten with citric acid to produce super absorbent materials. J. Appl. Pol. Sci. (2013) 3192-3197. DOI: 10.1002/app.39044. [71]

Y. Zhong, X. Song, Y. Li, Antimicrobial, Physical and Mechanical Properties of

Kudzu Starch–Chitosan Composite Films as a Function of Acid Solvent Types. Carb. Pol. 84 (2011) 335–342. DOI: 10.1016/j.carbpol.2010.11.041. [72]

M. Ahmad, N.M. Hani, N.P. Nirmal, F.F. Fazial, N.F. Mohtar, S.R. Romli, Optical

and thermo-mechanical properties of composite films based on fish gelatin/ rice flour fabricated by casting technique. Prog. Org. Coat. 84 (2015) 115-127. DOI: 10.1016/j.porgcoat.2015.02.016. [73]

M.A.S.P. Nur-Hazirah, M.I.N. Isa, N.M. Sarbon, N. M., Effect of xanthan gum on the

physical and mechanical properties of gelatin-carboxymethyl cellulose film blends. DOI: 9 (2016) 55–63. DOI: 10.1016/j.fpsl.2016.05.008. [74]

J. G. Gutierezz, P. Partal, M.G.

Morales, C. Gallegos, Development of highly

transparent protein/starch based bioplastics. Biores. Technol. 101 (2010) 2007-2013. DOI: 10.1016/j.biortech.2009.10.025. [75]

J. Uranga, I. Leceta, A. Etxabide, P. Guerrero, K. de la Cabo, Crosslinking of fish

gelatins to develop sustainable films with enhanced properties. Eur. Pol. J. 74 (2016) 8290. [76]

Y. W. In, J.J. Kim, H.J. Kim, S.W. Oh, Antimicrobial activities of acetic acid, citric

acid and lactic acid against Shigella species. J. Food Safe. 33 (2013)79–85. DOI: 10.1111/jfs.12025.

32

[77]

R.K. Pundir, P. Jain, Evaluation of five chemical food preservatives for their

antibacterial activity against bacterial isolates from bakery products and mango pickles. J. Chem. Pharma. Res. 3 (2011) 24–31.

33

List of Figure Captions Fig. 1. Trimming hydrolysate based transparent bioplastic a) & b) THPCA0 (no citric acid) c) THPCA40; d) Transparency of the film THPCA40 Fig. 2a. Thermograms of bioplastic films: THPCA10, THPCA20, THPCA30 and THPCA40 Fig. 2b. Thermograms of PVA-CA, PVA, TH-CA, TH Fig. 3. FTIR spectra of samples a) native THP hydrolysate; b) bioplastic THPCA40 Fig. 4. Micrographs of bioplastic films: a) & e) THPCA10; b) & f) THPCA20; c) & g) THPCA30; d) & h) THPCA40 Fig. 5. Zone of inhibition exhibited by bioplastics against organism a) P. aeruginosa; b) S. aureus; c) E. coli Fig. 6. Soil degradation profile of bioplastic films THPCA40 and PVA

34

Fig. 1.

Fig. 2a.

Fig. 2b.

Fig. 3.

Fig. 4.

Fig. 5. A- THPCA10; B- THPCA20; C- THPCA30; D- THPCA40

Fig. 6.

Tannery trimming waste based biodegradable bioplastic: Facile synthesis and characterization of properties

Highlights: •

Untanned tannery solid waste – trimming waste was utilized for preparing transparent bioplastic material.

•

Trimming hydrolysate based bioplastic was mechanically robust and highly extensible.

•

Citric acid acted as compatibilizer and as a flexible crosslinking agent providing multiple beneficial properties.

•

The bioplastic films were found to be antimicrobial in nature.

•

Trimming based bioplastic can become a prospective alternative to the fossil based plastic materials.

CRediT - Author contribution statement

Vimudha Muralidharan: Conceptualization, Methodolgy, Validation, Writing – Original draft preparation Michael Selvakumar Arokianathan: Resources, Software, Formal analysis Madhan Balaraman: Investigation, Supervision, Project administration, Writing – Review and editing Saravanan Palanivel: Supervision, Writing – Review and editing, Visualization, Investigation

Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: