Microbial Degradation of a Poly(lactic acid) - American Chemical Society

Chapter 14

Microbial Degradation of a Poly(lactic acid) as a Model of Synthetic Polymer Degradation Mechanisms in Outdoor Conditions 1

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A. Torres , S. M. Li2, S. Roussos , and M. Vert 1

GIRSA Research Center, 52000 Lerma, Mexico URA-CNRS 1465, CRBA, Faculty of Pharmacy, 34060 Montpellier, France Laboratory of Biotechnology, ORSTOM, 34032 Montpellier, France

Downloaded by UNIV OF GUELPH LIBRARY on February 24, 2013 | http://pubs.acs.org Publication Date: March 25, 1999 | doi: 10.1021/bk-1999-0723.ch014

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Poly(α-hydroxy alkanoates) are known to easily degrade hydrolitically in aqueous media. In the case of lactic acid polymers (PLA), microbial degradation mechanisms are not well known. The fate of a PLA in the presence of microorganisms was investigated. First, the assimilation of by-products, such as monomer, lactic acid, dimer and oligomers by two microorganisms, a fungus (Fusarium moniliforme) and a bacterium (Pseudomonas putida) was evaluated in aqueous culture media. Second, 1 cm plates of a racemic PLA (Mw=40,000) were allowed to age in an aqueous medium containing a mixed culture of the same microorganisms. Finally, racemic PLA plates were buried in a wood and recovered after 8 weeks to incubate them in laboratory conditions for 8 weeks more. Results showed that by-products of PLA chemical degradation can be assimilated by microorganisms thus demonstrating that PLA can be considered as a bioassimilable polymer. In contrast, it was observed that PLA plates must be initially degraded by chemical hydrolysis until the formation of such by-products and then the microorganisms are able to assimilate them, suggesting that PLA can not be considered as a biodegradable polymer. 2

Polymer materials developed during the last 50 years are generally resistant to microbial attack, a property that is now regarded as a problem with respect to solid waste management. As a source of alternative environment friendly material, the synthesis of degradable polymers is becoming of great interest. Among the few polymer families which have been identified so far as good candidates, aliphatic polyesters are the most attractive materials. These polymers, also named poly(hydroxy alkanoates), can be divided into two subgroups: poly(p-hydroxy alkanoates), such as poly(P-hydroxy butyrate) (PHB) or higher analogues and their copolymers; and poly(a-

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© 1999 American Chemical Society

In Biopolymers; Imam, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1999.


219 hydroxy alkanoates), such as poly(lactic acid) (PLA), poly(glycolic acid)(PGA) and their copolymers. Poly(P-hydroxy alkanoates) are microbial polymers and their biodegradation has been well documented (7, 2). In contrast, the biodegradability of PLA/GA polymers is still questioned, although these polymers do resorbfromcompost and in vivo after chemical degradation. It has been observed indeed that chemical hydrolysis of PLA/GA polymers yields oligomers that can be released to the aqueous medium once they become small enough to solubilize. Thefinalhydrolysis products are then lactic and glycolic acids (5-7). Whether or not these by-products can be totally assimilated by microorganisms has not been well demonstrated. Results dealing with the fate of PLA polymers under controlled or natural conditions are presented. First, two microorganisms able to use lactic acid or PLA by-products as their only source of carbon and energy were selected: a filamentous fungus, Fusarium moniliforme, and a bacterium, Pseudomonas putida. Second, the ability of selected microorganisms to assimilate PLA by-products, such as lactic acid (LA), lactyl lactic acid (dimer) and higher oligomers, was tested to demonstrate the PLA bioassimilation. The effect of enantiomeric composition on the assimilation rate was also studied by using two series of compounds: racemic and L-pure lactic acid derived forms. Finally, to analyze possible PLA biodegradation, i.e., direct microbial attack on high molecular weight polymer chains, racemic PLA (PLA50) plates were aged under controlled conditions in a mixed culture and in the natural soil. Screening of Microorganisms A screening of filamentous fungi strains was realized by using three different substrates: D, L-lactic acid, racemic PLA oligomers (Mw=1000) and a PLA/GA copolymer (Mw=l 50, 000). The first two substrates were used in a mineral liquid culture, whereas the copolymer was used on a solid culture (mineral agar medium). Racemic oligomers and the copolymer were added to the media after sterilization to avoid chemical degradation. Selection criteria in liquid culture were: total lactic acid consumption, residual nitrogen concentration and final biomass production, after 7 days at 28°C in shakenflasks(150 rpm). Only three of 14 fungi strains tested were able to use D, L-lactic acid as their only source of carbon and energy: two strains of F. moniliforme and one strain of Penicillium roqueforti. Besides, F. moniliforme strains totally assimilated the nitrogen source [(NH^SOJ and produced the highest biomass concentration (3 g/l of dry weight mass). These three strains could also assimilate racemic oligomers. In the case of the PLA/GA copolymer, only one F. moniliforme strain was able to grow at the surface after two months of incubation at 28°C. It was then concluded that F. moniliforme was able to assimilate PLA. However, it was not yet determined whether this assimilation occurred because of an enzymatic activity or because of a chemical hydrolysis followed by consumption of by-products.


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Furthermore, a strain of Pseudomonas putida was also selected as a microorganism able to consume these kinds of compounds, on the basis of its capability to assimilate a large variety of products.


Bioassimilation of PLA By-Products The ability of selected microorganisms to assimilate racemic and L-pure lactic acid compounds was tested in liquid cultures containing lactic acid, dimer and oligomers. In all cases, pure cultures of each microorganism were used and only in the case of oligomers was a mixed culture of both tested. Dimers and oligomers were added aseptically after media sterilization. D, Land L oligomers were 2000 and 4000 Mw, respectively. Lactic acid and dimer consumption were evaluated by direct HPLC of the liquid media. Figure 1 shows a typical HPLC chromatogram. It can be seen that the method detected not only lactic acid (17.205 min retention time), but also dimer and trimer (15.722 and 14.988 min retention time, respectively). In the case of oligomers, media were heterogeneous because some of them were not soluble. The soluble fraction was then evaluated by alkaline hydrolysis of a small homogeneous volume, followed by HPLC quantification, and the total residual oligomers were measured as lactic acid after complete alkaline hydrolysis of the whole flask volume at different culture times (Figure 2). Results showed that D,L and L monomers were totally assimilated by the two microorganisms without any influence of enantiomeric composition. Differences appeared with dimer assimilation: F. moniliforme started to consume dimers earlier than P. putida and the L-form was better assimilated (Figure 3). In contrast, D, L-oligomers were consumed faster than the L-form. It was interesting to note that the mixed cultures of both microorganisms had a synergistic effect on die D,L-oligomer assimilation: After 8 days of incubation, 80% of oligomers were consumed, whereas only 30% of them were consumed by the bacteria and 20% by the fungi in the same period (Figure 4). It was then supposed that both microorganisms possessed a complementary enzymatic system which permitted a faster assimilation of D,L-oligomers in a mixed culture. P. putida would be able to produce enzymes that hydrolyze chains randomly (endo-enzymes) and F. moniliforme would only be able to produce enzymes that attack the chain ends (exo-enzymes). In the case of L-oligomers, solubility was very poor in all media and general assimilation was limited, suggesting that chain stereoregularity restricted the microbial activity. In order to compare chemical and microbial degradation, a control without microorganisms was analyzed for dimer- and oligomer-containing media. Chemical hydrolysis was then observed by the gradual accumulation of typical by-products: the solublefractionof oligomers, which includes monomer (lactic acid),


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Figure 1. Typical HPLC analysis showing monomer (lactic acid), dimer and trimer.

oligomer media at "t" days (50 ml) 6 ml

direct HPLC analysis: lactic acid, dimer and trimer concentration

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HPLC after strong alkaline hydrolysis: oligomer assimilation as total residual lactic acid

HPLC analysis after alkaline hydrolysis: soluble oligomer concentration as lactic acid

Figure 2. Protocol used to analyze oligomer heterogeneous media.


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Figure 3. Assimilation of D,L- and L-dimers by the two microorganisms.

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Microbial Degradation of a Poly(lactic acid) - American Chemical Society

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