1

2

INDEX

Table Index

Figures Index

Abstract

Resumen

Chapter 1. Antecedents

1.1 Introduction

1.2 Plant-derived compounds are useful for biodegradable plastics preparations.

1.3 Polyethylene: ideal for biodegradable plastics preparation

1.4 Bio-composites.

1.5 Biodegradation and composting.

Chapter 2. Rationale, Hypothesis and Objectives.

Chapter 3. Material and methods

3.1 Biodegradable material: manufacture and characterization

3.2 Biodegradation assessment

Chapter 4. Results and discussion

4.1 Biodegradable material: manufacture and characterization

4.2 Biodegradation assay

Chapter 5. Conclusions

5.1 Material development conclusions

5.2 Biodegrading process assay conclusions

Chapter 6. Perspectives

References

3

FIGURES INDEX

Figure B1. Mechanisms involved in PE biodegradation

Figure B2. Preparation of the thermoplastic unripe banana flour

Figure B3. Preparation of the biodegradable plastic

Figure B4. Biodegradability assay

Figure 1. Viscosity of the regular and the thermoplastic green banana flour

Figure 2. Differential scanning calorimetry for regular (a) and thermoplastic (b) unripe banana flour

Figure 3. Differential scanning calorimetry. Depicted are the thermograms of the neat components and the four injected blends Figure 4. Mechanical properties of the injected blends and injected neat mPE. a) Tensile strength at break. b) Tensile strength at yield, and c) Elongation at break Figure 5. Scanning electron microscopy of regular and thermoplastic unripe banana flour: a,b) granule size and shape, c,d) distribution of flour particles, e,f) microstructure of extruded flour

Figure 6. Scanning electron microscopy of the mold injected material. Surface and transversal view of the different TPF preparations

Figure 7. Fourier-transformed infrared spectroscopy (FT-IR) analysis for regular green banana flour and TPF

Figure 8. FT-IR spectra of the blends from 50 to 80 TPF and mPE-g-MA. Circle encloses the three peaks from MA

Figure 9. Weight loss percentage after 125 days under compost

Figure 10. Chronological Microstructural analysis by SEM (500x) of the samples under compost during 125 days

Figure 11. Analysis of fungi found on the surface of the TPF samples Figure 12. DSC Thermograms of the samples at a) Day 0, b) Day 75 and c) Day 125

Figure 13. Mechanical properties of the blends and neat mPE a) Tensile resistance and b) Elongation at break

Figure 14. Tensile resistance, Total Elongation and Percentage of Elongation of the neat mPE and TPF blends before and after composting

Figure15. Infrared analysis before and after compost for the 50, 60, 70 and 80 TPF samples

4

TABLES INDEX

Table A1. Starch content according to the ripeness degree

Table A2. Mexico 2011 agricultural production ordered by the economic value

Table 1. Colorimetric analysis of regular and whitened unripe banana flour

Table 2. Melting Temperatures (Tm) and enthalpy (∆H) for regular and thermoplastic unripe banana flour

Table 3. Relative weight loss percentage data at 25 days intervals. The total weight lost at day 125 was set to 100%

5

Abstract

To meet the challenge of procuring new sources of natural polymers for biodegradable

material manufacturing without affecting the alimentary demands of main crops, we

produced and characterized a thermoplastic unripe banana flour (TPF) and blended it

with metallocene-catalyzed polyethylene (mPE). Both pulp and peel of unripe banana

formulated a flour with appropriate thermoplastic properties and stability for processing

in the extruder. This allowed the blending of the thermoplastic flour in high

concentrations (50, 60, 70, and 80%) with mPE at optimum temperature conditions. For

creating the polymers blend, the mPE was functionalized with maleic anhydride (MA).

The MA grafted into mPE (mPE-g-MA) promoted an inter-phase adhesion of the

synthetic polymer with the natural polymer. The adhesion generated a homogeneous

material and improved the mechanical, thermal, and microstructural properties of the

blends. Blends and neat mPE (as a control sample) were molded by injection. Weight

loss analysis, scanning differential calorimeter (DSC), mechanical properties testing

(Instron equipment), scanning electron microscopy (SEM) and infrared spectral

properties (FTIR) were applied to the molded material for characterization.

Biodegradability was assessed by burying samples in a 60 cm lower layer and a 30 cm

upper layer of compost. 50, 60, 70 and 80 TPF and neat mPE (as a control) samples

were fixed in five 15cm x 20cm plastic grids (one for each period). After 25, 50, 75,100

and 125 days samples were retrieved, washed, dried, acclimated and then weighed.

Weight loss analysis, mechanical, thermal, microstrucrural and infrared analysis also

were conducted after each 25 days period. Results contributed to demonstrate a degree

of biodegradability. In addition, fungi hyphae were detected on the surface of the blend

samples after each period of composting process, but not on the neat PE surface.

Details of the fungal structures were analyzed by light microscopy and scanning

electron microscopy (SEM).

The characteristics of the blends made them ideal in designing of plastics with

biodegradability potential while taking advantage of the banana peels, usually

considered an agricultural waste.

6

Resumen

Ante el reto de obtener nuevas fuentes de polímeros naturales para la obtención de

materiales biodegradables sin afectar la demanda alimentaria, producimos y

caracterizamos una harina de plátano verde termoplástica (TPF) y la mezclamos con

polietileno catalizado por metalocenos (mPE). La harina se formuló con la pulpa y la

cáscara del plátano inmaduro; las propiedades termoplásticas de la harina fueron

adecuadas y con estabilidad para su procesamiento en el extrusor. Esto permitió el

mezclado de la harina termoformable en altas concentraciones (50, 60,70 y 80%) con el

polietileno mPE en condiciones óptimas de temperatura. Para la conformación de la

mezcla de polímeros se funcionalizó el mPE con anhídrido maleico (AM). EL AM

injertado en el polietileno catalizado por metalocenos (mPE-g-MA) favoreció una

adhesión interfacial del polímero sintético con el polímero natural que generó un

material homogéneo en cada mezcla con mejores propiedades térmicas, mecánicas y

microestructurales. Las mezclas y el mPE puro fueron moldeados por inyección. Para

su caracterización se evaluaron la de pérdida de peso, calorimetría diferencial de

barrido (DSC), las propiedades mecánicas (Instron equipment), microscopia electrónica

de barrido (SEM) e infrarrojo (FTIR) en las muestras inyectadas.

Para las pruebas de biodegradabilidad se enterraron las muestras entre una capa

inferior de composta de 60 cm y una capa superior de 30 cm. Las muestras de las

mezclas, una de cada mezcla, fueron fijadas en cinco mallas de plástico de 15cm x

20cm (una para cada periodo). Después de 25, 50, 75, 100 y 125 días, una malla fue

retirada y las muestras fueron lavadas, secadas, ambientadas y posteriormente

pesadas. Para evaluar el grado de biodegradación, después de cada periodo se realizó

nuevamente un análisis de pérdida de peso, calorimetría diferencial de barrido,

microscopia electrónica de barrido e infrarrojo. Además, se detectaron hifas de hongos

en la superficie de las muestras después del composteo. Los detalles de la estructura

de los hongos fueron analizados en el microscopio óptico y en el microscopio

electrónico de barrido (SEM).

Las características de las mezclas resultaron ideales para el diseño de plásticos con

potencial biodegradable; aunando a esto, se toma ventaja de la cáscara de plátano

comúnmente considerada como basura orgánica. 7

CHAPTER 1

ANTECEDENTS

1. 1 Introduction

Solid waste management is an issue of enormous concern all over the world because of

factors that include oil consumption and CO2 production [1,2]. In developing countries,

additional factors like poverty, overpopulation, urbanization and lack of government

funding amplify the problem [3]. Because plastics are considered an ecological problem,

the design and development of biodegradable materials are being explored [4]. The

purpose of this work was to produce a flour to blend it with mPE to generate a

biodegradable plastic; therefore the background, methods and results sections are

going to be presented in two stages: the manufacturing and characterization of the

green banana flour-mPE blends and the demonstration of the biodegradability of the

resultant plastic. The biodegradability confirmation is of high importance because the

world requirement for biodegradable polymers and the scientific development has made

these materials easier to produce, and soon they will be cheaper to manufacture [5], but

their actual biodegradability remains obscure.

1.2 Plant-derived compounds are useful for biodegradable plastics preparations.

Starch from various sources (i.e., corn, potato, rice) is considered as one of the

biodegradable components that can be employed in the plastics industry [6]. The

structure and the physicochemical properties of corn, rice, wheat, and potato starches

have been well documented [7] as well as their ability to blend with synthetic polymers

to improve their biodegradability properties [8,9]. Nevertheless, because of the starch

widespread usage, especially for food applications, new sources of starch and other

natural polymers appropriate for industrial purposes are required.

8

The complete unripe banana is of great interest as a candidate source of biodegradable

materials. The unripe banana pulp contains up to 70–80% starch on a dry weight basis,

a percentage comparable to that in the endosperm of corn grain and the pulp of white

potato [10]; and the peel of banana, that represents approximately 40% of the total

weight of a banana [11], has a potential use in industry and it is usually considered a

waste. During the banana ripening the starch reserve rapidly transforms into sucrose

and further into glucose and fructose due to the activities of amylase, glycosidase,

phosphorylase, sucrose synthase and invertase [12,13]. Thus, it is necessary for starch

production to process the bananas while they are unripe. The approximate sugar

contents at different stages in the ripening process, classified by the color of banana

peel, appear in table A1. The starch content is different to that calculated on dry weight

basis because this percentage considers the whole banana and not just the pulp.

Stage Peel color Starch

(%) Reducing sugar

(%) Sucrose

(%)

1 Green 61.7 0.2 1.2 2 Green 58.6 1.3 6.0 3 Green/A trace of yelllow 42.4 10.8 18.4 4 More green than yelllow 39.8 11.5 21.4 5 More yellow than green 37.6 12.4 27.9 6 Yellow with a green tip 9.7 15 53.1 7 All yellow 6.3 31.2 51.9 8 Yellow / a few brown spots 3.3 33.8 52.0 9 Yellow / many brown spots 2.6 33.6 53.2

Table A1. Starch content according to the ripeness degree [14].

Although the main focus on the banana research is the pulp, there are potential

applications of banana peels, e.g. production of alcohol, methane, food for livestock,

and adsorbents for water purification [15]. In general, plant cell-wall polysaccharides are

9

an extremely diverse set of biopolymers that play an important role as structural

elements; they include soluble fibers (pectins, gums, etc.) and insoluble fibers

(cellulose, lignin, some hemicelluloses, etc.) [16]. Because the biopolymers of the

banana peels might contribute as filler and reinforcing material [17], in this study we

produced and characterized a thermoplastic unripe banana flour (TPF). The TPF was

blended with mPE by extrusion, molded by injection, and further characterized.

Bananas are the fourth worldwide most important crop in the food industry, only after

rice, wheat and corn [18]. It grows throughout the year and the most important thing for

the purpose of this investigation is that there is generally availability of the unconsumed

material [19]. In Mexico, banana crop ranks 15 among the 20 most important crops by

production value (Table A2) and Mexican banana production is the ninth most important

in the world [17].

CROP Plantings Harvested area Production Yield Production value

(Ha) (Ha) (Ton) (Ton/Ha) $(Thounsands of Pesos)

1 CORN 7,750,301.19 6,069,091.63 17,635,417.30 2.91 71,913,855.24 2 SUGAR CANE 774,243.18 713,824.00 49,735,273.26 69.67 30,369,914.67 3 SORGHUM 1,972,058.88 1,728,228.45 6,429,311.46 3.72 22,185,072.12 4 AVOCADO 142,146.10 126,597.89 1,264,141.46 9.98 18,136,404.25 5 PASTURES 2,548,924.35 2,461,500.66 46,173,564.77 18.76 17,179,282.28 6 LUCERNE 387,799.54 376,421.64 28,247,520.47 75.04 13,055,453.78 7 WHEAT 714,864.19 662,221.35 3,627,510.83 5.48 13,043,285.28 8 GREEN CHILE 152,742.37 144,390.60 2,131,739.73 14.76 12,099,213.70 9 TOMATOE 53,780.18 44,932.15 1,872,481.69 41.67 10,336,853.07 10 POTATO 69,054.26 54,551.05 1,433,239.14 26.27 9,069,647.13 11 COTTON 198,439.53 193,485.33 746,245.83 3.86 7,089,886.92 12 BEAN 1,506,033.82 894,972.06 567,779.15 0.63 6,889,765.84 13 COFFEE 760,974.05 688,208.41 1,287,642.69 1.87 6,815,878.60 14 LEMON 166,580.41 149,607.56 2,132,921.78 14.26 6,305,658.73 15 BANANA 77,303.66 74,284.16 2,138,686.85 28.79 6,163,079.29 16 NUT 96,277.27 68,008.91 96,476.35 1.42 6,027,379.78 17 ORANGE 335,471.72 330,174.59 4,079,677.74 12.36 5,903,847.70 18 GRAPE 27,830.86 27,209.75 281,144.98 10.33 4,736,829.58

10

19 ONION 48,638.91 47,126.16 1,398,851.21 29.68 4,566,352.58 20 CORN FODDER 489,152.52 311,225.44 9,605,147.84 30.86 4,402,710.47 Table A2. Mexico 2011 agricultural production ordered by the economic value [20].

In the present study, we aimed at preparing unripe banana flour with potential use as a

matrix, and not just as filler, for a theoretically biodegradable plastic material. Several

granular starches have been blended with plastics to develop biodegradable materials

[21]. However, the starch is not dispersed very finely within the plastic matrix owing to

its inherent hydrogen bonding [22]. To avoid the agglomeration and lack of

homogenization in blending with synthetic polymers, thermoplastic starches (TPS) have

been developed by incorporation of fitting amounts of water and/or plasticizers into the

pristine starch [23]. Blends containing TPS exhibit improved homogenization compared

with those containing only native starch in their formulation, even when the same plastic

matrix was used [24,25]. We used the thermoplasticization process in the preparation of

our unripe banana flour.

1.3 Polyethylene: ideal for biodegradable plastics preparation

Polyethylene (PE) has the biggest production volumes of synthetic polymers all over the

world [26]. This polymer poses many advantages e.g., low cost, excellent chemical

resistance, lightness, durability, and is non-toxic [27]. Polyethylene is synthesized by

using either Ziegler-Natta (ZN) catalysts or metallocene catalysts [28]. Ziegler–Natta

catalyst produces a relatively broad distribution of molecular weights [29], but ZN-

catalyzed PE products often show some problems like melt fracture, surface distortion,

and important deterioration in their mechanical properties. Conversely, metallocene-

catalyzed polyethylenes (mPEs) exhibit superior physical and mechanical properties

because the metallocene catalyst yields polyolefins with narrow molecular-weight

distribution and sparse long-chain branches [30]. Metallocene catalyst produces PE

11

with an excellent degree of control on molecular structure [31] and stereospecificity [32]

at a commercial scale [33,34]. In the manufacturing process, mPE is considered a good

polymer option among available plastics despite its high viscosity because it has good

mechanical properties.

The simple mixture of PE and starch lacks homogenization [35] due to the non-polar PE

condition. Grafting maleic anhydride to the backbone of the synthetic polymer can

provide polarity to the mPE [36]. Maleic anhydride increases compatibility and improves

inter-phase adhesion between the natural and the synthetic polymer [37].

1.4 Bio-composites.

Synthetic polymers like plastics are resistant to microbial attack; without previous waste

separation they have become a pollutant in soil or landfill because their degradation rate

is too slow. It has been reported that it requires 300 years for a 60 µm LDPE film to

degrade completely in soil [38]. Biodegradable polymers have been obtained by

incorporating natural polymers derived from cereals and tubers to plastics; this blending

has allowed reducing the polymer microbial resistance to degradation by attracting fungi

and bacteria from soil to attack both the synthetic and natural moieties [39].

A biodegradable polymer had been defined as a bio-composite of mostly a matrix of a

synthetic polymer complemented with cellulosic materials, which act as the reinforcing

filler [40]. Biodegradation is believed to release intermediate products toxic to the

environment, but when the matrix is based on a natural polymer the probability of

leaving toxic residues is low. In addition, the advantage of materials based on

renewable sources is that when these become waste, they can be taken directly to the

landfill or a composting process [41].

1.5 Biodegradation and composting. 12

The degradation of polymers and biopolymers in the environment occur in several ways:

by micro and macroorganisms degradation, photodegradation and chemical

degradation [42]. In the specific case of the compost, microbial organisms transform

biopolymer through metabolic or enzymatic action into new compounds generating

smaller fragments of the polymer blend enabling their reintegration to soil [43].

For a bio-composite production we selected metallocene catalyzed polyethylene (mPE);

mPE is an easy processing synthetic polymer with a high degree of customization

(stretch film, trash bags, heavy-duty bags, etc.), narrow molecular weight distribution,

and chemical homogeneity compared with those obtained with classical Ziegler-Natta

catalysts [44]. Besides, it exhibits other good properties in comparison with conventional

thermoplastics; for instance, low melting temperature of mPE is advantageous because

it can be blended with bio-fillers, which often have a lower thermal stability [45].

Several bio-fillers have been tried including starch, flour and cellulose [46,47,48]. This

natural organic material can be degraded by microorganisms in soil, which use it as a

carbon source [49]. Carbon serves primarily as an energy source for the

microorganisms, while a small fraction of the carbon is incorporated into their cells.

Some of the energy formed is used for microbial metabolism, and the rest is released as

heat [50]. During composting microorganisms transform organic matter into CO2,

biomass, and humus [51].

Figure B1 describe a detailed biodegradation process for a polyethylene film, which

actually may occur. Neat polyethylene biodegradation process is a heterogeneous

process. The lack of polyethylene water solubility prevents microorganisms to

incorporate the plastic material directly. Thus, a feeding stimulant for microorganism like

a starch or cellulose facilitates and shortens the biodegradation process.

13

Figure B1. Mechanisms involved in PE biodegradation. PE films can be fragmented by abiotic oxidation processes (1), but the fragments would be too big to get across the microbial cell wall; so the soluble extracellular or cell wall associated enzymes can mediate their further oxidation (2). Some microorganisms can release oxidant radicals (3) and mobilize smaller water insoluble PE degradation products that can pass through the cell wall (4). Inside the microorganism, PE fragments can be transformed by enzymes into smaller molecules and be completely assimilated through the oxidation pathway (5) [52].

Composting is being taken into consideration in waste management strategies as an

alternative technology to conventional disposal in landfills or incineration. This process

mixes biodegradable plastics with other natural organic compostable materials such as

yard, food, and agricultural waste that can generate carbon-rich material useful for soil

14

amendment products [53,54,55]. In the present work we use composting to assess the

biodegradability of our blends.

Previous works evaluated the biodegradability of bio-composites where the synthetic

polymer constituted the majority of the blend [56,57]; in the present work, we analyzed

the biodegradability of a plastic material where banana flour is acting as a matrix (up to

80%) and the metallocene catalyzed polyethylene (mPE) just as reinforcing filler. Within

this evaluation process, we evaluated the physical, mechanical and thermal properties

of the blends buried under compost for 50, 75, 100 and 125 days; we analyzed the

structural changes associated to degradation by SEM; additionally, we performed the

identification of the main microorganism that contributes to the biodegradation.

15

CHAPTER 2

RATIONALE

Durability, strength, lightness, and affordability make polyethylene one of the most

extensively used polymers all over the world. These qualities allow producing a wide

range of everyday products primarily for packaging. Despite the efforts of separation

and recycling, much of the polyethylene ends up in landfills, contributing to the soil

pollution. The interest in reducing contamination without putting aside polyethylene has

lead researchers to blend it with natural polymers like starch and cellulose to make

polyethylene degradable.

Due to their thermoplastic property, starches from corn, rice, and potato have been

successfully used for blending with polyethylene. However, crops supply is mostly

confined to nutritional and energetics purposes. Banana, unlike the other starchy crops,

has that surplus that could be used for manufacturing biodegradable polymers without

detriment of the nutritional market requirements, which make it good candidate for

blending with synthetic polymers. In addition, most of the vegetable peels are

considered garbage, but one of the purposes of this work is also to take advantage of

the fibers that banana peels would contribute to the plastic design. Thereby, the

complete unripe banana can be used as a component of a biodegradable plastic.

In the manufacturing of biodegradable polymers, the matrix is commonly made of the

synthetic polymer whereas the natural polymer is used as filler. The main contribution of

this work lies in reversing the roles by transforming the unripe banana flour into the

matrix of a material with lower proportions of polyethylene, with expectable shorter

degradation times. To prepare the biodegradable plastic we selected mPE because it

16

has better physical and mechanical properties that would prevent the lost of the

mechanical properties as the concentration of flour increases.

Biodegradable polymers offer a solution in plastic waste management, a contribution to

solve the world ecological problem. Thus, the preparation of a blend with the best

polyethylene and an available natural polymer whose potential applications exceed

those of the existing biodegradable plastics is a challenge that this study addresses.

HYPOTHESIS

The blend of thermoplastic flour from unripe banana and metallocene catalyzed

polyethylene (TPF-mPE) produce a biodegradable polymer that under compost

conditions reduces its mechanical, thermal, and structural properties with reduction in

the biodegradation time proportionally to the polyethylene concentration.

GENERAL OBJECTIVE

To manufacture an injection molded biodegradable polymer blend of thermoplastic flour

from unripe banana and metallocene-catalyzed polyethylene and to determine its

biodegradability under compost conditions.

STUDY OUTLINE

1. To manufacture thermoplastic flour from unripe banana.

2. To functionalize the mPE by maleic anhydride grafting.

17

3. To blend both polymers by extrusion.

4. To mold the biodegradable material by injection.

5. To characterize the biodegradable polymer: mechanical properties, thermal

properties, microstructural analysis, and infrared spectral analysis.

6. To assay biodegradation by subjecting the material to a compost process during

125 days.

7. To evaluate the degradation degree every 25 days period by DSC, SEM,

INSTRON and FTIR techniques.

8. To find the microorganism responsible for the biodegradation by optical and

scanning electronic microscopy techniques.

18

CHAPTER 3

3. Material and methods

3.1 Biodegradable material: manufacture and characterization

3.1.1 Unripe banana flour

Unripe banana (Musa cavendishii AAA), which was not exposed to ethylene gas for

ripening acceleration, was purchased in Mexico City’s supplies center. This kind of

banana is cultivated in the Gulf of Mexico and Ocean Pacific slopes (14°15' and 22°30'

latitude north). Unripe bananas were sanitized with 1% sodium hypochlorite, cut into 2

mm thick longitudinally slices, and sprayed with 5% acetic acid to prevent enzymatic

oxidation. Banana slices were dehydrated at 60 °C for 36 h in a F.J. Stokes vacuum

chamber (Philadelphia, USA) followed by powdering in a milling disc, sifting in a 40

mesh, and re-sifting in a 100 mesh to recover a fine-grained flour. The flour was

subjected to a whitening process by mixing it with 40% hydrogen peroxide at a weight

ratio of 6:4 in a Teledyne Lab internal mixer (York Pennsylvania, USA) at a speed of 20

rpm during 60 min. This blend was dried for 24 h at 50 °C followed by an additional

milling and sifting process. To generate thermoplastic material, flour, water and glycerol

were mixed in the internal mixer at a 5:3:2 weight ratio for 30 min at 50 rpm. A fraction

of the obtained bulk was dehydrated at 60 °C for 36 h for characterization, and the rest

was reserved for blending preparation (Figure B2).

19

Figure B2. Preparation of the thermoplastic unripe banana flour.

3.1.2 Functionalization of the metallocene-catalyzed polyethylene

Metallocene-catalyzed polyethylene Engage 8402 (Dow Chemical, Midland Michigan

USA), density 0.902 g/cm3 (ASTM D 792) and melt flow index of 30 dg/min (190 °C/2.16

Kg, ASTM D 1238) was mixed with 1% of maleic anhydride (99% purity from Sigma-

Aldrich, St Louis MO, USA) and 1% of benzoyl peroxide (Luperox A75, 75% purity,

Sigma-Aldrich) for 40 min. Maleic anhydride grafted into mPE (mPE-g-MA) was

20

extruded (60, 80,120,130 °C in the heating zones and 45 rpm for the screw) and then it

was milled.

3.1.3 Preparation of TPF and mPE blends

Thermoplastic unripe banana flour (TPF), mPE and mPE-g-MA were blended, extruded

and injected in the following proportions: 50% TPF and 45% mPE (herein termed 50

TPF), 60% TPF and 35% mPE (herein termed 60 TPF), 70% TPF and 25% mPE

(herein termed 70 TPF), 80% TPF and 15% mPE (herein termed 80 TPF), 0% TPF and

100% mPE (herein termed neat mPE). All blends contained 5% mPE-g-MA.

3.1.4 Extrusion

Thermoplastic unripe banana flour and the blends described above were extruded in a

single-screw extrusor (CICATA-IPN, Mexico City, Mexico). Extrusion was performed at

80,120,180 °C in the three heating zones and 200 °C in the exit die. Mass flow

110g/min and screw speed 30 rpm were set. A feeder (Baldor Electric, Arkansas, USA)

controlled the mass flow (Figure B3).

21

Figure B3. Preparation of the biodegradable plastic.

22

3.1.5 Flour colorimetric analysis

Five samples of the regular unripe banana flour and the whitened flour were randomly

selected for the color index analysis. A Chroma meter CR-400 (Minolta, Osaka, Japan)

was employed. The color, represented as color difference ∆E* was calculated as:

(1) ∆E*= [(∆L*) 2 + (∆a*) 2 + (∆b*) 2] 1/2

Where ∆L*, ∆a*, and ∆b* are the differentials between the color parameters of the

samples and the white standard (L* = 93.49, a* =−0.77, b* = 1.40) [58]. L*, a* and b*

are parameters of the CIELAB system [59]. Where L* represents lightness, and its value

extends from 0 (black) to 100 (white); a* Indicates the color ranging from red to green;

and b* indicates the color ranging from yellow to blue. [60].

3.1.6 Viscosity analysis

Samples of 3.37 g of regular green banana flour and TPF (14% humidity) were

supplemented with distilled water to reach a weight of 28.5 g before analyzing them in a

Rapid Visco Analyzer (RVA), (Newport Scientific, Australia). The established settings

were: 10 s for homogenization and 5 min of heating from 50 °C up to 90 °C followed by

5 min constant temperature at 90 °C. The samples were then cooled at 50 °C for 6 min

before analyzing viscosity. Total elapsed time was 16 min at 160 rpm constant speed.

3.1.7 Thermal properties measurement

Flour and injected samples were weighted and placed in hermetically sealed aluminum

capsules. Samples were subjected to increasing heating from 30 °C up to 400 °C at a

rate of 10 °C/min. Melting temperature, gelatinization temperature, and enthalpy were

determined in a Pyris 1, Perkin Elmer calorimeter (Norwalk, CT, USA). Calibration was

performed with indium and an empty pan was used as the reference.

23

3.1.8 Mold Injection

Injection of five samples of each blend and neat mPE were performed into a Demag

Ergotech 50-200 system (Düsseldorf, Germany) of 50 tons of closing force. The

samples were injected using an eight cavities mold for mechanical properties test

specimens. The injection temperatures were 140,150,160,160 °C for the heating zones

and the speed of the screw was 100 rpm. The injection mass flow was 30 cm3/s, and

the total volume for each injection was 75 cm3. Cooling time between cycles was 20 s.

3.1.9 Mechanical properties

Standard test method ASTM D638-10 for tensile properties of plastics was performed

on the injected samples. All samples were conditioned during 96 h at 23 °C and 50%

relative humidity according with ASTM D618-08 prior testing. Five specimens for each

sample (in the form of standard dumbbell-shaped test specimens) were submitted to

tensile resistance and elongation test. Testing was performed in an Instron equipment

(model 5583, Massachusetts, USA), load capacity of 150 kN, and weight of 500 kg. A

speed of testing of 50mm/min was established. Mechanical testing software Bluehill

3.11 (Illinois, USA) was used to analyze the results.

3.1.10 Microstructural analysis

Samples of unripe banana flour, TPF and samples of injected specimens were mounted

on brass stubs with double-sided graphite-filled tape and were vacuum coated with

silver by sputtering (Desk IV, Denton Vacuum, USA). SEM micrographs were obtained

at magnifications of 100 x, 250 x, 500 x, 1000 x and 1500 x (JSM-6390LV, JEOL,

Japan).

24

3.1.11 Fourier transform infrared spectroscopy (FT-IR) analysis

FT-IR spectra of banana flour, TPF, neat mPE and injected samples of each blend were

obtained using an Attenuated Total Reflectance FT-IR spectrophotometer (Spectrum

Two, Perkin Elmer, Massachusetts USA). The tested wavelength range was from 500 to

4000 cm-1. The samples were pressed against the objective lens and analyzed directly.

3.2 Biodegradation assessment

3.2.1 Composting

Thermoplastic flour and metallocene catalyzed polyethylene injected blends samples

(80/20, 70/30, 60/40, 50/50 and 0/100, TPF/mPE respectively) were fixed in a 15cm x

20cm plastic grid. Five plastic grids were prepared (one for each period). The grids were

buried between two layers of Organodel compost (Agroformuladora Delta, Mexico), a

lower layer of 60 cm and an upper layer of 30 cm. To facilitate further localization every

plastic grid was settled according to the period to be withdrawn and a labeled flag

indicated their location. The compost contained organic matter, humus, and humic

acids. According to the producer specifications it was supplemented with N, P, K, Ca.

Compost was conditioned to 50% humidity. After 25, 50, 75, 100, and 125 days beneath

the compost, grids were removed (Figure B4).

25

Figure B4. Biodegradability assay

3.2.2 Weight loss

Samples were retrieved from the grids and washed with deionized water. Retrieved

specimens were dried for 24 h at 50ºC and rested for 12 h at room temperature. All

26

samples were weighed in an analytical balance and the percentage of weight loss was

calculated as follows:

Weight loss (%)= Initial weight-Final weight x 100

Initial weight

3.2.3 Mechanical properties

ASTM D638-10 Standard test method for tensile properties of plastics was performed

on the samples after composting as described in 2.1.9.

3.2.4 Thermal properties measurement (DSC)

Samples from every composting period (~8mg) were placed in hermetically sealed

aluminum capsules. Melting temperature, gelatinization temperature, and enthalpy were

determined as described in 2.1.7.

3.2.5 SEM Analysis

All the samples were chronologically observed at 0 days (as a control) and at 25, 50,

75, 100, and 125 days after finished their period under compost. SEM (5 mm x 5 mm)

coupons obtained from samples were mounted on brass stubs with double-sided

graphite-filled tape and were vacuum coated with silver by sputtering (Desk IV, Denton

Vacuum). SEM micrographs were obtained at magnifications of 500X, 1000X and

1500X (JSM-6390LV, JEOL, Japan).

27

3.2.6 Fourier Transform Infrared Spectroscopy Analysis

FTIR spectrum of each sample after composting was obtained using an attenuated total

reflectance–FTIR spectrophotometer (Spectrum Two, Perkin Elmer, Waltham MA USA)

as described in 2.1.11.

3.2.7 Fungi morphology characterization

Visible mycelia from the surface of the composting samples were recovered in a 0.09

NaCl solution. The suspension was stirred vigorously. An aliquot of 900 µl of the

suspension was placed in a test tube and then centrifuged to 800 rpm. The dirty-free

supernatant was transferred to another tube. This suspension contained fungi without

compost remains. A 200 µl portion of the clean suspension was placed in a slide and it

was air-dried. The slide preparation was stained with methylene blue and then

examined under an optical microscope (Binocular Sehkraft M08 series, Brea, CA USA).

Details of the fungal structures were observed by SEM analysis.

CHAPTER 4

4. Results and discussion

4.1 Biodegradable material: manufacture and characterization

4.1.1 Colorimetric analysis

Because the color of the final product is a critical quality parameter for consumer’s

acceptance, its measurement in the unripe banana flour would be of relevance for

industrial making of decisions [61]. Unlike starch from corn, rice, and potato, the color of

our unripe banana flour is brownish. Table 1 showed the color determinations of the

flour. Taking into account the CIELAB parameters of the white standard, the mean L* 28

value 14.65±0.28 correlated with the low lightness of the flour. The high a* 0.46±0.04

showed that the material tended to red color. The average b* value 3.15±0.2 (deeper

yellow) also correlated with the brownish color of the flour. In addition, the large ∆E

value observed (78.87±0.28) is consistent with the lack of whiteness in our flour [62]. ∆E

value indicates the numerical difference between white and the flour color [63]. Similarly

to the regular starch, it is possible to apply either a bleaching treatment or a coloring

process in a blend with a synthetic polymer. We used a whitening process that resulted

in an improvement in the color parameters L* (27.96±0.6), a*(-0.68±0.1), b* (5.45±0.12),

and in the color difference ∆E (65.65±0.6) of the flour versus the white standard. The

whitened flour acquired an acceptable yellowish color instead of the original brownish

one.

Although the raw material was sprayed with 5% acetic acid solution to prevent

oxidation, the fact that the fruit is very susceptible to enzymatic browning may explain

the color of the flour; this process occurs because cutting or peeling the fruit exposes

enzymes such as polyphenol oxidase to the presence of oxygen [64]. The brownish

color of the flour could also be attributed to the fibrous moiety of the flour, the banana

peel. In terms of color appearance, the flour may be of interest for blending with

polyolefins to create recycled and biodegradable products whose appearance is not the

most important attribute.

Regular flour Whitened flour

Sample L* a* b* ∆E* L* a* b* ∆E* 1 14.38 0.53 3.13 79.14 27.5 -0.77 5.32 66.07

2 15.11 0.44 3.17 78.41 27.9 -0.7 5.51 65.73

3 14.66 0.43 3.14 78.86 28.8 -0.73 5.57 64.81

4 14.46 0.47 3.16 79.06 28.3 -0.68 5.52 65.33

5 14.65 0.44 3.15 78.87 27.3 -0.51 5.32 66.32

29

Mean 14.65 0.46 3.15 78.87 27.96 -0.68 5.45 65.65

Standard deviation 0.28 0.04 0.02 0.28 0.60 0.10 0.12 0.60

Table 1. Colorimetric analysis of regular and whitened unripe banana flour.

4.1.2 Viscosity analysis

Figure 1. Viscosity of the regular and the thermoplastic green banana flour.

Since our flour is composed mostly of starch, we determined the flour capacity to form a

viscous paste or a gel by performing a viscosity analysis after a heating and cooling

process. We observed a difference between the maximum viscosity peaks of the regular

flour compared with the TPF (1726 and 5669 respectively, Fig.1). This difference of

30

3493 centipoises (cP) indicated that the glycerol added to the flour for

thermoplasticization could be acting as a coat that prevented water from permeating

into the starch granules and thus avoiding the maximum swelling that leads to granular

breakage, which determines the viscosity. Despite the difference, both samples had the

capability of forming gels.

Viscosity of both our flour preparations differed from the viscosity of the normal banana

starch, which has a viscosity maximum peak of 2880 cP previously reported by others

[65,66]. It is remarkable that the addition of glycerol contributed to a two fold change in

the viscosity of the TPF. This large difference in viscosity may be due to the interaction

of the amylose and amylopectin moieties of the starch with glycerol. The hydroxyl

groups of starch form hydrogen bonds with the glycerol ones reinforcing the structure of

the blend; this behavior would decrease the hydrating capability of the material by

neutralizing the hydration sites and therefore producing a viscous fluid whose viscosity

could correlate with the amount of glycerol in the preparation [67,68]. The rather small

amount of cellulose fibers from the banana peels may have little influence in viscosity.

Because temperature, humidity and velocity are involved in the analysis, this

characterization is useful for predicting the rheological behavior that TPF would have in

the extrusion process.

31

4.1.3 DSC Analysis

Figure 2. Differential scanning calorimetry for regular (a) and thermoplastic (b) unripe banana flour.

Table 2. Melting Temperatures (Tm) and enthalpy (∆H) for regular and thermoplastic unripe banana flour.

In polymers, DSC can reveal first order thermal transitions as melting temperature (Tm),

and second order thermal transitions like crystallization temperature (Tc), and glass

transition temperature (Tg) [69]. Gelatinization is a very important phase transition and

Regular flour TPF Endothermic peak Endothermic peak Endothermic peak

Sample Tm (°C) ∆Η (J/g) Tm (°C) ∆Η (J/g) Tm (°C) ∆Η (J/g)

1 168 220 130 157 299 46

2 170 172 155 127 303 62

3 165 220 161 57 300 72

4 170 221 170 71 302 23

5 161 185 115 199 296 38 Mean 166.8 203.6 146.2 122.2 300 48.2

Standard deviation 3.8 23.4 22.9 59.2 2.7 19.4

32

is used to describe the molecular behavior of starch related with heat and moisture

content [70].

When analyzing the wholemeal banana flour, we observed well-defined peaks

(endothermic events in the DSC curves) occurring between 165 and 170 °C (Fig. 2a).

These peaks are attributable to the melting temperature of the flour. Banana starch

gelatinization has been reported to yield a peak between 70–74.5 °C and an enthalpy

value of 17.5 J/g [71]. However, we did not observe events at that temperature range.

The DSC analysis also indicates the amount of energy required for a state transition,

namely enthalpy. We observed that the enthalpy of the regular flour (Table 2) was

higher than that reported for common starch [72] (203.6 and 17.5 J/g respectively).

Considering that we are analyzing banana flour, whose starch content can reach up to

80%, the inability of the flour to reach gelatinization and enthalpy at the reported

temperature for starch, may be the result of the constituents of the banana peel as

cellulose, hemicelluloses, lignin and others that modify the temperature and the energy

required for fusion [73].

Thermal transitions of the thermoplastic flour were very different; thermograms

appeared affected by the glycerol and endothermic transformations. Fig. 2b showed two

peaks, the first peak occurred between 115 and 170 °C, which was slightly lower than

that of regular flour but higher than that reported for common starch, and the second

peak occurred at 300 °C. Enthalpy was also lower than that of regular flour but higher

than that expected for common starch (Table 2). The thermoplasticization may have

influenced the thermal stability of the flour, and the exothermic peaks that appeared at

300 °C, barely detectable in the regular flour, can be attributed to thermic degradation of

the cellulose [74,75]. These results may have been advantageous because we intended

33

to use the flour as the base for a biodegradable material manufactured by extrusion and

molded by injection, and in such cases an excess of humidity would make the extrusion

process difficult. In addition, the analysis of the melting temperatures aided to set the

extrusion differential heating program.

Figure 3. Differential scanning calorimetry. Depicted are the thermograms of the neat components and the four injected blends. Further, we analyzed the blends of TPF and mPE. Figure 3 showed a comparative

analysis of the two main components, the grafting reactive, and the blends. Neat mPE

has been reported to have a melting peak at 65 °C [76], our result from neat mPE

thermo scan was 67 °C. The blend scans slightly shifted to the right, and the higher the

flour concentration the lower the intensity of mPE melting peak. The TPF yielded two

peaks at about 150 and 300 °C as previously observed. Though barely perceptible, the

34

150 °C peak remained in the blends, except for 50 TPF that had the highest mPE

content. Maleic anhydride showed three very well defined peaks. First MA peak varied

about 10 °C to the left in the blend material. In the range of 250 and 300 °C a very

interesting phenomenon appeared for each blend. The two MA melting peaks (aprox.

230-260 °C), the second flour peak and the neat mPE seemed to interact and yield a

singular thermal behavior. This behavior could be generated for the grafting process.

Maleic anhydride grafted into mPE (mPE-g-MA) had the purpose to induce polarity and

increase compatibility with the flour, especially with banana biopolymers. For that

reason the peculiar blends thermograms were assumed to be the result of the grafting

process but also for the cellulose and the extra constituents of the flour (pectins, gums,

lignin and the glycerol added in the process of thermoplasticization), which would

demand extra energy for their melting. The thermal stability analysis of a previous study

reported that degradation of mPE and corn starch blends initiates at 320 °C [1]. This

information is important to discard a thermal degradation of our material in the

phenomenon observed in the 250–320 °C interval of the calorimetric study.

4.1.4 Mechanical properties

The mechanical properties of biodegradable materials define their primary applications.

The biodegradable materials prepared with synthetic polymer-starch blends and those

originated from renewable organic sources commonly possess diminished mechanical

properties such as tensile resistance, compression resistance and elongation compared

to regular plastics [77]. Improvement in mechanical properties is necessary for materials

that are designed to be biodegradable plastics.

35

Figure 4. Mechanical properties of the injected blends and injected neat mPE. a) Tensile strength at break. b) Tensile strength at yield, and c) Elongation at break.

To assess the effectiveness of our polymer blends, we evaluated the mechanical

properties of the injected neat mPE as well as those of the blends. Figure 4a showed a

difference of 0.153 MPa (11.71%) in the tensile strength at break between neat mPE 36

mean and 50 TPF. For biodegradable materials design, the difference in tensile strength

between neat mPE and 50 TPF is promising taken into account the amount of flour in its

preparation. Unexpectedly, the tensile strength of 60 TPF is greater than that of neat

mPE, but it is a positive result considering its content of TPF. The tensile resistance at

break of 80 and 70 TPF blends was slightly lower; the difference only reached 0.377

MPa (28.86%) versus neat mPE. Lower values of tensile resistance were expected for

these two last concentrations due to the larger content of flour.

We also evaluated the tensile resistance at yield, which is the maximum load that a

given material resists. As expected, neat mPE had the best tensile resistance, 1.792

MPa. However, a mean of 2.296 MPa for 50 TPF is a good result for a natural-synthetic

polymer blend considering that this tensile resistance value indicates an average load

resistance of 95.53 N. The tensile resistance at yield decreases when the percentage of

TPF increases in the blend.

It has been reported that the addition of a natural polymer such as starch or flour to a

synthetic polymer generally results in a decrement of elongation at break [78]. This is

due to the immiscibility between them. The addition of maleic anhydride, the

thermoplasticization of the unripe banana flour, and the homogenization process have

minimized the immiscibility of our blends. We observed practically the same elongation

for neat mPE and 50 TPF (figure 4c). Chemical interaction among biopolymers from

flour, glycerol, and mPE-g-MA generated a compact polymeric matrix that could have

helped to improve the elongation at break. When the flour content increases the

elongation capacity of the material lessens, but remains a good result for 60 and 70

TPF (794.69 and 570.79% respectively). The resulted elongation for 80 TPF is not

enough to consider that blend adequate for any future utilization.

37

4.1.5 Microstructure characteristics

Figure 5. Scanning electron microscopy of regular and thermoplastic unripe banana flour: a,b) granule size and shape, c,d) distribution of flour particles, e,f) microstructure of extruded flour.

Morphology and size of starch granules depend on the source plant [79]. Starch

granules range between 1 and 110 µ. The granules of our unripe banana flour (Fig.

5a,b) averaged 30 µ, similar to the potato starch granule [80] and larger than the

granules of rice and corn starch. The flour SEM micrographs showed oval and irregular 38

granules as well as amorphous structures consistent with insoluble fiber. Although the

proportion of fiber observed is low, DSC measurements revealed that fiber exerts

influence in the thermal properties of the flour raising the fusion temperature of the

banana starch [81,82]. In addition, we observed that the thermoplasticization process

had no detrimental effect on morphology and microstructure of the unripe banana flour

(Fig. 5c,d).

The behavior of the starch and the flour when subjected to temperature depends on the

moisture level [2,83]. Extrusion of the regular banana flour, which is performed in

moisturizing conditions, produced a homogeneous material (Fig. 5e); thermoplastic

flour, which is performed in drying conditions, produced a porous material (Fig. 5f).

Porous were probably generated because in absence of humidity the glycerol prevents

the gelatinization of the starch granules generating a non-compact structure. Moreover,

the resultant extruded material was rigid, hard and frail. In contrast, when the banana

flour was humidified before extrusion (Fig. 5e), the heat made the water to flow through

starch granules improving gelatinization and further plasticization. Thus, the extruded

flour was compact and fibers appeared to be integrated.

39

Figure 6. Scanning electron microscopy of the mold injected material. Surface and transversal view of the different TPF preparations.

40

Because microstructure analysis is important for relating and explaining the mechanical

properties, we performed SEM analysis of the injected blends, both at the surface and

transversal level. For neat mPE, the direction of the flux of injected material was

evident; it was practically a smooth surface with no discontinuities or cavities that could

affect the mechanical properties, and the transversal view revealed a uniform texture

(Fig. 6). The 50 TPF blend exhibited a slightly rough surface, the injection flux appeared

scarcely discontinuous, though the direction was well defined; the transversal view

revealed a porous structure that could be explained by the glycerol content and

insufficient humidity level that prevented starch granules to complete gelatinization. This

porosity at this flour concentration probably contributed to the characteristics of the final

material, which was robust, elastic and non-brittle.

More irregularities were visible in 60 TPF surface. Although no major visual differences

existed between 50 and 60 TPF (transversal view), their mechanical properties differed

in a TPF-mPE ratio-dependent manner. The surface of 70 TPF was a uniformly oriented

fibrous structure, and the transversal view showed a honeycomb pattern probably

originated by the greater proportion of starch and cellulose fibers of the unripe banana

flour and its respective humidity level. The 80 TPF was disorganized and had cavities

and discontinuities and no layer formation was seen; this may be the reason for its poor

mechanical properties.

It is worth mentioning that even at the greater flour proportion starch cumulus never

appeared. This suggests not only a physical or heterogeneous blending but also an

interaction among the starch, cellulose and mPE induced by the maleic anhydride. Both

superficial and transverse micrographs elucidate the mechanical properties observed in

our blends because the level of porosity, the size of the cavities, the absence of a

41

pattern and the disoriented microstructure correlated with the reduction of the tensile

resistance and the elongation.

4.1.6 FT-IR Analysis

Figure 7. Fourier-transformed infrared spectroscopy (FT-IR) analysis for regular green banana flour and TPF.

FT-IR spectra of the regular and thermoplastic unripe banana flour were obtained

(Fig.7). The spectra of both materials were similar. We observed a peak between 3275

and 3300 cm-1 that is characteristic of the stretching vibration of the hydroxyl group (–

OH) in cellulose and starch [84]. Spectrum of the TPF showed an increase in the width

of this peak because the three –OH groups of the glycerol (C3H5 (OH) 3) contributed to

the peak intensity. The peak at 2925–2932cm-1 is attributable to the vibration of the 42

asymmetric tension of the –CH2 group [85]. The peak between 1622 and 1630 cm-1

corresponds to linked water absorption. The crest at 1330–1430 cm-1 is associated to

the bending vibration for the –OH group. Three peaks appeared in the fingerprint region

from 400 to 1250 cm-1 which are common in the banana starch: the first one (923 y

1150 cm-1) and the second one (1148–1150 cm-1) are attributed to C-O stretching

vibration mode [86], and the third peak is characteristic of the C-O-C groups in the

glycosidic units or the β-(1-4) glycosidic linkages between glucoses in the cellulose

(1160 cm-1). The band observed in 860 cm-1 corresponds to the harmonic vibration of C-

H in starch [87] and is larger in TPF.

Figure 8. FT-IR spectra of the blends from 50 to 80 TPF and mPE-g-MA. Circle encloses the three peaks from MA.

43

When analyzing the blends of TPF and mPE we observed a similar pattern for all blends

that resemble the TPF spectrum with the incorporation of PE characteristics to the

spectra (Fig. 8). The peaks characteristic of the TPF were the broad absorption band of

the stretching vibration of hydroxyl group (–OH) in cellulose, starch, and glycerol (3307–

3292 cm-1); the hemiacetal bonds from the starch flour (1638–1650 cm-1); the vibration

peak corresponding to the cyclic -C-C- of the glucose (1030–1018 cm-1) in the starch;

and the 1151, 924 and 860 cm-1 peaks corresponding to the fingerprint region of the

banana starch. The mPE in the blends contributed additional peaks to the spectra: the

symmetric and anti-symmetric (2918 and 2850 cm-1) stretching bond vibrations of the

groups -CH2 and CH3 in mPE [25]; the characteristic -CH2 stretching for oxidation of

synthetic polymers (1460 cm-1); the angular deformation absorption group CH3 (1375

cm-1) and the 719 cm-1 band, typical of the polyethylene, also appeared.

We included the mPE-g-MA spectrum and circled the absorption peaks of anhydride

group (1791 and 1714 cm-1) to highlight them. The MA carbonyl stretching absorption

band at 1791 cm-1 indicated that a specific quantity of MA group has been grafted on

the molecular chains of mPE. Another carbonyl symmetric stretching absorption band at

1714 cm-1 was expressed and confirmed the MA presence. Although we did not

observe these MA peaks in the blends (mPE, banana starch and mPE-g-MA) spectra,

the homogeneity of the blends can be attributed to the polymers inter-phase adhesion

promoted by MA grafting that influenced the mechanical, thermal, and microstructural

properties of the blends.

44

4.2 Biodegradation assay

4.2.1 Weight loss

Figure 9. Weight loss percentage after 125 days under compost.

Weight loss is one of the indicators that denote biodegradation [88]. Figure 9 showed

the weight loss monitoring of the samples under compost. The control sample (neat

mPE, 0 TPF) had the smallest weight loss after 125 days. This minimum effect of the

composting over the mPE is probably due to its natural resistance to microbial attack

[89]. The lower weight loss of all blends was observed for 50 TPF; perhaps the

homogeneity of the material due to the functionalization of the mPE avoided the

enzymatic action of the microorganisms for this blend. The blends 60, 70 and 80 TPF

lost a considerable amount of weight. The 70 TPF lost 40% of its weight in a short time,

but the 80 TPF lost half of its weight as early as 50 days under compost. The thickness

of the samples was 4 mm thereby the observed weight loss rate revealed a good

45

tendency of deterioration. It is interesting to note that all samples, including the neat

mPE, lost more than half the weight they would eventually lose in the first 50 days

(Table 3). The results implicated that the higher flour content increased the microbial

enzymatic attack therefore granting biodegradation [90].

Relative weight loss (%) SAMPLE (TPF) Day 25 Day 50 Day 75 Day 100 Day 125

0 0.11 0.63(52.5) 1.04 0.04 1.20(100) 50 0.10 3.71(74.9) 1.21 4.12 4.95(100) 60 6.01 10.60(52.29) 16.65 23.94 20.27(100) 70 20.86 24.61(54) 41.39 41.75 45.23(100) 80 34.48 49.29(83.4) 48.78 54.76 59.09(100)

Table 3. Relative weight loss percentage data at 25 days intervals. The total weight lost at day 125 was set to 100%.

4.2.2 SEM Microstructural analysis

Day 0 Day 25 Day 50 Day 75 Day 100 Day 125

0

50

60

46

70

80

Figure 10. Chronological Microstructural analysis by SEM (500x) of the samples under compost during 125 days.

Biodegradation under compost begins with an erosion process [91] caused by

microorganisms and the environment. There are erosion indicators such as fractures,

breaches, cavities and holes. All these indicators can be monitoring by the scanning

electron microscopy. SEM analysis allows a visual tracing of biodegradation when

material has been submitted to compost process [92].

We monitored the changes in the microstructure of all samples subjected to composting

(Figure 10). The neat mPE sample showed a smooth surface practically intact in the

micrographs from 0 to 100 days, only the last observation at 125 days exhibited a little

deterioration marked by very small pores. This observation correlates with the weight

loss for this sample. All TPF/mPE blends underwent intense changes along the

composting time. The 50 TPF sample showed a semi-flat surface and only the flux of

the injection could be perceived at day 0. However, deterioration within 100 days

caused some breaches and holes, and deterioration was even clearer at day 125. The

60 TPF did not show a smooth surface at day 0; the flux of the injection was very

marked, though a compact material was perceived; it was notorious that it was not a 47

neat material but an injected blend. Micrographs from 25 to 125 days showed the

erosion caused by the microorganisms present in the compost. At day 75, we observed

a large hole in the surface, this kind of spoilage impacts on the weight loss. The 70 TPF

also presented a compact structure at the beginning but in the compost period SEM

manifested deterioration since the first 25 days towards the 100 days observation. At

day 125, the sample lost its structure. Finally, the 80 TPF did not have a homogeneous

surface since the beginning; the great weight loss could be corroborated with the 125-

day micrograph, where the loss of structure and lack of solidity were evident. Also in this

micrograph we could observe fungal conidia, although all the samples were washed

with deionized water. Fungi are present in the compost, and the spread of the conidia

over the surface of our samples can be explained by their high content of TPF.

4.2.3 Fungi microscopic analysis

The active component involved in the biodegradation and conversion processes during

composting is the resident microbial community, among which fungi play a very

important role. In addition, fungi can use many carbon sources, mainly lignocellulosic

polymers, and can survive in extreme conditions. Fungi are responsible for compost

maturation. The genera with the highest load and number of species in composts are

Penicillium, Aspergillus, Cladosporium, Acremonium, and Mortierella [93].

We observed mycelia spread over the mPE-banana flour blends samples buried in

compost since the day 50. Mycelia were present in all blends but not over the neat

mPE. We analyzed the mycelia to identify the fungus by morphology, and because the

mycelia were visible over the plastic samples, we also investigated whether the fungi

formed a biofilm directly on the surface of the material by SEM analysis of samples that

were not washed. We found a single fungus growing over the plastic material 48

throughout the composting time (Fig. 11). Under light microscopy (Fig. 11A-B) conidia

exhibited irregular morphology probably caused by diminishing turgidity and

dehydration, cell structure unusual, with a small sort of cavity in the cells, somewhat

thickened and darkened-refractive. SEM higher magnifications (not shown) further

defined the morphologic characteristics as follows:

Hyphae: thin, unbranched or sparingly branched, approximately 1−5 μm wide, septate,

without any swellings and compressions, smooth, with thin walls.

Conidiophores: solitary, macronematous or semimacronematous, arising terminally from

ascending hyphae or laterally from plagiotropous hyphae, straight to barely flexuous,

narrowly cylindrical to cylindrical-oblong, sometimes filiform, non-nodulose, unbranched,

smooth to minutely verruculose towards the base.

Conidiogenous cells: integrated, terminal, cylindrical-oblong, not geniculate, non-

nodulose, crowned at the apex.

Conidia: numerous, catenate, in long branched chains, up to 10 conidia in the upper

unbranched part, branching in all directions, small terminal conidia subglobose,

obovoid, ovoid to limoniform, approximately between 3−7 μm, aseptate, with a small

cavity or a scar in the cells. Intercalary conidia were limoniform, ellipsoid-ovoid,

aseptate, smooth under SEM.

The microscopic morphology was compatible with Cladosporium species; species

belonging to Cladosporium are characterized by having a unique coronate scar

structure. Cladosporium cladosporioides is a very common, cosmopolitan, saprobic

species. It often occurs as a secondary invader on necrotic parts of many different host

plants, has been isolated from air, soil, textiles and several other substrates [94,95,96].

49

Figure 11. Analysis of fungi found on the surface of the TPF samples.

50

Figure. 11C-F showed the fungus growing over the sample buried 75 days in compost

indicating the presence of a biofilm widely distributed in the blends. In Fig. 11E the

conidia were evident inside cavities of the partially degraded polymer. By day 125 there

were no visible hyphae, only spores, probably due to the loss of humidity in the compost

(Fig. 11F).

Although being acknowledged as non-biodegradable, soil fungi have been reported to

degrade synthetic polymers, especially by stimulation of microbial communities with

addition of exogenous nutrients [97,98,99]. Commercially available LDPE

biodegradation has also been demonstrated in different experimental settings:

1) Carrier bags films were buried in natural soil for up to 2 years. After 7-9 months

of soil exposure, microbial colonization was evident on the film surface and

progressive changes were observed towards degradation after 17-22 months

[100].

2) LDPE films were buried for 100 days in a controlled solid waste medium at 28º C

and were separately inoculated with landfill-source isolated Aspergillus

fumigatus, Aspergillus terreus and Fusarium solani. These fungi could degrade

LDPE in the sterilized solid waste medium by a mechanism dependent on

formation of a biofilm. Photo-oxidation (25 days under UV-irradiation) was used

as a pretreatment of the LDPE samples. A. terreus and A. fumigatus, despite the

availability of other organic carbon of materials, could utilize LDPE as carbon

source [101].

3) LDPE films buried in sterile soil or LPDE powder cultured in liquid medium both

inoculated with bacterial consortia, whose synthetic polymers degradability had 51

been demonstrated, were degraded starting at 1 month of incubation. SEM

analysis revealed cracks and fissures appearing after 2 months that further

increased in the following month. The addition of bacterial growth enhancing

nanoparticles accelerated the process [102,103].

Thus, microorganisms can degrade LDPE but it needs additional factors to stimulate

microbial communities or a limited carbon source pre-requisite. We did not observe

mycelia growing over neat mPE, indicating that the fungus responsible for the

degradation of our plastic samples was not degrading PE itself, but the organic

component of the blends. So the candidate fungal species (because we observed a

single fungus throughout the experimental composting time) degrading our blends was

likely to be a saprobe, probably Cladosporium cladosporioides. Cladosporium sp. can

colonize several substrates such as paper, fabric, wood, and plastics [104]. Although

microscopic morphology suggested Cladosporium as the fungus forming a biofilm over

our samples, molecular analyses are needed to confirm its identity.

Saprobe fungi are relevant for degradation of plant debris. Plant biomass, and the

product of its decomposition in soil, is mainly composed of three types of polymers:

cellulose, hemicellulose, and lignin that are strongly intermeshed and chemically

bonded by non-covalent forces and by covalent cross-linkages. Species of saprobe

anamorphic fungi are important in decomposing plat debris; its degradation is important

for carbon recycling of the biosphere, bioremediation of soils and water, and pollutant

treatments [105,106,107]. Soil fungi possess the ability to produce alkaline cellulases,

ligninolytic enzymes, cutinase. Microorganisms that can degrade plant cell wall produce

a variety of plant cell wall-degrading enzymes, which include not only carbohydrate-

degrading enzymes but also lipolytic/esterolytic enzymes (i.e. esterases from

52

Xanthomonas oryzae and Thermobifida fusca or xylanases from Cellulomonas fimi,

Micromonospora aurantiaca, and Aspergillus fumigatus [(Sulaiman, 2012), 108].

Moreover, biodegradation of films of commercial biodegradable plastics by fungal

strains isolated from plant surface (phylloplane) and from farm soils has been reported

to occur within 6 days. With fungi growing on the surface of films, producing breaks

along the direction of hyphal growth indicating that fungi secreted a degrading enzyme

that directly contribute to accelerating the degradation of film. Among the fungi

responsible of the degradation were Paraphoma chrysanthemicola, Asperigillus sp.,

Penicillium sp., and Acanthamoeba sp., Fusarium solani [109,110].

Taking into account that 1) the activity of fungal species improves with humidity, gas

concentrations and penetration degree on the substrate [(Borrego, 2012)], and 2)

amylases produced by soil microorganisms are enzymes that hydrolyze starch and

some of them perform transglycosylation or condensation as well as hydrolysis [111],

we propose that the deep penetration of the fungus into our blends greatly contributed

to its degradation by digesting their plant-derived moiety. Although the diversity of

natural microbial populations poses that the potential for biodegradation of synthetic

polymers exists, factors such as absence of electron acceptors or donors, low nitrogen

or phosphorus availability, or a lack of induction of the metabolic pathways responsible

for degradation can inhibit waste remediation [(Cosgrove, 2010)]. Therefore, the

addition of plant-derived compounds to biodegradable plastic designing would

accelerate their degradation, even in thick materials and not only in films.

53

4.2.4 Thermal properties

54

Figure 12. DSC Thermograms of the samples at a) Day 0, b) Day 75 and c) Day 125.

The thermal behavior of the neat mPE, TPF and the maleic anhydride (the

functionalizing agent), and the blends are depicted (Figure 12). For comparison,

thermograms corresponding to the single components and the blends are depicted

separately, the single components appear at the top of all panels and the different TPF

blends are depicted at the bottom. Peak melting temperatures (Tm) of the single

components were identified and extended to the temperature axis with a vertical line. At

day 0, we observed the peak melt temperatures of the single components appearing in

some TPF blends meaning the neat components were not chemically affected when

they were mixed and functionalized (Figure 12a). Some samples did not show these

melting peaks probably because of the reduced amount of these components in the

sample. Some new peaks at different temperatures appeared in the TPF blends

probably because they were chemically affected by the functionalization of the mPE

creating new bonds and influencing their melting points. Beyond 250ºC all blends

thermograms displayed a peculiar seismogram behavior produced by the thermal

degradation at this temperature. This degradation was confirmed when the aluminum

55

pans that contained the samples were opened. Only TPF blends exhibited this behavior.

Polyethylene has a wide temperature window that allows temperatures to get beyond

300ºC. When the polyethylene-containing pans were opened after DSC assay, we

observed only an amber coloration without visual sample deterioration. The TPF

sample, however, did show a visual deterioration near to calcination.

After 75 days of composting, the first prominent thermal change was the diminution of

the thermal inconsistency after 250ºC (Figure 12b). This is an expected result if we

consider that the weight loss may be basically owed to the losing of the thermoplastic

flour in the blends. Some other melting peaks appeared in the TPF blends associated to

the thermal behavior of the basic components. The thermogram of the 80 TPF was

similar to that of the neat mPE and it can be explained by the loss of mass observed at

this time.

At day 125 (Figure 12C), the thermal behavior of TPF samples beyond 250ºC practically

disappeared. The DSC thermograms indicate that the weight loss (Figure 9, Table 3)

was associated to the loss of the TPF moiety of the blend. The 80 TPF lost more than

half its weight and its thermogram is more like the neat mPE. Although mPE fraction

prevailed, in this sample the mPE was the smallest portion. In the 50, 60, and 70 TPF

the melting peaks associated to the new bonds created by the functionalization of the

mPE remained over the single components melting peaks.

56

4.2.5 Mechanical properties

a) Tensile resistance

b) Elongation at break

Figure 13. Mechanical properties of the blends and neat mPE a) Tensile resistance and b) Elongation at break.

Loss in mechanical properties has been considered a factor in biodegradation [112].

The tensile resistance at maximum load was in accordance to the TPF content

(Figure 13a). As expected, neat mPE had the best tensile resistance. At the end of

the 125 days under compost, all the blends showed little changes with respect to the

original tensile resistance and the reduction in tensile resistance inversely correlated

with the TPF content.

We expected the tensile resistance of all the samples to decrease by the end of the

125 days, but it was maintained at the maximum possible load even for the blends

containing a high amount of TPF. It is possible that functionalization allowed the

57

blends to be a homogeneous material. Despite weight loss and visual deterioration

of the samples, these were capable to maintain the strength to resist the load

applied.

When polysaccharides like starch are blended with non-functionalized synthetic

polymers, clusters of the material are formed producing fragile blends; if they are

subjected to compost, the diminution of the tensile resistance is notable after a few

weeks [112]. Whenever a grafting process was performed, a greater tensile strength

was observed [113]. Thus, modification of the main components prevents the

formation of heterogeneous blends and enhances the plasticization effect [114],

which may explain our results.

As for elongation, no major changes were observed in the neat mPE during the 125

days under compost (Figure 13b), which was also congruent with the absence of

weight loss and the undamaged sample after composting. In the 50 and 60 TPF

cases, the elongation was variable from the beginning to the end of the 125 days.

However, at the end, the diminution of the elongation was of 20%. For the 70 and 80

TPF samples there was not lessening of elongation. The former result is similar to

the tensile resistance evaluation and could have the same explanation.

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE Tensile resistance (MPa) 2.32 1.85 1.52 1.36 4.14 Elongation (mm) 523.16 519.17 237.34 43.59 472.09 Elongation (%) 554 549 197 54 490

58

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE Tensile resistance (MPa) 2.59 1.89 1.66 1.59 3.67 Elongation (mm) 362.67 323.62 255.58 50.14 431.67

Elongation (%) 453 405 319 63 540

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE

Tensile resistance (MPa) 2.68 2.25 1.92 1.51 3.52

Elongation (mm) 415.67 470.5 208.42 41.25 439.42

Elongation (%) 520 588 261 52 549

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE Tensile resistance (MPa) 3.01 2.45 2.12 1.63 3.9 Elongation (mm) 497.5 421.53 176.75 70.59 438.74

Elongation (%) 622 527 221 88 548

59

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE

Tensile resistance (MPa) 2.64 2.11 1.75 1.32 3.8

Elongation (mm) 187.02 339.42 149.27 83.69 397.52

Elongation (%) 234 424 187 105 497

Sample 50 TPF 60 TPF 70 TPF 80 TPF neat mPE Tensile resistance (MPa) 3.35 3.56 2.98 2.5 4.95

Elongation (mm) 397.68 401.09 190.75 114.32 527.92

Elongation (%) 497 501 238 143 660

Figure 14. Instron Output. Tensile resistance, Total Elongation and Percentage of Elongation of the neat mPE and TPF blends before and after composting.

The Instron software used to analyze mechanical properties generated curves like

those depicted in Figure 14. Samples and blends were identified by capital letters.

The maximum load is indicated with a little filled black triangle located at the highest

point of every curve. The elongation value was the point when the curve fell abruptly.

When there was not an abrupt fall, it meant that the sample reached the maximum

60

elongation of the Instron equipment (500 mm). The table at the bottom of each

graphic summarized the main mechanical properties. An 80 mm gage length of the

sample, indicated that the specimens neat mPE, 50 and 60 TPF were elastic. The

lower elongation of 70 and 80 TPF indicate they were not elastic material.

4.2.6 FT-IR Analysis)

a) Infrared analysis for 50% of thermoplastic flour content.

b) Infrared analysis for 60% of thermoplastic flour content.

61

c) Infrared analysis for 70% of thermoplastic content.

d) Infrared analysis for 80% of thermoplastic content.

Figure15. Infrared analysis before and after compost for the 50, 60, 70 and 80 TPF samples.

Infrared spectroscopy is considered an important technique for polymers biodegradation

assessment [115]. Figure 15 showed the FTIR spectra in the range of 4000–400 cm-1

for the 50-80 TPF samples. The day 0 (beginning) of all the blends was depicted in

black and day 125 (ending of the composting) in red in each panel.

All TPF blends spectra showed no attenuation or weaknesses in their individual bond

vibrations before composting (Figure 15 a-d, black spectra). The vibration of the group -

62

OH (3423 cm-1), a broad peak attributable to the starch and the glycerol of the TPF was

unchanged. The symmetric and antisymmetric stretching bond vibrations of the groups

-CH2 and CH3 in mPE (2970–2860 cm-1) exhibited two peaks without any intensity

reduction through the time. Hemiacetal bonds of starch in the flour (1650 cm-1) and -

CH2 stretching (1460–1440 cm-1) also remained unaffected. Nevertheless, the intensity

of vibration peak (1010 cm-1) belonging to the cyclic -C-C- of the glucose in the starch

increased with TPF concentration.

Once the samples were buried in compost for 125 days, the infrared spectra for all the

samples changed (Figure 15a-d, red spectra). The broad peak of the group -OH (3423

cm-1) markedly softened. Flour constituents like starch and cellulose are excellent

source of carbon for microorganisms [39]. We assumed that microorganisms, basically

fungi, consumed the flour moiety of our blends as carbon source and this was

evidenced by the reduction of -OH group in the spectrum. The two peaks of the bond

vibrations of the groups -CH2 and CH3 in mPE (2970–2860 cm-1) were intensified and

sharpened. In the samples with lower concentrations of TPF these two peaks turned

more evident because of mPE biodegradation resistance. Hemiacetal bonds bands

stayed without changing. The peak (1010 cm-1) of the cyclic -C-C- of the glucose in the

starch also weakened. Thus, the changes in the infrared spectra of the samples after

125 days of composting were not different among the samples.

63

CHAPTER 5

5. Conclusions

5.1 Material development conclusions

1. The characteristics of the thermoplastic flour made it feasible to blend it with synthetic

polymers to produce biodegradable materials.

2. We successfully prepared a plastic material generated by blending whitened

thermoplastic unripe banana flour with metallocene-catalyzed polyethylene at

differential proportions.

3. Blends containing up to 80% of thermoplastic banana flour were molded without any

injection problems.

4. Grafting MA on the molecular chain of the synthetic polymer allowed the homogeneity

of the blends evaluated by SEM.

5. One of our plastic blends, the 50 TPF, exhibited mechanical properties (before

compost) similar to those of the neat mPE.

5.2 Biodegrading process assay conclusions

6. All the TPF-mPE blends lost weight proportionally to its TPF content after 125 day of

compost. 80 TPF blend lost more than its half weight in this period.

7. All the samples exposed structural deterioration after the compost period evaluated

by scanning electric microscopy.

64

8. All the TPF-mPE blends lost mechanical properties (elongation at break and tensile

resistance) according to its TPF content after the composting process.

9. The 50 TPF mechanical properties exhibited before and after compost makes this

blend suitable for industrial purposes similar to the neat mPE, with the advantage of a

shorter degradation time.

10. 60 and 70 TPF blends exhibited properties to be assigned to specific products with

medium mechanical requirements like carriage bags, small containers and general

packaging.

11. Finally for 80 TPF blend material could be used for the design of disposable plastics

(short product life, minimal mechanical properties and ready for landfill).

65

CHAPTER 6

PERSPECTIVES

Based on our results, we propose to continue this research with the following schemes:

1.- The identification of the fungi found on the surface of the material is important to

design bioremediation protocols and to implement biodegradability tests. Polymerase

Chain Reaction technique deserves further consideration.

2.- There were no difficulties with molding by injection the 80 TPF; however, an

improvement of the mechanical properties would be desirable. The search of methods

to produce a 100% natural polymer plastic based on renewable sources without

affecting these properties is a matter of future investigations.

3.- Though we have developed a biodegradable material, we have not tested whether

the addition of color would affect the biodegradability of the plastic, and most of the

plastic items in the market are pigmented according to their usage. The approach would

be to determine if biodegradation is affected in the end product by the pigmentation

process and, if so, to produce a natural pigment specific for this material.

4.- When translating this research to the manufacture of an end product of industrial

interest, to use smaller amounts of the natural polymer might be necessary to gain in

mechanical properties. In such cases, nanotechnology advantages can be used by the

incorporation of nanoparticles (like pro-oxidants) as stimulants of biodegradation. In

addition, the incorporation of silver nanoparticles can be used as a timer in the

biodegradability process.

66

5.- Homogeneity can be improved by blending nano-components. Nanopolymers can be

synthesized and mixed to obtain stronger biodegradable plastics. The incorporation of

mixing techniques like ultrasound can be useful.

67

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