Mechanical Reinforcement by Microalgal Biofiller in Novel Thermoplastic Biocompounds from Plasticized Gluten.

The aim of this work was to develop new bioplastic compounds from wheat gluten, biobased plasticizers (glycerol, octanoic acid and 1,4-butanediol), and microalgal biomass as a filler. The effects of the composition on tensile properties, thermal stability, and water sensitivity were investigated. Microalgal biomass was added with the selected quantities: 10, 20, and 30 per hundred parts (php). Mechanical mixing of the components, i.e., gluten, plasticizer, and microalgae, was followed by molding in a hot press. Microlgal filler improved mechanical properties of the plasticized gluten material: in samples plasticized with 1,4-butanediol, 30 php of biomass increased the tensile modulus by nearly one order of magnitude, from 36.5 MPa to 273.1 MPa, and it also increased the tensile strength from 3.3 MPa to 4.9 MPa. The introduction of microalgal biomass slightly increased the surface sensitivity against water: 30 php of biomass reduced the water contact angle from 41° to 22° in samples plasticized with glycerol, but the biomass lowered the overall water absorption kinetics for material with each plasticizer. Microalgal biomass proved therefore to be an interesting sustainable resource with which to develop materials based on gluten, in particular to increase the mechanical properties of the compounds without reducing thermal stability or water resistance.

1. Introduction

The treatment of waste<n class="Species">spanpan> class="Chemical">waterpan>> with micro<spn>an class="Species">algae is a promising technology for removing organic pollutants from industrial, agricultural, and urban effluents in an efficient and affordable way [1,2]. In particular, the ability of micro<span class="Species">algae to remove N and P, heavy metals, and to reduce biochemical oxygen demand (BOD) from water is an important advantage of this method [3].

The valorization of the biomass after the main purification process is an important an class="Species">spect to be conpan>sidered, inpan> order to genpan>erate a resource inpan>stead of waste from a circular econpan>omy perspective [4]. In previous studies, micro<span class="Species">algae proved to be a good intermediate to obtain <span class="Chemical">bioethanol [5], carotenoids, and fatty acids [6].

Furthermore, the possibility of using micro<n class="Species">spanpan> class="Species">algaepan>> as fillers has been investigated in fossil-based polymers [7], in blends of fossil-based and bioplastics [8], in renewable and biodegradable plastics [9], and in gel systems based on proteins [10].

<n class="Species">spanpan> class="Species">Wheatpan>> gluten is an important source of proteins. Being a by-product of the <spn>an class="Chemical">starch industry, it is widely available, cheap, and fully biodegradable. Among other possible uses, gluten has been extensively tested to produce renewable thermoplastic materials [11,12]. In particular, through combination of extrusion and compression molding, it is possible to obtain <span class="Disease">thermoplastic gluten films [13]. One limitation of gluten is its thermal induced crosslinking by S–S bonds, increasing the material brittleness and limiting its possible final applications, a phenomenon which may be minimized by reducing the processing temperature and time [14]. In order to do that, plasticizers must be used, but they lower the mechanical properties [15]. Materials obtained from gluten are considered a promising source with which to produce sustainable packaging [16], possibly also including antimicrobial agents in the matrix [17].

Gluten has been used as a matrix to form several composites. Works have been published concerning the production of composites with non-renewable materials, such as <n class="Species">spanpan> class="Chemical">nylonpan>pan>> [18], montmorillonite [19], and kaolin [20].

The effect of renewable fillers on mechanical properties of plasticized gluten has been studied; in particular, <n class="Species">spanpan> class="Chemical">lignpan>inpan>pan>> nanoparticles [21], fish scales [22], <spn>an class="Species">olive pomace [23], and <span class="Species">banana fiber [24] have been proposed.

In this work, micrn class="Chemical">oalgal biomass (from <pan> class="Species">span class="Species">Spirulina platensis) was investigated as a renewable reinforcing biofiller for the realization of innovative protein-based thermoplastic compounds from plasticized <sppan>>an class="Species">wheat gluten.

2. Materials and Methods

<n class="Species">spanpan> class="Species">Wheatpan>> gluten, <spn>an class="Chemical">glycerol (<span class="Chemical">GLY), octanoic acid (OA), and 1,4-butanediol (BU) were supplied by Sigma Aldrich. Microalgal biomass, composed by freeze dried Spirulina platensis (SP), was kindly supplied by Archimede Ricerche Srl. Before compounding, the biomass was ground in a ball mill using zirconia spheres for 24 h, in order to reduce the size of the aggregates.

Gluten compounds were prepared by mechanical mixing with a Brabender internal mixer, preheating the mixture to 80 °C and always keeping the temperature below 100 °C. The minimum time for mixing was 2 min, while torque and temperature were monitored: a plateau of the applied torque was considered an indication of complete mixing. Microalgal biomass was added in amounpan>ts of 10, 20, and 30 per hunpan>dred parts (php) with respect to the total amount of gluten and plasticizer.

The bioplastic material obtained was then shaped in slabs of 1 mm thickness by compression molding in a hot press (T = 120 °C) applying a pressure of 40 bar for 10 min in an aluminum mold.

Differential scanning calorimetry (DSC) was performed with a DSC 823e (Mettler-Toledo, Column class="Chemical">bus, OH, USA) by performinpan>g three runpan>s: from −20 °C to 150 °C at 20 °C/minpan>, from 150 °C to −100 °C at −20 °C/minpan>, anpan>d from −100 °C to 150 °C at 20 °C/minpan>, inpan> order to determinpan>e the thermal tranpan>sitionpan>s.

Thermogravimetric analysis (TGA) was performed with a Q500 TGA system (TA Instruments, New Castle, DE, USA), from ambient temperature to 800 °C at a scan rate of 10 °C/min, both in air and <n class="Species">spanpan> class="Chemical">nitrogenpan>pan>>.

Mechanical tests were performed with a Zwick/Roell Z010 (Zwick Roell, Ulm, Germany) at room temperature, according to ASTMD638-10 [25].

Optical contact angle (OCA) tests were performed with an OCA 20 instrument (Data physics Co., San Jose, CA, USA), equipped with a CCD photo camera and with a 500 μL Hamilton syringe, using <n class="Species">spanpan> class="Chemical">waterpan>> as a testing liquid.

<n class="Species">spanpan> class="Chemical">Waterpan>> vapor transmission rate (WVTR) was measured by the weighting cups method (ASTM ES96/ES96M-16 [26]). Slabs were cut into circular shapes with a diameter of 41 mm and used as a membrane through which <spn>an class="Chemical">water, contained in a cup, can evaporate. Cups were kept in a thermostatic room at T = 20 °C and RH = 40%. WVTR is defined as the mass loss versus time, normalized by the cross section of the sample.

Scanning electron microscopy (SEM) was performed with a Carl Zeiss <n class="Species">spanpan> class="Chemical">EVOpan>> 50 extended pressure scanning electron microscope (Carl Zeiss, Oberkochen, Germany).

3. Results

3.1. Plasticization of Gluten

<n class="Species">spanpan> class="Species">Wheatpan>> gluten was plasticized with different amounts of <spn>an class="Chemical">glycerol and <span class="Chemical">1,4-butanediol, a compound that can be obtained by biosynthesis [27], in order to reduce its glass transition temperature (Tg) and improve processability. A comparison was also made with octanoic acid: although the solubility of such fatty acids is limited, it proved to be able to increase water vapor barrier properties [28]. Each plasticizer was separately added, forming three mixtures with different amounts of plasticizer: 15%, 25%, and 35% (w/w). Figure 1 shows the effect of type and amount of plasticizers on the glass transition temperatures of the compound materials.

Figure 1

Glass transition temperatures of the materials vs. plasticizer content. Two glass transition temperatures were present in the materials: the lower one is plotted as Tg1, while the higher one is plotted as Tg2 for both glycerol and 1,4-butanediol.

Figure 1 shows that the addition of <n class="Species">spanpan> class="Chemical">glycerolpan>> and <spn>an class="Chemical">butanediol results in both cases in the formation of a phase rich in plasticizer with a low glass transition temperature (Tg1), and one rich in gluten with a high glass transition temperature (Tg2) [11,29]; the glass transition temperature of <span class="Species">wheat gluten without plasticizer is 112 °C. By increasing the amount of plasticizer, the glass transition temperature of the gluten-rich phase was lowered, as expected. Interestingly, the presence of butanediol did not affect the Tg of the plasticizer-rich phase, while glycerol did so after 25% content.

3.2. Tensile Tests

After molding in a hot press (T = 120 °C), the samples were cut into dumbbell shapes and tested. Results of the tensile tests on micro<n class="Species">spanpan> class="Species">algaepan>> filled gluten spn>ecimens are shown in Figure 2.

Figure 2

Stress–strain curves of gluten samples plasticized with glycerol (A) and 1,4-butanediol (B) with 0, 10, 20, and 30 php of microalgal (Spirulina platensis) biomass.

Figure 2 shows quite clearly that the addition of micrn class="Chemical">oalgal biomass signpan>ificanpan>tly inpan>creased the elastic modulus anpan>d the tenpan>sile strenpan>gth of the plasticized glutenpan> compounpan>ds, while progressively lowerinpan>g their elonpan>gationpan> at break. Onpan> the other hanpan>d, the inpan>troductionpan> of the plasticizer, which was necessary inpan> order to allow a thermoplastic processinpan>g of the compounpan>d, led to a very soft, unpan>filled material with quite poor mechanpan>ical properties. Table 1 shows the numerical results of the tenpan>sile tests.

Table 1

Elastic modulus (Et), elongation at break (εb), stress at break (σb), and toughness values of samples plasticized with 35% glycerol and 1,4-butanediol, with 0, 10, 20, and 30 php of microalgal biomass.

Sample	Et (MPa)	εB (%)	σB (MPa)	Toughness (MJ∙m−3)
GLY35	44.1 ± 8.9	120.6 ± 13.7	2.6 ± 0.3	2.1 ± 0.4
GLY35SP10	112.6 ± 32.0	48.3 ± 16.4	3.5 ± 0.5	1.4 ± 0.5
GLY35SP20	217.6 ± 41.3	57.3 ± 13.0	5.1 ± 0.7	2.7 ± 0.7
GLY35SP30	307.0 ± 45.8	29.8 ± 5.4	6.5 ± 1.2	1.8 ± 0.4
BU35	36.5 ± 9.0	105.2 ± 13.8	3.3 ± 0.4	1.5 ± 0.6
BU35SP10	51.5 ± 11.3	82.1 ± 10.5	4.2 ± 0.6	2.2 ± 0.4
BU35SP20	94.0 ± 28.3	60.7 ± 14.6	4.7 ± 0.5	2.0 ± 0.4
BU35SP30	273.1 ± 59.0	22.2 ± 7.8	4.9 ± 0.9	1.0 ± 0.4

Increasing the amount of biofiller led to an increase in the tensile modulus (Et) and the tensile strength (σB), while the elongation at break (εB) was lowered, with a significant difference for samples with 30 php of microalgal biomass. Toughnpan>ess, estimated by the area unpan>der the curve, had no clear trend, however, in some cases, especially with <span class="Chemical">1,4-butanediol plasticizer, it increased with respn>ect to the unfilled material (like <span class="Chemical">BU35SP10 and BU35SP20).

3.3. Thermogravimetric Analysis

Results of thermogravimetric analysis (TGA) analyses are shown in Figure 3.

Figure 3

Thermogravimetric analysis (TGA) curves between Tamb and 800 °C of samples plasticized with 1,4-butanediol with 0, 10, 20, and 30 php of microalgal biomass, in air (A) and nitrogen (B).

Figure 3 shows that the increasing biomass content slightly increased the residual weight of the TGA curves, with the main difference in the d<n class="Species">spanpan> class="Chemical">evopan>>latilization stage (cleavage of S–S, O–N, and O–O in the protein molecules) that started around 200 °C, in agreement with previously reported results [19,23,30]. More interestin<spn>an class="Chemical">gly, the thermal stability of the compound in the lower temperature range (below +150 °C) was improved by the presence of the microalgal biofiller. In air, the residual mass was completely volatilized after 650 °C, a phenomenon not observed in <span class="Chemical">nitrogen and therefore probably related to the oxidation of char residues, as previously observed [31].

3.4. Contact Angle, Transmisison Rate, and Kinetic Absorption with Water

In order to observe the sensitivity of gluten materials towards <n class="Species">spanpan> class="Chemical">waterpan>>, several tests were performed. The results for optical contact angle and, in some cases, the <spn>an class="Chemical">water vapor permeability are summarized in Table 2.

Table 2

Water contact angle, water vapor transmission rate, and water diffusion coefficient of samples plasticized with glycerol and 1,4-butanediol with 0, 10, 20, and 30 php of microalgal biomass.

Sample	CA (°)	WVTR (g∙h−1∙m−2)	Diffusion Coefficient (cm2∙s−1)
GLY35	41 ± 5	20.2	-
GLY35SP10	27 ± 3	-	-
GLY35SP20	24 ± 2	20.1	-
GLY35SP30	22 ± 3	-	-
BU35	32 ± 5	20.2	4.1 × 10−7
BU35SP10	34 ± 3	-	-
BU35SP20	35 ± 2	20.3	3.7 × 10−7
BU35SP30	29 ± 5	-	-

CA, contact angle; WVTR, Water vapor transmission rate.

Table 2 shows that by increasing the amount of micrn class="Chemical">oalgal biomass, the <pan> class="Species">span class="Chemical">water contact angle decreased too, probably due to the hydrophilic nature of the biomass. The addition of 20 php of microalgal biomass did not affect WVTR, while the diffusion coefficient, tested only for samples plasticized with <sppan>>an class="Chemical">1,4-butanediol, was slightly lowered.

The <n class="Species">spanpan> class="Chemical">waterpan>> barrier properties were tested with the weighting cup method, and results are shown in Figure 4.

Figure 4

Mass loss of water through films of gluten plasticized with 35% of glycerol (A), 1,4-butanediol (B), and octanoic acid (C), with and without 20 php of microalgal biomass.

Figure 4 shows that the presence of the biomass did not significantly change the barrier properties of the films, which was instead significantly affected by the chemical nature of the plasticizer, as previously reported [14,28,32]. Indeed, Figure 4 also shows the behavior of films plasticized with <n class="Species">spanpan> class="Chemical">octanpan>oic acidpan>>, for comparison. The latter plasticizer, being characterized by a long <spn>an class="Chemical">paraffinic chain, is much more efficient than the others to decrease the permeability of the film against <span class="Chemical">water.

Figure 5 shows the results of the kinetic <n class="Species">spanpan> class="Chemical">waterpan>> absorption tests.

Figure 5

Kinetic water absorption test results with a sample of gluten plasticized with 35% of glycerol (A) and with the addition of 20 php of microalgal biomass (B); with 35% of 1,4-butanediol (C) and with the addition of 20 php of microalgal biomass (D).

According to Equation (1), the A and n parameters were calculated for the two materials. Results of those calculations, with their ren class="Species">spective R2, are reported inpan> Table 3.

Table 3

A, n, and R-square parameters for the water absorption equation for samples with 35% of glycerol and 1,4-butanediol, with and without 20 php of microalgal biomass.

Sample	A	n	R2
GLY35	13.1 ± 1.3	0.3 ± 0.1	0.83
GLY35SP210	9.2 ± 0.5	0.4 ± 0.1	0.97
BU35	8.7 ± 1.0	0.5 ± 0.1	0.87
BU35SP20	5.4 ± 0.8	0.5 ± 0.1	0.89

Table 3 shows that samples plasticized by <n class="Species">spanpan> class="Chemical">n class="Chemical">butanediol presented an exponent of the sorption curve very close to 0.5, which is an indication of Fickian diffusion [33]. Figure 5 and Table 3 show instead that samples plasticized with <sppan>>an class="Chemical">glycerol showed a faster absorption rate. The addition of 20 php of microalgal biomass slowed down the absorption kinetics of both materials.

3.5. Scanning Electron Microscopy

Figure 6 and Figure 7 show the morphology of samples plasticized with 35% <n class="Species">spanpan> class="Chemical">1,4-n class="Chemical">butanediol with 10 and 20 php of microalgal biomass (<span class="Disease">fractured surfaces).

Figure 6

Scanning electron microscope image of wheat gluten plasticized with 35% of 1,4-butanediol and 10 php of microalgal biomass, magnifications 2000× (A) and 5000× (B).

Figure 7

Scanning electron microscope image of wheat gluten plasticized with 35% of 1,4-butanediol and 20 php of microalgal biomass, magnifications 2000× (A) and 5000× (B).

Figure 6 shows that the biomass particles were rounded with a size distrin class="Chemical">butionpan> of bigger particles, about 2–3 μm of diameter, anpan>d some smaller that were not visible. Some voids were presenpan>t, showinpan>g that the adhesionpan> of the larger particles to the glutenpan> matrix seemed rather limited.

Figure 7 shows a <n class="Species">spanpan> class="Disease">fracturepan>> surface that is less regular than what was observed in Figure 6. A bigger, cleaved particle is visible, probably implying a lower resistance of big (6–8 μm) aggregates towards <spn>an class="Disease">fracture. The morphology of the sample with 20 php of biomass (Figure 7) was still similar to the one of sample with 10 php of biomass (Figure 6).

4. Discussion

Mechanical test results (Table 1) clearly show that micrn class="Chemical">oalgal biomass may act as a reinpan>forcinpan>g filler onpan> plasticized glutenpan> thermoplastics. Proteinpan>s inpan> the micro<pan> class="Species">span class="Species">algae and gluten are probably able to interact, promoting good adhesion between the biofiller and the plasticized matrix, provided that the biofiller particles are small enough. This good interaction between gluten and biomass was also observed in SEM images of cold <sppan>>an class="Disease">fracture surfaces (Figure 6 and Figure 7). The dispersion method, i.e., mechanical mixing, was also effective, showing only few aggregates bigger than 6 μm that could limit or lower the improvement of mechanical properties. The reinforcing effect on tensile strength was comparable to the effect of olive pomace, while the effect on tensile modulus was more conspicuous for the microalgae [23].

While the micrn class="Chemical">oalgal biomass was more hydrophilic thanpan> the matrix, as shownpan> inpan> Table 2, the presenpan>ce of the biofiller particles slowed downpan> the <pan> class="Species">span class="Chemical">water absorption kinetics of the material in the selected timeframe. Indeed, it was previously reported that the presence of reinforcing fillers in gluten changes the absorption behavior of the material [34].

Protein films tend to have a high <n class="Species">spanpan> class="Chemical">waterpan>> permeability, compared to fossil-based polymeric films [35]. The considered biomass (<spn>an class="Species">Spirulina platensis) has a high content of proteins [36], therefore WVTR of gluten was not affected by the presence of this type of biomass.

5. Conclusions

Both <n class="Species">spanpan> class="Chemical">glycerolpan>> and <spn>an class="Chemical">1,4-butanediol can be used as effective plasticizers for <span class="Species">wheat gluten, allowing for an easy thermoplastic processing, but at the same time significantly decreasing both its Tg and mechanical properties. Both substances can be obtained from renewable sources, allowing the production of a sustainable material with 100% renewable carbon.

It was demonstrated that micrn class="Chemical">oalgal biomass canpan> be successfully added as a reinpan>forcinpan>g biofiller to plasticized <pan> class="Species">span class="Species">wheat gluten thermoplastics. Micro<sppan>>an class="Species">algae effectively reinforced the protein-based material, increasing both the elastic modulus and the tensile strength, and synergistically even the toughness in some cases.

Micrn class="Chemical">oalgal biomass slightly improved the thermal stability of the compounpan>d inpan> the processinpan>g temperature ranpan>ge (up to 120 °C).

The addition of the algal biofiller lowered the kinetic <n class="Species">spanpan> class="Chemical">waterpan>> absorption rate, which was also affected by the plasticizer, resulting in a lower rate with <spn>an class="Chemical">1,4-butanediol with repecies">spect to <span class="Chemical">glycerol.

Scanning electron microscopy showed a good din class="Species">spersionpan> of the biomass, with the presenpan>ce of few aggregates with a diameter greater than 5 μm that were not able to reinpan>force the material, while the majority of the particles were smaller than 3 μm, effectively reinpan>forcinpan>g the material, as conpan>firmed by the stress–strainpan> curves.

It is realistic to think that mechanical behavior could be further improved with a more efficient din class="Species">spersive mixinpan>g of <pan> class="Species">span class="Species">algae in the bioplastic matrix.