Combination and processing keratin with lignin as biocomposite materials for additive manufacturing technology

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Combination and processing keratin with lignin as biocomposite materials for additive manufacturing technology Warren J. Grigsby , Sonya M. Scott , Matthew I. Plowman-Holmes , Paul G. Middlewood , Kimberly Recabar PII: DOI: Reference:

S1742-7061(19)30855-4 https://doi.org/10.1016/j.actbio.2019.12.026 ACTBIO 6509

To appear in:

Acta Biomaterialia

Received date: Revised date: Accepted date:

23 August 2019 13 December 2019 19 December 2019

Please cite this article as: Warren J. Grigsby , Sonya M. Scott , Matthew I. Plowman-Holmes , Paul G. Middlewood , Kimberly Recabar , Combination and processing keratin with lignin as biocomposite materials for additive manufacturing technology, Acta Biomaterialia (2019), doi: https://doi.org/10.1016/j.actbio.2019.12.026

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Combination and processing keratin with lignin as biocomposite materials for additive manufacturing technology *a

b

b

b

Warren J. Grigsby, Sonya M. Scott, Matthew I. Plowman-Holmes , Paul G. Middlewood and Kimberly Recabar

c

aScion, Private Bag 3020, Rotorua 3010, New Zealand. bAgResearch Ltd, Lincoln Research Centre, Private Bag 4749, Christchurch 8140, New Zealand. cUniversity of Waikato, Private Bag 3105, Hamilton 3240, New Zealand. *Corresponding author: e-mail: [email protected]

Abstract Additive manufacturing using Nature’s resources is a desirable goal. In this work we examine how the inherent macromolecular properties of keratin and lignin can be utilised and developed using green chemistry principles to form 4D functional materials. A new methodology utilising protein complexation by lignin was applied to form copolymers and reinforce keratin cross-linking networks on aqueous and solid state processing. Solubility, chemical and processing characteristics found a favoured 4:1 ratio of keratin to lignin was most desired for effective further processing as 3D printed paste forms. Thermally processing keratin-lignin with plasticisers and processing aids demonstrated extruded FDM filaments could be formed at temperatures >130˚C, but degradation of keratin-lignin materials was observed. Employing paste printing strategies, keratin-lignin hydrogels could successfully print 3D skirt outlines. This was achieved with aqueous hydrogels prepared at 30-40% solids content with and without plasticizers over a defined processing timeframe. Mechanical response to moisture stimuli was successfully demonstrated for the 4:1 keratin-lignin printed material on water soaking, realising the ability of these keratin-lignin biocomposite materials to introduce a 4th dimensional response after 3D printing.

Statement of Significance In this paper we describe new perspectives for how biopolymers can be used and processed to develop (co)polymers as 3D & 4D printed responsive materials without the need for synthetic chemical modifications. We utilise a novel methodology employing bioconjugation to synthesise and develop co-polymer materials from keratin and lignin and demonstrate this can be achieved in both water and solid state. We manipulate the inherent chemical attributes of both biopolymers to develop these new functional materials under green chemistry processing conditions. This is a practical example how the chemical coupling of two biopolymers at molecular-scale can be leveraged to give co-polymer materials which retain their inherent macromolecular properties to behave as functional, 4D responsive biomaterials.

Keywords: keratin, lignin, biomaterials, conjugates, biocomposites, green 3D printing, additive manufacturing

1. Introduction Additive manufacturing is redefining the manufacturing paradigms for production of customised parts, prototyping and material design. A rapidly growing area of additive manufacturing is the combination of renewables and natural fibres with synthetics to modify polymer properties for application across a range of 1

3D printing techniques and products . In addition to sustainability advantages, renewable materials can possess inherent properties, particularly at nano- or molecular-level where their use can introduce 4D attributes to products. 4D printing offers the potential for a 3D printed material to respond in a programmed 2-3

way to external stimuli such as temperature or moisture

. It would be advantageous to base such 4D

responses on the inherent attributes of renewable biopolymers. A cellulose-derived smart material can be 4

programmed to give mechanical responses to moisture . Proteins and polyphenolic lignins, commonly available resources, could also be used in smart design due to their range of inherent properties including response to moisture

5-8

9

Keratin is a fibrous protein, commonly sourced from wool, hair and feathers . Keratin can be used in applications such as personal care products

10

11

and wound dressings . As a biomaterial, keratin can be

processed in aqueous media and deposited into film or fibre forms either alone or in combination with synthetic or other biopolymers. In combination with thermoplastic urethanes keratin can modify rheological behaviours

12

13

or form copolymer scaffolds with poly(lactic-co-glycolic) acid . In the case of lignin, this

endemic biopolymer can be recovered from pulp and paper manufacture and emerging biorefinery facilities. Lignin has been studied extensively in applications research including examining its processability into filaments

14-15

or as a thermoplastic for modification of polymer systems

15-16

. For additive manufacturing with

natural-derived biopolymers, a focus has been on biomass polysaccharides

17

. Applications of keratin in 3D

18

printing have typically been with casted scaffolds and bio-printing . Such constructs utilise keratin selfassembly and can be tuned through molecular composition or cross-linking into hydrogels

19

. With lignin, 3D

printing has been restricted to combinations with thermoplastics in inks or FDM for example based filament materials are typically less than 70% lignin content

16

20

where lignin-

. Nonetheless, FDM-based printing is 20

projecting vast applications for lignin-based materials as biobased plastics . However, neither keratin nor lignin have been reported in the production of 4D smart materials and product applications.

Keratin and lignin each possess a range of inherent biopolymer properties experience changes with moisture such as plasticisation

22-24

9, 21

. These polymer properties

. In the current study their combination has been

explored as novel 3D printed materials for potential 4D applications. While keratin and lignin have some commonality in alkaline solubility, each biopolymer is technically challenging to process or use individually, let alone together. This work explored the combination of lignin with keratin to obtain materials suitable for 3D printing using green chemistry principles. Initially, aqueous processing was employed to form copolymers 25

through protein complexation and cross-linking of keratin with lignin . This developed an understanding of processing conditions for copolymer formation and optimisation of the combinations. Keratin-lignin copolymer materials were then assessed for processing as filaments in FDM approaches or their deposition as hydrogels

using 3D paste printing. Ultimately, a goal is to produce keratin-lignin copolymer smart materials with defined environmental responses for 4D applications.

2.

Materials & methods

2.1 Materials Keratin powder was obtained via an established procedure employing aqueous extraction of wool to give an intermediate filament protein (IFP) powder. IFP was obtained by treating wool with copper sulfate and sodium 26

sulphite following a standard oxidative sufitolysis procedure . Where stated, a 10% keratin stock solution was prepared by dissolving IFP keratin in water while maintaining the pH at 8.3 using 1 M NaOH. The lignin used was Indulin AT kraft lignin obtained from Ingevity (USA). Similarly, both 10% or 20% lignin stock solutions were prepared by suspending kraft lignin in water and adding 40% sodium hydroxide solution to achieve pH 10.2, with additional water then added to achieve the final % lignin solids content. Glycerol (BDH), acetic acid (BDH), guar gum (Sigma), PEG400 (Sigma) and kappa-carageenan (Sigma) were used as supplied.

2.2 Keratin-lignin aqueous suspensions A general procedure was used to produce a range of keratin-lignin copolymers typically at <10 g scale (Table 1). Firstly, 10% lignin solution (e.g. 65 g for 1) was weighed into a 200 mL flask, and, where necessary, then diluted with water to give ca. 80 mL reaction solution. In a separate flask, 10% keratin solution (e.g. 35 g for 1) was weighed out, then slowly poured into the stirred lignin solution. Stirring continued for 10 minutes after which the pH of the combined solution was adjusted with 20% acetic acid to achieve the target pH (e.g. pH 4 for 1), typically giving a heterogenous mixture. This aqueous mixture was then centrifuged, the resulting supernatant decanted and the precipitate washed with water at least twice with centrifuging to obtain a colourless supernatant. The washed precipitate was freeze-dried to give a light-dark brown solid.

2.3 Compounding and filament extrusion of keratin-lignin (4:1) copolymer

2.3.1 Thermomechanical processing Initial powder mixing was performed on a Thermo Scientific™ HAAKE Polylab OS torque rheometer equipped with a Thermo Scientific™ HAAKE Rheomix 600 OS electrically heated laboratory batch mixer. Keratin and lignin (4:1 ratio, 27-40g, Table 2) powders were combined using roller rotors operating at a constant speed of 50 rpm for 10 min at temperatures of 45, 80, 130 and 150 °C (11-14). Process parameters including torque and temperature were monitored using Thermo Scientific™ HAAKE Polysoft OS Software. Keratin-lignin (4:1) was also combined with polyethylene glycol (PEG-400) or guar gum. PEG-400 was added at 5, 10 and 15 weight% with guar gum added at 30 weight% (wt%, based on keratin, Table 2). These

combinations (15-18) were similarly processed on the HAAKE Polylab OS torque rheometer at the stated temperatures.

2.3.2 Reactive Compounding Extrusion Filament extrusion of 4:1 keratin-lignin copolymers containing 15, 20 and 30 wt% PEG-400 (19-22) was conducted on a small-scale OMC twin screw extruder fitted to the HAAKE Polylab OS torque rheometer and batch mixer operating at 150 rpm. Extrusions were typically over 10 mins and, where stated, water, glycerol, sodium hydroxide (NaOH) (10 M) or zinc stearate with the 30 wt% PEG-400 keratin-lignin combination (Table 2). Sample powder batches (ca. 12-40 g) were loaded into the extruder and extruder head temperature and extrusion pressure monitored. Where extruded filaments were produced, the filament strength and integrity were assessed by their physical form and ease of recovery and handling post-extrusion.

2.4 Keratin-lignin hydrogel formation and 3D printing trials The general procedure for preparing a low solids aqueous hydrogel pastes was as follows. A 9.5 % keratin solution (20 g, pH 8.47) was heated to 60°C. Guar gum (0.6 g) was mixed in to form a creamy white slurry. Lignin solution (20%, 2 mL) was then stirred in to yield a dark chocolate brown paste which was set aside to cool to ambient temperature over 30 min before proceeding to paste printing. In this formulation, glycerol, kappa-carageenan or PEG400 can be used in place of guar gum.

To prepare high solids hydrogel pastes, sodium sulfite (0.375 g) was dissolved in water (8.4 g) and briefly heated (85°C). Guar gum (0.43 g) was added to yield a pale-yellow paste. A mixture of 4:1 keratin-lignin powders (3.5 g formed from keratin 2.8 g and lignin 0.7 g) was stirred in to form a thick brown slurry. This was briefly heated for 1 min before setting aside to cool. With this formulation, the guar gum content was varied using 20, 30 and 40 total wt% of guar gum of keratin and similarly with PEG400 substitution. Collectively, these formulations were used to prepare single, double and triple-layered multi-beam skirt outlines.

All hydrogel preparations were printed onto polytetrafluoroethylene (PTFE) Teflon™ sheets using a PrinterBot model gel printer and Simplify3D® software. Hydrogels were loaded into a 60 mL extruder syringe capped with a nozzle tip (diameter: 1.5 mm) and maintained at a constant temperature of 35°C. The gel printer was primed for extrusion by ‘skirting’: extrusion-tracing the print bed site perimeter, in duplicate, prior to commencing printing. Extrusion multiplier (0.30) and primary height layer (1.36 mm) settings were used and nozzle tip height was adjusted with the G-code offset: z-axis parameter as appropriate to allow space for extrusion, particularly when additional/multiple layering was performed. The printing speed was ranged between 300-750 mm/min to generate 3D printed patterns of five single-strand beams. Upon determining optimal printing speed, five multi-strand beams were generated as singular and double successive layering of the keratin-lignin hydrogel materials.

2.5. Materials characterisation Solid state 13C NMR spectra were obtained on a Bruker Avance DRX 200 instrument with a 5 mm doubly tuned 1H/X MAS probe (Bruker). Hartmann-Hahn matching was conducted using glycine. Samples were packed into a zirconia rotor fitted with a Kel-F cap and spun at 5 kHz. A standard cross polarisation-magic angle spinning (CPMAS) pulse programme was used with a 1H preparation pulse of 5.56 µs, 1H decoupling field of 47 kHz, and an acquisition time of 20 ms to acquire 5000 scans.

Thermogravimetric analysis (TGA) was performed using a Q500 TGA from TA Instruments (USA). Samples (1215 mg) were weighed into platinum pans before being transferred to the TGA and heated to 600 °C at 10°C/min under a nitrogen atmosphere.

Differential scanning calorimetry (DSC) was performed on a TA Instruments (USA) Q1000. Samples (~2-4 mg) were placed in aluminium hermetic pans and crimp sealed with a lid. Samples were heated under a nitrogen purge using a heat/cool/heat cycle, initially heating to 200 °C before cooling to -20 °C and then heating to 200 °C at a temperature ramp rate of 10 °C/min.

Rheology profiles were assessed on a AR2000 rheometer from TA Instruments using parallel plate geometry (25 mm dia. plates) in oscillation mode. Hydrogel pastes were applied directly onto the rheometer plate and then compressed to a gap width of 500. A normal force (0.5 N) was additionally applied to accommodate any variance in gap width. An isothermal hold (23˚C) was applied using a frequency of 1 Hz and strain of 0.050.10%. Viscosity was monitored over 90-400 mins.

Dynamic mechanical thermal analysis (DMTA) was conducted in three-point bending mode (40 mm span) using a TA Instruments RSA-G2 instrument. Compressed keratin-lignin samples (10 x 4mm cross-section compressed bars) were trimmed to a nominal length. After conditioning (23˚C, 50% RH) samples were mounted in the DMTA and immersion cup filled with water (23˚C). Samples were evaluated isothermally (23˚C) employing a 1 Hz frequency and 0.01% strain until sample failure or excessive swelling observed.

3.

Results

3.1 Keratin-lignin copolymer combinations A range of binary keratin-lignin combinations were produced by aqueous processing with varied keratin:lignin ratio (1-10, Table 1). After combining at alkaline pH, the yields of copolymer materials were dependent on both final solution pH and lignin content. It was evident that solution pH and lignin content jointly control the formation and solubility of these combinations. Adjustment of the initial alkaline solution to pH 4 induced quantitative precipitation of all combinations consistent with the isoelectric point of keratin

27

and lignin

28

solubility . This was similarly the case for including calcium ions at acidic pH (8) to induce protein binding

29-31

However, at more neutral pH (pH 6) the aqueous solubility characteristics of keratin begin to dominate with the high-content keratin combinations more soluble (7-10). In contrast, those combinations with higher lignin 28

content (1-4) were associated with insolubility at pH 6 consistent with lignin physiochemical behaviours . In

.

alkaline solution (pH >9) the keratin-lignin combinations were deemed soluble, consistent with solubility factors for both keratin and native lignin

27-28

as well as the inhibition of protein complexation at higher pHs

32-

33

. Practically, this relationship with pH can be used to mix and produce aqueous hydrogels, suspensions and

precipitated forms of the keratin-lignin copolymers for further processing.

Table 1. Results for aqueous processing of keratin with lignin to generate copolymer materials as hydrogels, suspensions and precipitated forms suitable for powder-based and paste 3D printing. Sample

Keratin:Lignin

Protein

Lignin

number

Ratio

(g)

(g)

pH

%Yield

1

1:2

3.5

6.5

4

86

2

1:2

3.5

6.5

6

83

3

1:1

3.2

3.2

4

89

4

1:1

3.2

3.2

6

61

5

3:2

6.0

4.0

4

88

6

3:2

6.0

4.0

6

81

7

3:1

4.6

1.5

4

91

8

3:1Ca#

2.3

0.8

4

82

9

3:1

4.6

1.5

5

73

10

3:1

4.6

1.5

6

25

Where: # is addition of calcium carbonate to induce protein binding

Chemical analysis of isolated keratin-lignin copolymers from aqueous processing provided evidence for complexation of keratin by lignin. Shown in Figure 1A are solid state NMR spectra of samples precipitated at pH 4. Chemical shift comparisons show the keratin amide carbonyl peak (176 ppm) was broadened with shoulders emerging at 173 and 183 ppm at higher lignin contents consistent with shielding and -bonding 32

characteristics associated with protein complexation . In contrast, for samples with lower lignin contents, the amido 173 ppm peak profile remained more similar to the peak profile of pure keratin. The lignin phenolic peak at 147 ppm showed evidence of a downfield shoulder also consistent with protein complexation through this phenolic group. For the other key lignin aryl peaks (110-140 ppm) there were no peak shifts evident with only keratin peaks emerging in this region at higher keratin contents. For the aliphatic peaks (10-50 ppm) combination of lignin with keratin did not impact the chemical shift or intensity of peaks in this range.

A lignin

B

1

2

3

4

5

6

7

10

keratin 13

Figure 1. A. C NMR of the keratin-lignin copolymer combinations isolated at pH 4. From bottom to top: Keratin (bottom), 3:1 (7), 3:2 (5), 1:1 (3), 1:2 (1), and lignin (top). B.

13

C NMR of the keratin-lignin copolymer combinations

isolated at pH 6. From bottom to top: Keratin (bottom), 3:1 (10), 3:2 (6), 1:1 (4), 1:2 (2), and lignin (top).

For the keratin-lignin combinations isolated at pH 6, the NMR spectra were generally comparable to copolymer materials obtained at pH 4 (Figure 1B). As above, the greatest distinction in spectra was the emergence of the amide carbonyl shoulder at 183 ppm and the lignin phenolic peak at 153 ppm which both have greater intensity with higher lignin content. In contrast, at higher keratin contents the amide peak (175 ppm) was shifted toward 173 ppm with this upfield shift likely induced by the higher pH at which the sample was isolated (pH 6) compared to precipitation at pH 4. Additionally, with higher keratin content there is a sharpening of the lignin aryl peak at 110 ppm peak with this change distinct from the keratin aryl peaks (115-140 ppm). As above at pH 4, there were no differences in the chemical shift or intensity of the methoxy (56 ppm) or aliphatic peaks of lignin and keratin components.

With chemical evidence for keratin complexation by lignin, the impacts to resulting polymer thermal characteristics of the keratin-lignin copolymer materials were evaluated. Thermogravimetric analysis (TGA) revealed the expected inherent moisture loss of each combination between 90 and 120˚C as well as decomposition characteristics (>200˚C) which appear a hybrid of each component (Figure 2). This was most evident above 250˚C where a differing degradation profile for the keratin component was observed. Moreover, there was also evidence for some degree of thermal instability <200˚C for samples which may be derived from this complexation of keratin with lignin established by NMR. This thermal instability below 200˚C was most evident in the 3:2 (5) combination with the onset of degradation evident from ca. 130˚C and also observed in the higher lignin content samples (1:1 and 1:2). Overall, this analysis suggests complexation of keratin by lignin, even at 25% lignin content, will induce some thermal instability at temperatures above 130˚C.

1

lignin 3 5 7 keratin

Figure 2. Thermogravimetric analysis of various keratin-lignin copolymer combinations (pH 4) showing differing degradation onset and weight loss with temperature. Curves are: keratin (red), lignin (black), and keratin-lignin combinations 3:1 (7, grey), 3:2 (5, blue), 1:1 (3, green) and 1:2 (1, purple).

Complementary differential scanning calorimetry (DSC) analysis revealed minimal distinctions between thermograms of the keratin-lignin combinations (Figure 3). As found with TGA, on the first DSC heating cycle, all samples lose adsorbed water inherently common to biomaterials with distinctions between samples due to the degree of bound and unbound water. For the second heating cycle, above 120˚C it was evident the lignin glass transition (Tg) was shifted to higher 34

temperature on complexation with protein . This was generally observed across all samples and varying lignin contents. This shift in lignin Tg may be evidence to further corroborate the chemical conjugation and copolymerization of lignin with keratin established by NMR, as a simple blend or mixture of these two materials would not influence Tg 35

. Any initial degradation observed by TGA from ca. 130˚C for higher lignin content samples (1, 3, Figure 2) does not

appear to manifest in DSC thermograms.

Figure 3. Differential scanning calorimetry of various keratin-lignin copolymer combinations showing the first heating cycle (left) with moisture loss and the second heating cycle (right) with the onset of lignin glass transition. Curves are: keratin (red), lignin (black), and keratin-lignin combinations 3:1 (7, grey), 3:2 (5, blue), 1:1 (3, green) and 1:2 (1, purple)..

Collectively, chemical analysis corroborates lignin complexation of keratin and that this protein conjugation results in altered solubility with pH and differing thermal stability. Physiochemically, the acid precipitated copolymers did not result in fused, or film-like materials, but friable materials particularly with higher lignin contents. Overall, in mildly acidic conditions, a lower lignin content maintained keratin-lignin aqueous solubility whereas >40% lignin promoted

insolubility. Similarly, a low lignin content did not detrimentally impact thermal stability when processed below 200˚C. Therefore a lower lignin ratio (<25%) was desirable suggestive of using 4:1 or 9:1 keratin-lignin combinations.

3.2 Keratin-lignin filament formation by thermal processing Compound extrusion blending of keratin with lignin was evaluated at moderate processing temperatures to produce materials suitable for powder SLS and FDM printing (Table 2). Initially, thermomechanical mixing of keratin with lignin (ratio 4:1) powders at 45°C or 80°C yielded fine, pale brown homogeneous powders, 11 and 12, respectively. At 130°C a semi-consolidated material (13) was produced suggesting processing achieved some plasticisation and material flow. However, materials processed at this temperature were heterogeneous and marred by some keratin separation. At 150°C, greater material flow behaviour was evident (14) which produced fused materials, but their compounding at this temperature rapidly exceeded equipment torque settings (Supplementary Materials).

Table 2. Selected samples produced by thermomechanical processing of keratin with lignin (ratio 4:1) in powder form to give copolymers and filament materials using reactive compounding. Sample

Processing

number

temperature (°C)

Additive and Loading (%) *

compounding extrusion product

11

45

-

Homogenous, pale brown powder

12

80

-

Homogenous, pale brown powder

13

130

-

Homogenous, pale brown consolidated powder

14

150

-

Homogenous, consolidated pale brown powder

15

80

PEG (5)

Homogenous, pale brown powder

16

130

PEG (5)

Homogenous, pale brown powder

17

80

PEG (10)

Coarse, heterogeneous pale powder

18

130

PEG (10)

Strong, brittle, heterogeneous consolidated lumps

19

130

PEG (15)

Hard, heterogeneous clumps,

20

80

GG (30)

Fine, homogenous, pale brown powder

21

130

GG (30)

Homogenous, dark brown powder

22

130

PEG (15)

Extruded as fine homogenous powder

23

150

PEG (20)

Crumbly powder

24

160

PEG (30)

Brittle brown/black filaments (55 bar)

25

170

PEG (30)

Brittle, uniform brown/black

26

170

PEG (30) + Water (10)

Brittle dark brown filaments, more uniform

27

170

PEG (30) + Glycerol (10)

Brittle brown filaments (less uniform than 26)

28

170

PEG (30) + NaOH (10)

Brittle dark brown filaments

29

160

PEG (30) + ZnSt2 (10)

Black filaments with some strength, brittle, very slow extrusion

30

170

PEG (30) + ZnSt2 (10)

Black filaments with strength, integrity

*

Description of thermomechanical mixing or reactive

-1

Note: Plasticiser (wt% with respect to keratin); *PEG-400 = polyethylene glycol (average Mw: 400 gmol ); *GG = Guar -1 gum; ZnSt2 = zinc stearate; and NaOH = 1 molL sodium hydroxide. Samples 11-21 produced by thermomechanical mixing; and samples 22-30 by reactive compounding and extrusion

A chemical analysis revealed only subtle changes between thermomechanical processed samples (Figure 4). For example, NMR found the lignin aryl peaks at 143 and 149 ppm (11) shift to a single peak at 147 ppm (14). This change occurred from 80˚C and was more prevalent at higher temperatures. Similarly, the aryl peak at 157 ppm broadens to a multiplet (153-162 ppm) with higher temperature, consistent with Figure 1. At 150˚C (14) the 175 ppm amide carbonyl peak of keratin was broadened and shifted to 173 ppm, as observed in Figure 1. Samples show the dominant keratin

CH peak at 55 ppm to broaden at higher processing temperatures, perhaps indicative of both conjugation and cross36

linking . With DSC, the first heating cycle revealed a broad endothermic peak below 120°C in all thermograms (Figure 5) associated with loss of residual, absorbed water. However, unlike the aqueous-based samples (Figure 3), on thermomechanically processing at 45°C there was a defined peak which was likely attributable to the loss of unbound, adsorbed water which was not present in higher temperature samples (>80°C). TGA indicated small (~5 %), initial weight losses on heating to 100 °C, consistent with samples having minimal hygroscopicity after thermal processing (Figure 6). Above 210°C, TGA revealed rapid weight loss occurs with these samples generally losing 50 % weight by 300°C. This degradation profile above 200°C was also comparable to the aqueous-based copolymers (Figure 2). This, together with NMR and DSC suggest keratin-lignin copolymerization could be achieved by thermomechanical

processing and the reactive compounding

37

of powders.

13

Figure 4. Solid state C NMR spectra for thermomechanically processed keratin-lignin (4:1) powder composites – (a) 11 (base), 12, 13 and 14 (top); (b) 11 (base), 13, 16, 18 and 19 (top); and (c) 11 (base), 13, 20 and 21 (top) (a) (b)

(c)

(f)

(e)

(d)

Figure 5. Thermal analysis data: (a-c) thermogravimetric analysis (top) and (d-f) differential scanning calorimetry (bottom) for keratin-lignin (4:1) powder composites. (a,d) 11, 12, 13 and 14, (b,e) 12, 15, 16, 18 and 19 and (c,f) 11, 12, 13, 20 and 21.

Addition of plasticizers was investigated to improve the thermal processability of the keratin - lignin powders. Polyethylene glycol (PEG-400) and polysaccharide (guar) gum additions were compared at processing temperatures of 80 and 130 °C (Table 2). PEG-400 at 10% wt content (17 and 18) promoted formation of brown clumps at 130 °C with 15 wt% PEG-400 (19) giving a relatively hard extrudate formed around the rotors (Table 2). This suggests both higher temperature and increased PEG-400 content contributed to improved plasticization and processing of the keratin-lignin material. At 30 wt% guar gum (20 and 21) no plasticization was evident at 130 °C. The heterogeneity of samples 17 19 suggested that plasticization, whilst improving processability, also promoted phase separations of the different materials under rheological forces generated during mixing (Table 2).

Based on the thermomechanical processing observations (Table 2), 15 wt% PEG-400 and a processing temperature of 130 °C (TP-5) was used to attempt keratin-lignin filament formation. Firstly, increasing the PEG-400 content from 15 to 30 wt% improved filament formation (22 – 25). However, use of PEG-400 alone produced extruded filaments which lacked sufficient filament strength and integrity on exiting the extruder (Table 2, and also see Supplementary Materials). Other additives were trialled with PEG-400 to aid plasticization (26 – 28). Water appeared to improve filament uniformity (26), but no improvement to the extruded material integrity was found with these additives (Table 2). Zinc stearate, traditionally used as a lubricant within plastic processing, led to some heterogeneity but greater filament integrity and strength was notable (29-30) (Table 2, Supplementary Materials). While a favourable result, processing was marred by inconsistent processing residence times and partial degradation due in part to pressure build and jamming of the extruder screw which became frequent on extended processing times.

Analysis of NMR spectra revealed protein complexation by lignin together with some oxidation promoted by higher processing temperatures. For 16, the keratin amido peak showed evidence of broadening consistent with complexation with lignin (Figure 4b). However, due to the high keratin content, it was difficult to observe such minor chemical changes around this 173 ppm peak. Moreover, it was likely only a small proportion of the keratin component was involved in any chemical coupling with lignin or other additives compared to the bulk keratin content in samples. The 21

broadening of the lignin 143, 149 and 157 ppm peaks can be attributable to oxidation and PEG adsorption to lignin . Use of guar gum at both 80 and 130˚C led to changes in the lignin aryl peaks between 140 and 160 ppm together with the keratin 173 ppm peak (Figure 4c). Overall, NMR tended to show downfield chemical shifts consistent with both

lignin oxidation or hydrogen-bond formation through the amido groups of the keratin with these changes most pronounced when processing between 80 and 150 ˚C.

3.3 Keratin-lignin-based hydrogel paste printing and compression moulding Keratin in aqueous solution was combined with lignin to form hydrogels suitable for processing in paste printing applications. The keratin:lignin ratio was 4:1 with the resulting hydrogels formed by processing solutions with 20-40% keratin-based solids contents under mildly alkaline pH (pH 8-9) to retain solubility through adapting processing conditions of formulations described in Table 1. Figure 6 demonstrates examples of the varying quality of hydrogel deposition and morphology achievable by paste printing keratin-lignin solutions (see also Supplementary Materials). Typically, initial skirt tracings could be readily achieved with multiple (3-4) layering possible (Figure 6a and Supplementary Materials). On standing for a short period (ca. 100 min), the keratin-lignin hydrogel becomes increasingly difficult to extrude and deposit as a continuous skirt or film (Figure 6b). This was likely evidence for cross38

linking within the hydrogel and network formation . The introduction of plasticizers, e.g. guar gum, promoted more uniform printing, particularly at higher solids content (40%) with timeframes extending up to ca. 3 h.

Figure 6. Time dependency of paste printed keratin-lignin (4:1) hydrogels (30% keratin solids content). Left: Trace printed 60 minutes after hydrogel preparation. Right: Trace printed 95 minutes after hydrogel preparation.

Complementary rheology evaluations were undertaken to understand hydrogel formation, cross-linking and the influence of additives on this process (Figure 7, Supplementary Materials). Shown in Figure 7 are rheology profiles of keratin and keratin-lignin hydrogels formed in pH 8 solution. The rheology demonstrates the complexity of the effect of the additives on the keratin and keratin-lignin systems. Evident for keratin-only plasticized with guar gum was a viscosity build over 3 h where both sulfite and plasticizer additions can impact formation of hydrogen-bonding 38

networks within the developing hydrogel (see also Supplementary Materials) . After this time, this cross-linking becomes more extensive contributing to greater viscosity build and a final hydrogel network. Similarly, the presence of

lignin led to comparable viscosity build which was distinguished by combination of guar gum without sulfite. Evident here was minimal influence of protein complexation by lignin to the rate of cross-linking as the hydrogel network developed. These rheology behaviours and viscosity build were consistent with the observed timeframes for satisfactory paste printing (Figure 6).

Keratin-lignin/guar gum Keratin-lignin/guar gum/sulfite Keratin/guar gum/sulfite

Figure 7. Strain-controlled viscosity profiles of keratin and keratin-lignin (4:1) hydrogels at 30% solids keratin content. Keratin/guar gum/sulfite (red), keratin-lignin/guar gum (green) and keratin-lignin/guar gum/sulfite (blue).

Although the mixing of keratin and lignin created demonstratively usable skirt tracings and ability to layer these (Figure 6a), their subsequent dehydration on air-drying led to collapse and shrinkage of these paste-printed hydrogels (Supplementary Materials). While this was not unanticipated and widely known for other biobased printed materials, adaptations to these initial paste printing procedures were evaluated to improve upon the deposition of the keratinlignin materials. Progression to higher solids led to lower rates of shrinking and deformation on drying. However, qualitatively, a maximum of 40% solids could be employed before the pasting viscosity was also considered too high for continuous printing as above (see for example Figure 6b). Inclusion of plasticizers were also viewed as beneficial to maintaining skirt integrity on deposition and drying, but dimensional instability was still observed. Nonetheless, these approaches have led to some understanding of the requirements for paste printing these capricious keratin-lignin copolymer materials. Currently, this study is still developing the processing window for paste printing keratin-lignin hydrogel materials which involves an optimal combination of solid/liquid ratio, printing temperature and drying regime (Supplementary Materials) to minimise the detrimental aspects observed with shrinkage. Moreover, we believe the directed design and deposition of printed materials can positively incorporate and utilise the benefits of this inherent shrinkage when fabricating constructs of the keratin-lignin copolymer materials.

While extrusion and hydrogel processing proved challenging, combination of keratin and lignin powders as a low water content aqueous paste followed by compression moulding allowed formation of dimensionally stable rectangular bars.

After allowing the compressed bars to equilibrate and dry in ambient conditions, these bars were considered relatively rigid, consistent with the inherent properties of the starting materials

5-8

(Supplementary Materials). Water immersion

of the bars resulted in softening due to absorption of water, demonstrating potential 4D characteristics. Measurement of the keratin-lignin material softening by dynamic mechanical analysis (DMA) on water immersion is given in Figure 8. Evident here was a general reduction in flexural modulus of the compressed bar as the keratin-lignin material absorbed water with time. Initial wetting of the material gave an initial increase in modulus, consistent with water ingress within the voids of the bar. Subsequently, with water beginning to plasticise the material components, a decrease in stiffness was observed. The profile of softening was initially rapid before approaching a minimum. At extended time, this led to sample swelling which impacted the integrity of the sample after >4 h. Interestingly, on adding glycerol to the keratinlignin paste prior to consolidation, the extent of softening was considered lower with greater integrity of the bar observed (>7 h). Overall, by demonstrating keratin-lignin materials exhibit softening on exposure to moisture or water immersion, such inherent behaviour

21-22, 24

of the combined materials provides a practical example of how 3D printed

copolymers may be employed to provide transitionary phases from rigid to soft materials for 4D applications. Currently, these study outcomes are being optimised and adapted into prosthetic applications.

Keratin-lignin/glycerol

Keratin-lignin-only

Figure 8. Dynamic mechanical analysis of keratin-lignin (4:1) compressed bars on water immersion at ambient temperature (23˚C). Keratin-lignin only (green) and keratin-lignin-glycerol (black).

3.4 Discussion Combination of keratin with lignin formed copolymers through protein complexation and was established by both chemical and polymer property evaluations. Such keratin-lignin copolymers can be achieved over a range of lignin contents in aqueous solution (Table 1). This complexation of keratin with lignin appeared primarily through keratin amido and lignin aryl hydroxyl interactions (Figure 1). However, hydrogen-bonding and aryl -bonding interactions may 25

also be involved and likely influenced by lignin ratio and aqueous protein conformations. Moreover, a shift in the

lignin Tg to higher temperature was further evidence of copolymer formation between keratin and lignin. While keratin conjugation was established over different combinations, aqueous processing was dictated by solution pH with keratin complexation induced in acid or mildly alkaline pH. In this study, a preference for a 4:1 ratio of keratin with lignin was a balance of conjugation and physiochemical properties with this ratio taken forward for thermomechanical processing for FDM filaments and hydrogel paste printing applications.

Thermomechanical approaches to processing lignin with keratin as powders led to conjugated materials, but this reactive compounding approach was dependent on the processing temperature and a need for plasticizers to avoid detrimental impacts of intra- and inter-cross-linking of these components and onsets of degradation at the temperatures employed. Using plasticizers such as guar gum promoted improved reactive compounding of powders and integrity of extruded filaments. However, temperatures greater than 150˚C were required for filament production, but filaments produced at these temperatures were marred by some degradation consistent with TGA (Figure 2). This observed degradation and difficulties with extrusion do not make thermomechanical processing and reactive compounding of keratin with lignin attractive to forming conjugated materials above 130˚C. Processing keratin-lignin copolymers as aqueous hydrogels led to 3D printed outlines using paste printing methodology. While dependent on solids content, keratin-lignin-only has a limited processing window (<120 mins) after mixing. This processing window for hydrogels can be significantly extended by using plasticizers similarly found beneficial in reactive compounding (Supplementary Materials). By balancing aqueous solids content, deposition temperatures and drying regime, improvements in the dimensional stability of paste printed outlines can be managed.

4.

Conclusions

For the first time, the conjugation of keratin protein by polyphenolic lignin using green chemistry principles has been employed to form novel copolymer materials intended for additive manufacturing. Keratin conjugation by lignin was established by chemical and polymer analysis. This protein complexation appears primarily through protein amides and lignin aryl hydroxyls and further corroborated by a common glass transition and similar thermal decomposition profiles of the protein-lignin conjugates across differing keratin-lignin combinations heated above 150˚C.

Conjugate formation was developed by processing keratin and lignin in powder form and aqueous solution. In powder form, keratin-lignin cross-linking increased with temperature and, above 130˚C, plasticizers were required to adapt reactive compounding to form keratin-lignin filaments. Processing keratin and lignin as an aqueous hydrogel solution established the copolymers could be deposited by 3D paste printing. While initially marred by dimensional instability and collapse as keratin-lignin copolymer skirts undergo drying, this inherent response to moisture loss could be minimised and also potentially adapted by incorporating this 4D material response into the printed product design. The potential attributes of such 4D printed keratin-lignin copolymers was demonstrated by mechanically responsive behaviours and material softening on water immersion.

Acknowledgements

The authors are grateful to the Science for Technological Innovation National Science Challenge, and the Strategic Science Investment Funding (SSIF) at AgResearch Limited and Scion for funding of this research. The authors would also like to acknowledge the assistance and guidance provided by Maxime Barbier, Marie-Joo Le Guen, Marc Gaugler and Beatrix Theobald (Scion) for this study. K.R. was a recipient of a University of Waikato summer studentship scholarship supported and resourced by Scion.

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Graphical Abstract

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:

There are no competing interests