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Romanian Biotechnological Letters Vol. 16, No.1, 2011, Supplement Copyright © 2011 University of Bucharest Printed in Romania. All rights reserved

ORIGINAL PAPER

9

Evaluation of the biodegrading action of the Penicillium Sp. on some composites based on PHB

Received for publication, October 21, 2010

Accepted, January 18, 2011

MARIA RÂPĂ1*, MONA ELENA POPA2, ELENA GROSU1, MIHAELA GEICU2, PETRUŢA STOICA1 1 S.C. INCERPLAST S.A., 23 Ziduri Moşi Str., 021203, Bucharest, Romania 2University of Agronomic Sciences and Veterinary Medicine Bucharest, Biotechnology Faculty 59 Mărăsti Bd., 011464, Bucharest, Romania *Corresponding author: Tel/Fax: +40212522381/+40212522115,email: [email protected]

Abstract

Biodegradation studies of PHB with wood fibers/celluloze fibers composites were performed. These materials are of interest for food packaging applications, which presume to solve the problems concerning accumulation, disposal and degradation.

The samples in the form of composite films were prepared by blending in BRABENDER Plastograph. Examined films were incubated in the presence of the Penicillium sp. isolated from soil. Degree of colonization and surface morphology of the films with an optical microscope were observed. The loss of mass was estimated. Fourier Transform Infrared (FTIR) spectroscopy was used to monitor the development of degradation products, the structural change by effect of microorganism on the samples.

The results of the study have shown that Penicillium sp. was capable of degrading PHB samples.

Key words: biodegradation, polyhydroxybutyrate, composite, Penicillium sp. Introduction

Novel polymer materials have revolutionized the polymer applications. Serious efforts

are directed to the development of biopolymers with appropriate properties and processing, the so-called ‘‘green’’ polymers, in contrast to the conventional polymers of petrochemical origin [1, 2].

Poly-3-hydroxybutyrate (PHB) is the best known representative of PHA family which has been produced from renewable resources via biosynthesis by bacterial fermentation [3, 4].

These polyesters have attracted widespread attention, as environmentally friendly polymers which can be used in a wide range of agricultural, industrial, and medical applications. Perspective areas of PHAs applications are following: the use of PHA as filler for non-biodegradable plastics (production of biodegradable plastics); disposable packages; in agriculture - systems for prolonged release of fertilizers and agrochemicals; in medicine – medical devices and systems of sustained drug delivery [5].

PHB shows thermoplastics properties and is regarded as a potentially useful biodegradable natural plastic, which is biodegradable in the environment [6, 7].

However, the PHB use in a wide field of applications is limited because of its high costs [8]. Use of biodegradable polymer matrices with fibers derived from wood is an effective method to reduce final product costs and to improve the mechanical properties [9].

Microscopic fungi, because of their great capacity to adapt to the various sources of nutrition and living conditions play an important role in the deterioration of plastics when the ambient temperature and humidity are conducting to growth and development.

There are known research regarding degradation of films based on PHB by soil microorganisms at different pH values [6, 10 - 14]. It is found that PHB and its blends, the overall degradation rate was increased with higher pH values, and they were totally degraded

MARIA RÂPĂ, MONA ELENA POPA, ELENA GROSU, MIHAELA GEICU, PETRUŢA STOICA

Romanian Biotechnological Letters, Vol. 16, No. 1, Supplement (2011) 10

after 20-30 days of incubation in highly alkaline medium. The bacteria detected on the degraded PHB films were dominated by the genera Pseudomonas, Bacillus, Azospirillum, Mycobacterium and Streptomyces. The fungi were dominated by the genus Penicillium. The aim of the present study is to investigate the biodegradable properties by exposure of plastic epruvettes to the action of Penicillium sp. for a limited period of time, in given temperature and humidity conditions. The methods for assessing the degree of biodegradability were: visual inspection, determining the change of mass by exposure to fungi and determination of structural change by FT-IR analysis. Materials and Methods Chemicals and Reagents

Thirteen types of PHB type P226 based materials were used in this study, having the

composition indicated in Table 1. Poly (3-hydroxybutyrate) (PHB), type P226 was used as the polymer matrix, supplied

by Biomer, Germany. The material has a density of 1.17 g/cm3 and melting point of 173 0C. Prior to blending, PHB was dried in the oven at 80 0C, for 2 hours.

Cellulose fibers type EFC 1000 (Rettenmeier & Söhne AG, Germany) were supplied by CARTIF, Spain. The properties of the fibers are: water content = 4.5 ± 0.1(%); lignin = 28.7 ± 0.1%; holocellulose = 87.3 ± 1.3 %; free –OH content on the surface = 322 ± 14 mg KOH/g and aspect ratio = 6.7.

Wood fibers type LSL 200/150 (La.So.Le. Est SRL, Italy) were supplied by CARTIF, Spain. These shows following properties: water content = 8.7 ± 0.1 %; lignin = 28.2 ± 0.1%; holocellulose = 85.0 ± 1.2 %; free –OH content on the surface = 230 ± 15 mg KOH/g and aspect ratio = 5.4.

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, K2HPO4 7 g/l, peptone 3 g/l, agar 15 g/l, distilled water. Penicillium sp. was isolated from soil. Table 1. Composition of evaluated composite formulations

Formulation a Mixture Reinforcing agent content

(% vol.)

Resin content (% vol.)

B PHB 0 100 BRMA-5 PHB-Cellulose Fiber 5% -MA 5 95 BRMA-10 PHB-Cellulose Fiber 10% -MA 10 90 BRMA-20 PHB-Cellulose Fiber 20% - MA 20 80 BLMA-5 PHB-WF 5% - MA 5 95 BLMA-10 PHB-WF 10% - MA 10 90 BLMA-20 PHB-WF 20% - MA 20 80 BRSA-5 PHB-Cellulose Fiber 5% - SA 5 95 BRSA-10 PHB-Cellulose Fiber 10% - SA 10 90 BRSA-20 PHB-Cellulose Fiber 20% - SA 20 80 BLSA-5 PHB-WF 5% - SA 5 95 BLSA-10 PHB-WF 10% - SA 10 90 BLSA-20 PHB-WF 20% - SA 20 80

a) B = pure PHB; R = Cellulose Fiber from Retenmaier; WF = Wood fibers from La.So.Le.; MA = maleic anhydride; SA = succinic anhydride

Evaluation of the biodegrading action of the Penicillium Sp. on some composites based on phb


Preparation of test epruvettes After blending in BRABENDER Plastograph, the samples were pressed into thin

plates by a laboratory press type POLYSTAT 200 at the following conditions: temperature: 160 0C, pressing time: 5 minutes and pressure of 200 bars. Films with the thickness of 0.1 mm were obtained. Sample incubation

The samples were inoculated with Penicillium sp., incubated at 24 0C and humidity up to 90%. No inoculated samples (controls) were incubated in the same conditions such as the inoculated ones.

Working method

Test method was performed according to SR EN ISO 846:2000, Method B. The principle of the method consists of samples exposed to the microorganisms action, for a certain period of time at constant temperature and favorable relative humidity. Biodegradation of the films was performed in Petri dishes containing minimal culture media.

From the composite materials listed above, the samples were made with dimensions of about (65 x 35 x 0.1) mm.

The minimal culture media was sterilized, cooled down to a temperature of 45 0C and poured in the Petri plates. Before the media solidified, each film was then aseptically transferred and individually placed into sterile medium. Two replicates were used for each film. The suspension of microorganism isolated from the soil was prepared by taking the isolated Penicillium spores with a loop and immersing them in a 9 ml tube of distilled water. Afterwards, the microbial load was determined in a Thoma room. After the solidification of the culture media, the mould spores suspension was inoculated on samples. In every Petri dish, 2 μl suspensions of 106

spores UFC/ml were inoculated in six points of each plastic specimen, as shown in Figure 1. The Petri dishes were insulated with Parafilm, then incubated at 24 0C and monitored for 50 days (Figure 2).

Figure 1. Scheme of working protocol



Figure 2. Inoculated samples of PHB Biodegradability Assessment Degree of colonization

To evaluate the degree of colonization, the number of conidia and hyphae invading the samples was quantified, weekly. Five-grade scales of invasion ranging from 0 to 4 were established as a function of fungi observed on the surface of the films. Grade 0 indicated absence of invasion; grade 1 was considered to correspond to a low attack with a maximum 25% of the film surface covered with fungi, grade 2 indicated an expansion of moderate intensity with a maximum 50% of the film covered with fungi, grade 3 expressed a high degree of colonization over 50%, and grade 4 denoted the growth of fungi occupying the whole surface of the specimen. Weight loss

The strips were washed with distilled water to remove the much cell mass from the residual film as possible, submerged in ethylic alcohol for 5 min. to halt further action and thoroughly washed again then blotted dry and dried until a constant weight was obtained and the results were compared with visual assessment of fungal growth on the samples. Originally masses were noted each specimen with m1, m2, m3, and after testing noted m1′, m2′, m3′. For each specimen was determined the weight change, Δm = (m′-m) and then calculated the arithmetic mean obtained for each batch: Δm0, Δmi şi Δms. The weight change was calculated using the formula:

Δm biol.= 100)( xm

mmm

e

osi Δ+Δ−Δ (1)

where: Δmi is average mass of inoculated samples; Δms is average mass of uninoculated control; Δm0 is average mass of zero control; me is average mass of initial samples.



Optical Analysis After a period of incubation with Penicillium sp., polymeric films were observed with

an Olympus BX51 optical microscope, at different magnifications to identify cracks, holes and other changes on the surface during the degradation process. FTIR Analysis

Spectra measurements were carried out with a FTLA2000-104 spectrometer. Transmittance spectra were collected at wave number values between 4000 and 600 cm−1 with spectral resolution of 2 cm−1 and 10 scans were recorded and averaged. Findings are compared with the spectra of zero controls preserved in laboratory. Results and Discussions Degree of colonization

Colonization grade of composites based on PHB and wood fibers/cellulose fibers by visual observation is reported in Table 2.

Table 2. Colonization grade of composites based on PHB

Penicillium sp. Incubation time (days)

Sample

10 20 30 40 50 PHB 1 1 2 3 3

BRMA-5 1 2 3 3 3 BRMA-10 1 2 3 3 4 BRMA-20 1 2 3 3 3 BRSA-5 1 2 2 3 3

BRSA-10 1 2 2 3 3 BRSA-20 1 2 3 4 4 BLMA-5 1 2 3 3 3

BLMA-10 1 2 2 3 3 BLMA-20 1 2 2 3 3 BLSA-5 1 2 2 3 3

BLSA-10 1 2 3 4 4 BLSA-20 1 2 2 3 4

Generally the composites based on PHB presented a degree of colonization around 3

and sometimes 4. We can say that after 40 days, the degree of colonization is high for all samples. At the end of incubation, a high density of colonies of Penicillium sp. covers 100 % of the BRMA-10, BRSA-20, BLSA-10 and BLSA-20 samples. Weight loss

Figure 3 shows the mass loss due to the action of Penicillium sp. It is obvious that

mass loss for all composites performed increased with increased fiber content. The greater mass loss is observed for BLSA-20 and BRMA-20 samples.

As it has been shown by Spyros et al. [15] PHB contains amorphous and crystalline regions, of which the former are much more susceptible to microbial attack. The microbial degradation of PHB must be associated with a decrease in its molecular weight and an increase in its crystallinity. Moreover, microbial degradation of the amorphous regions of PHB films made them more rigid. As consequence, PHB films became more rigid and fragile; the surface of PHB films became distended, micro cracks appeared.



A visual observation on composites based PHB incubated with Penicillium sp. revealed color change and holes on the surface of the samples.

Figure 3. Weight loss (%) for samples tested after 5 weeks exposure to Penicillium sp.

Optical Analysis

In Figure 4 shows the optical microscope pictures of composites colonization. We can see a steady growth of fungi on the samples tested, the degree of fungal growth is dependent on the content of biodegradation agent. Micrographs reveal micelle growth and filament networks covering the surface of the polymer. Microorganism Penicillium sp. shows a good growth on all samples tested, indicating the ease of this organism to adapt to growing conditions. The samples based on PHB and cellulose/wood fibers serve as a good substrate for fungal growth.

Kim and others [16] showed that the chemical composition, structure and hydrophylicity of polymer affect fungal growth on artificial culture medium. It is also known that fungi produce generative forms under conditions of stress, mainly such as conidia [17].



Figure 4. Optical microscope observations of samples (10X; 40X) inoculated with Penicillium sp.after 50 days of incubation; (a) PHB; (b) BRSA-5; (c) BRSA-10; (d) BRSA-20; (e) BRMA-5; (f) BRMA-10; (g) BRMA-20; (h) BLSA-5; (i) BLSA-10; (j) BLSA-20; (k) BLMA-5; (l) BLMA-10; (m) BLMA-20 FTIR Analysis

FTIR analysis was performed only for composites with 20% filler content. The FTIR spectra for zero controls and inoculated composites based on PHB are presented in Figures 5- 9.

FTIR analysis presented the evidence of a chemical bridge between the cellulose/wood fiber and polymeric matrix through esterification for BLMA-20 (Figure 6), BLSA-20 (Figure 7), BRMA-20 (Figure 8) and BRSA-20 (Figure 9) samples. The feature peak of esterification occurred in the range between 1800 and 1650 cm−1 at FTIR spectra [18, 19, 20]. As shown in Figures 5 - 9, FTIR spectra of bio based composites after 50 days of incubation with Penicillium sp. indicated slightly increased absorption C=O in 1750-1700 cm-

1 region. At the same time a new weak band near 1410 cm-1 and wide O-H stretching band near 3600-3000 cm-1 was observed. Bands that exhibit visible variations between zero controls and inoculated samples are 3350 cm-1 (O-H stretching), 2895 cm-1 indicating C-H stretching absorption and of –C-O- (1100 cm-1) caused by alcohol, which indicate faster degradation rates of the carbon chains.



Analyzing using FTIR spectroscopy confirms qualitative changes in the chemical structure of composites due to the biodegradation action of the Penicillium sp. of composites based on PHB.

Figure 5. FTIR spectra of PHB; Zero control; Inoculated sample

Figure 6. FTIR spectra of BLMA-20 sample; Zero control; Inoculated sample

Figure 7. FTIR spectra of BLSA-20 sample; Zero control; Inoculated sample



Figure 8. FTIR spectra of BRMA-20 sample: Zero control; Inoculated sample

Figure 9. FTIR spectra of BRSA-20 sample; Zero control; Inoculated sample

Conclusions It was studied the biodegradability of same samples based on PHB and two different

reinforcing agents such as cellulose fibers and wood fibers under Penicillium sp. action. It was performed tests regarding to ability of growing Penicillium sp. on the polymer surface.

It is found that composites based on PHB were colonized and biodegraded by Penicillium sp. The results of FTIR spectroscopy confirm qualitative changes in the chemical structure of composites due to the biodegradation action of the Penicillium sp. Acknowledgements

The work described in this paper was supported by the European Union through the

FP 7 project 212239 FORBIOPLAST, “Forest Resource Sustainability through Bio-Based Composite Development”.



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evaluation of the biodegrading action of the penicillium … mona popa.pdf · spores ufc/ml were...

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