Degradation study by trichoderma spp. of poly (3 ... 13_Maria Rapa...MARIA RÂPĂ, MONA ELENA POPA, PETRUŢA CĂLINA CORNEA,VLAD IOAN POPA, ELENA GROSU, MIHAELA GEICU-CRISTEA, …

Romanian Biotechnological Letters Vol.19, No3, 2014 Copyright © 2014 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

9390 Romanian Biotechnological Letters, Vol. 19, No. 3, 2014

Degradation study by trichoderma spp. of poly (3-hydroxybuthyrate) and wood fibers composites

Received for publication, April 04, 2014 Accepted, May 15, 2014

Maria RÂPĂ1*, Mona Elena POPA2, Petruţa Călina CORNEA2, Vlad Ioan

POPA2, Elena GROSU1, Mihaela GEICU-CRISTEA2, Petruţa STOICA1, Elisabeta Elena TĂNASE2

1- S.C. I.C.P.E. BISTRITA S.A., Parcului Street, no. 7, 420035, Bistrita, Romania 2-UNIVERSITY OF AGRONOMIC SCIENCES AND VETERINARY MEDICINE

BUCHAREST, Faculty of Biotechnology, Blv., Mărăşti, no. 59, Sector 1, 011464, Bucharest, Romania

*Corresponding author: Maria RÂPĂ, e-mail: [email protected],

Tel.: +40744967559 Abstract

Poly(3-hydroxybutyrate) (PHB) is a typical biodegradable thermoplastic polymer from PHA class and is recognised as a potentially useful natural plastic, which is biodegradable in the environment. For overcome the stiffness and britlless of PHB, green composites based on PHB/cellulose fiber and PHB/wood fiber were prepared by melting technique. Maleic anhydride and succinic anhydride were used as the modifying agents for fibers. The biodegradation ability of the prepared green composites was investigated by estimation of degree of colonization by exposure to Trichoderma spp. action and by Fourier transform infrared spectra scanning. Attack of Trichoderma spp. to PHB composites led to physical and chemical changes. The results of the study have proved that Trichoderma spp. is able to colonize and degrade the studied PHB composites.

Keywords: biocomposites, fungi, colonization degree, chemical changes Introduction

Polyhydroxyalkanoates (PHA) represent a family of biodegradable polymers produced from renewable resources and are ideal candidates to replace petroleum-based plastics, with multiple applications: surgical sutures, meshes, implants and tissue engineering scaffolds ([1], [2],[3],[4],[5]) controlled drug [6], packaging, fibers, waste water treatment ([7],[8],[9]).

Poly(3-hydroxybutyrate) (PHB) is a typical biodegradable thermoplastic polymer from PHA class and is recognised as a potentially useful natural plastic, which is biodegradable in the environment ([10],[11]). PHB has a high degree of crystallinity because of highly stereo regular structure which leads to a rather stiff and brittle material (Tg of approximately 1-5 °C) thus limiting its applications [11]. Several attempts have been made to improve the physical properties of PHB by blending with other biodegradable polymers. Some authors investigates effects of increasing wood fiber weight contents on mechanical, thermo-mechanical and morphological properties of PHA based biocomposites ([4],[12],[13],[14],[15]). Wood fiber offers several advantages like low density, high specific properties, non-abrasive to


Romanian Biotechnological Letters, Vol. 19, No. 3, 2014 9391

processing equipment, low cost and most importantly biodegradability ([14],[15],[16],[17],[18],[19]). However the primary drawback of using wood fibers for reinforcement PHB is the poor interfacial adhesion between polar-hydrophilic wood fibers and nonpolar-hydrophobic plastics. The interfacial adhesion can be improved by using of compatibilizers or coupling agents [12]. In this work, maleic anhydride and succinic anhydride were chosen as the modifying agents for fibers based composites.

Microbial degradation of PHB and its composites was described in a number of publications: in natural ecosystems, such as forest soil, in the sandy soil, in the activated sludge soil ([20],[21]), compost ([16],[22]), by the thermo gravimetric method under dynamic conditions ([23],[24],[25],[26],[27]). The main factors contributing to the biodegradation of polymer blends are: chemical structure, phase structure (amorphous or crystalline) of the components, molecular mass, miscibility properties between components, presence of hydrolysable and oxidation groups, hydrophilicity / hydrophobicity ratio, chain orientation, and roughness of blend surface and environmental factors (microorganisms, temperature, humidity) [28]. According to Boyandin [21], PHA degradation is influenced by weather conditions, polymer chemical composition, specimen shape, and microbial characteristics. Lovera [29] studied the biodegradation of PHB)/poly(ε-caprolactone) (PCL) by a exposure to A. flavus and showed that the increased miscibility between the components caused a reduction in the degradation rate. Reduction in crystallinity leads to increase in degradation rates of composite films [5]. Recent researches have shown that the higher hydrophilicity and concentration of oxygenated functional groups at the surface of treated PHB films possibly improved the biodegradation of films by entomopathogenic fungi [30]. Kim [20] reported the biodegradability of PHB by fungi isolated by soil and concluded that Penicillium simplicissimum LAR 13 and Paecilomyces farinosus LAR 10 degraded PHB relatively well, while the degradation rate by Aspergillus fumigatus LAR 9 was lower than expected. Boyandin [21] found that representatives of the bacterial genera Burkholderia, Bacillus, Cupriavidus, Mycobacterium, and Nocardiopsis and such micromycetes as Acremonium, Gongronella, Paecilomyces, Penicillium, and Trichoderma have been identified as major PHA degraders.

The aim of this paper is to characterize green composites based on PHB and cellulose/wood fibers by assessing their degree of biodegradability against Trichoderma spp. for 50 days and studying the chemical changes by FTIR. Materials and Methods

Poly(3-hydroxybutyrate)(PHB), type P226 was used as the polymer matrix. It was

supplied by BIOMER, Germany. The material has a density of 1.2294 g/cm3, melting point of 173 0C, MFI, 6.372 g/10 minute (1700C/2.16 kg), tensile strength, 10.6 MPa and elongation at break 7 % [31].

Cellulose fiber type EFC 1000 (Rettenmeier & Söhne AG, Germany) and wood fiber type LSL 200/150 (La.So.Le. Est SRL, Italy) both were supplied by CARTIF, Spain.

Coupling agents as maleic anhydride (MA) were supplied by FLUKA and succinic anhydride (SA) by ALDRICH, both with purity ≥ 99%.

Chemicals and reagents were purchased as follows: (NH4)2SO4 7 g L-1, K2HPO4 7 g L-

1, peptone 3 g L-1, agar 15 g L-1, distilled water. Fungus Trichoderma viride 456 was isolated from soil and maintained on PDA medium (Potato Dextrose Agar) in the collection of Faculty of Biotechnology from Bucharest.

MARIA RÂPĂ, MONA ELENA POPA, PETRUŢA CĂLINA CORNEA, VLAD IOAN POPA, ELENA GROSU, MIHAELA GEICU-CRISTEA, PETRUŢA STOICA, ELISABETA ELENA TĂNASE


Preparation of biodegradable PHB blends

PHB and wood/cellulose treated fibers were initially weighed and melted according to the various fiber contents indicated in Table 1. The composition of each formulation is also shown. The components were preparing using a BRABENDER Plastograph, under a mixing temperature of 180 0C for 10 minutes and a rotation speed of screws of 40/70 rpm. After blending, melted formulations were pressed into thin films by a laboratory press type POLYSTAT 200 at the following conditions: temperature: 1600C, pressing time: 5 minutes and pressure of 200 bars. Films with thickness max. 100 µm were obtained.

Table 1 – Composition of green composites

Formulation a Mixture Filler content,

wt.% Resin content,

wt.% B PHB 0 100 BLMA-5 PHB-WF 5% - MA 5 94 BLMA-10 PHB-WF 10% - MA 10 89 BLMA-20 PHB-WF 20% - MA 20 79 BLSA-5 PHB-WF 5% - SA 5 94 BLSA-10 PHB-WF 10% - SA 10 89 BLSA-20 PHB-WF 20% - SA 20 79 BRMA-5 PHB-Cellulose Fiber 5% -MA 5 94 BRMA-10 PHB-Cellulose Fiber 10% -MA 10 89 BRMA-20 PHB-Cellulose Fiber 20% - MA 20 79 BRSA-5 PHB-Cellulose Fiber 5% - SA 5 94 BRSA-10 PHB-Cellulose Fiber 10% - SA 10 89 BRSA-20 PHB-Cellulose Fiber 20% - SA 20 79

a) B = pure PHB; R = Cellulose Fiber; WF = Wood Fiber; MA = maleic anhydride; SA = succinic anhydride Colonization of PHB composites Media composition and preparation

Ammonium sulphate (NH4)2SO4 7 g L-1, potassium di-hydrogen phosphate K2HPO4 7 g L-1, agar 15 g L-1, and distillated water were used to prepare the minimal media. A minimal nutrient medium was sterilized and was poured in Petri dishes.

Sample preparation consisted of cutting the specimens, measuring approximately 3.5 cm x 6.5 cm and depositing on this medium in Petri dishes before its solidification. 2 µl spores 106 UFC of T. viride 456 were inoculated on the each six points of specimen surface. Two assays were carried out in Petri dishes, following the procedure previously described [32]. Petri dishes were isolated with parafilm and incubated at 27 0C and the degradation of the PHB/wood fibers composites was periodically monitored for fifty days. Similarly, control samples made under the same conditions were incubated. Degree of colonization

In order to assess the degree of colonization, the number of hyphae that invaded the

samples was quantified. Visual examination has been done according to the standard ISO 868 and the results are shown in scores from 0 to 4: grade 0 indicated no microbial colonization; grade 1 indicated microbial colonization which is not visible by eye, but can be seen by



optical microscope, and corresponds to 25 % microorganisms covered surface; grade 2 expressed average microorganisms development also visible by eye; specimens surface is almost 50 % covered; grade 3 indicated a broad colonization by microorganisms; specimens surface is above 50 % covered and grade 4 denoted a very high degree of colonization; specimens surface is completely covered. Investigation of microbial growth was performed using the NOVEX HOLLAND microscope equipped with a camera, at magnification of 40 X. Chemical changes by FT-IR

In order to estimate the biodegradation of PHB/cellulose and wood fibers composites, the concentration of C-OH groups was determined by FT-IR spectroscopy. Analyses were carried out with a spectrometer FTLA 2000-104 in the range of 3600 cm-1 - 1000 cm-1, resolution 4 cm-1, in transmission mode. Using the Beer-Lambert law, the concentration of C-OH bonded groups can be determined from the absorbance peak by the following relationship:

ρεM

lAC OHC ⋅

=− 21 %, (1)

where: A is absorbance of C-OH at 3436 cm-1, εC-OH is the molar extinction coefficient (estimated at 72 kg/mol·cm) [33], l, the thickness of the sample (cm), M, the molar mass of COOH (45 g/mol) and ρ, the density of the sample (g/cm3). Results and discussions Colonization of PHB

Growth rate of T. viride 456 on the specimens is according to Table 3. From Table 2 it

can be seen a good growth rate of fungus on specimens and an increasing of it proportional with wood fibers contents. Initially, the growth of T. viride 456 appeared to be concentrated near the edges of test specimens of PHB that were originally directly exposed to the test fungus. After 20 days of incubation, fungal growth was observed in an area exceeding 25 % of tested specimens. After 50 days of incubation, a high density of colonies covering almost 50 % from samples surfaces was observed for BLMA-5, BLMA-10, BLMA-20, BRMA-5, BRMA-10, BRMA-20, BLSA-10 samples, meanwhile BLSA5, BLSA-20, BRSA-5, BRSA-10 and BRSA-20 samples showed less growth of fungi. Thickness of samples can be assumed as an explanation for this behavior. We can conclude that PHB composites represent a good substrate for growth of fungus, so good substrate for colonization.

Table 2. The fungal growth rate on the film samples

Code sample Incubation time (days)

10 20 30 40 50 PHB 1 1 1 2 2 BLMA-5 1 2 2 3 3 BLMA-10 1 2 2 2 3 BLMA-20 1 2 2 3 3 BLSA-5 1 2 2 2 2



BLSA-10 1 2 2 3 3 BLSA-20 1 2 2 2 2 BRMA-5 1 1 2 2 3 BRMA-10 1 2 2 2 3 BRMA-20 1 2 2 3 3 BRSA-5 1 2 2 2 2 BRSA-10 1 2 2 2 2 BRSA-20 1 2 2 2 2

Similar results were obtained in a previous work in the case of action of Penicillium

spp. on PHB samples [32]. Also, after 10 days exposure to the action of T. viride 456, formation of holes or cracks on the surface of specimens, changes in color that increase with increasing exposure time and increasing of biodegradation agent were visually observed. These processes lead, as it is known, to chemical modification - damages caused by digestion processes, in which the fungi use the constituents of the degraded material as nutrients. Frequently, physical changes initiate or are associated with chemical changes, an obvious aspect for fungi that perforate external structures before developing of hyphae and starting the biodegradation processes. These changes prove a first indication of fungal attack.

Figure 1 shows the expansion of the fungal hyphae on the surface of specimens after 50 days of incubation. During incubation period, white hyphae, long filaments and green conidiophores were observed on specimens. It is well known that T. viride 456 is able to synthesize and release in culture media a variety of enzymes such as lipases, esterases, ureases, depolymerases and hydrolases that could be involved in the degradation processes of these materials.



Figure 1. Trichoderma spp. growth on PHB composites, after 50 days of incubation FTIR spectra

The biodegradability of formulations is shown by FT-IR spectra of the specimens subjected to T. viride 456 degradation, during 10 days, 20 days and 30 days of incubation compared with initial samples (Figure 2). Due to cracks of the test pieces subjected to the process of biodegradation, it was not possible to analyze by FT-IR spectra all the samples exposed to the action of fungus more than 30 days.

The major PHB bands are the intense ester carbonyl stretch at 1738 to 1728 cm−1 and a number of strong bands at wave number values between 1450 and 1000 cm−1 due to asymmetric and symmetric methyl (CH3) and methylene (CH2) groups deformations and C-O-C stretching [5]. Wood fibers showed characteristic peaks at 1632 cm-1 for -OH bending from the absorbed water, at 3400 cm-1 for hydrogen bonded -OH stretching and at 2900 cm−1 for C–H stretching [34]. Blending PHB with wood/cellulose fibers gradually reduced the peak intensities in the spectra belonging to cystalline PHB as the amorphous content of composites increased. Hydroxyl sites on the surface of natural fibers are favorable for hydrogen bonding. Hydrogen bonds are weak bonds which can occur between hydrogen atoms and electronegatively charged atoms such as oxygen [13]. As PHB is a hydrophobic polymer, it generally shows little attraction to hydrophilic fibers. Natural fibers are generally hydrophilic in nature due to their chemical structure. Succinic and maleic anhydrides change the structure of fibers and these changes can increase carboxyl group [35].

From Figure 3 it can be seen that the loading of the composites with wood /cellulose fibers leads to lowest peak intensity in region 3436 cm-1 in comparison with PHB, which can be correlated with the scission of the intra- and intermolecular hydrogen bonds. Also, the degraded PHB/wood fibers composites exhibit a decrease of transmittance intensity at 3436 cm−1 attributed to O–H vibration with respect to the initial samples due to the increment of O–H groups produced by the rupture of ester bridges.

Based on the FT-IR spectra of PHB/cellulosic fiber and wood fiber composites presented in Figure 2 and the relationship (1) it was calculated the concentration of C-OH bounded groups by integrated peak area in 3436 cm-1 (Figure 3 and Figure 4).



Figure 2. FTIR spectra for PHB/wood fibers composites recorded up to 30 days during incubation



Initial After 10 days After 20 days After 30 days

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

C-O

H b

onde

d co

ncen

trat

ion

(x10

-2, %

)

PHB BRMA-5 BRMA-10 BRMA-20 BRSA-5 BRSA-10 BRSA-20

Figure 3. C-OH bonded concentration of

PHB/cellulose fiber composites

Initial After 10 days After 20 days After 30 days0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

C-O

H b

onde

d co

ncen

trat

ion

(x10

-2, %

)

PHB BLMA-5 BLMA-10 BLMA-20 BLSA-5 BLSA-10 BLSA-20

Figure 4. C-OH bonded concentration of

PHB/wood fiber composites From Figures 3 and 4 it is clear that the initial concentration related to the bounded

hydroxyl groups is greater for the BRMA-5, BRSA-20, BLMA-5, BLSA-5 samples, then, after 30 days, the BRMA-5, BRMA-10, BLMA-5 samples show higher values than the one of PHB. After 30 days, the samples containing 20% reinforcing agent show the lowest values of the C-OH. These data are correlated with colonization degree for BLSA-20, BRSA-5, BRSA-10 and BRSA-20 samples that exhibit colonization degree noted 2. Generally, compared to the originally C-OH concentrations, a decrease of CC-OH with increasing of duration of exposure to the action of microorganism can be seen. It is known that an increase in bounded hydroxyl groups after exposure of samples to microorganism’s action is proportional with the number of chain changes which are produced. The results indicate that, although CC-OH decreases with increasing of exposure time, the degradation has still occurred, as a result of increasing the level of colonization and visual changes (holes, cracks) increasing. One explanation for this could be connected with the fact that hydroxyl compounds are soluble in the culture medium. Bikiaris [28] has studied biodegradation of polyesters by enzymes and found that the ester bonds which have sufficient mobility were cleaved enzymatically and the generated chain fragments finally were dissolved into the surrounding water phase. As a consequence of the polyester hydrolysis mechanism by enzymes, the molar mass of the polymers can be sufficiently reduced to generate water-soluble intermediates, which can be transported into the microorganisms and fed into the appropriate metabolic pathways.

According to the investigation of biodegradation ability of the green composites by estimation of degree of colonization by exposure to Trichoderma spp. action and by Fourier transform infrared spectra scanning, it can be said that T. viride 456 strain is able to colonize and degrade the PHB composites. Conclusions

Green composites made on the basis of PHB/ cellulose fibers and PHB/wood fibers, chemically treated with maleic anhydride / succinic anhydride were subjected to the action of T. viride 456 for 50 days. Biodegradation was assessed by estimating the degree of colonization and calculation of C-OH groups bounded. PHB composites represented a good substrate for growth of T.viride 456, the growth rate increased with wood fibers loading increasing. Physical changes associated with the



chemical modification, were observed, most probably due to the fungal action (hyphae development, etc.), colonization of the PHB composites and production of visual changes (holes, craters). The evaluation of C-OH bounded groups by FTIR spectra indicated a decrease of CC-OH with increasing of exposure time. The obtained results have proved that T. viride 456 strain is able to colonize and degrade the PHB composites. Acknowledgements

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Degradation study by trichoderma spp. of poly (3 ... 13_Maria Rapa...MARIA RÂPĂ, MONA ELENA POPA, PETRUŢA CĂLINA CORNEA,VLAD IOAN POPA, ELENA GROSU, MIHAELA GEICU-CRISTEA, …