Advertisement

Journal of Polymer Research

, 24:159 | Cite as

Identification of Cellulosimicrobium sp., a poly(3-hydroxybutyrate)-degrading bacterium isolated from washed rind cheese, Pont-l’évêque lait cru

  • Yohko Tachibana
  • Senri Hayashi
  • Miwa Suzuki
  • Phouvilay Soulenthone
  • Yuya Tachibana
  • Ken-ichi Kasuya
ORIGINAL PAPER
Part of the following topical collections:
  1. Topical Collection on Bio-Based Polymers

Abstract

Environmental pollution caused by commodity plastics has become a global issue. As a result, biodegradable plastics have found multiple applications in recent years, particularly in the manufacture of food packaging containers. However, microbes present in fermented foods have been found to degrade biodegradable plastics. In this study, we report, for the first time, a poly(3-hydroxybutyrate) (P(3HB))-degrading bacterium isolated from a type of washed rind cheese, Pont-l'évêque lait cru. The P(3HB)-degrading isolate, designated as PONα, was characterized in detail. The strain was found to be gram-positive and filamentous-shaped; the DNA G+C content was 71.6 mol%, and anteiso-C15:0 was found to be the major fatty acid. The strain grew well in the range of 37–40 °C and formed a large clear zone on P(3HB) medium at 37 °C. The phenotypic properties and phylogenetic inference indicated that strain PONα is closely related to Cellulosimicrobium cellulans. Strain PONα formed a clear zone on P(3HB), LB with P(3HB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and chitin media plates, whereas no clear zone was found on poly(ethylene succinate) (PESu), poly(butylene succinate) (PBSu), poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(butylene adipate-co-butylene terephthalate) (PBAT), poly(butylene succinate-co-butylene adipate) (PBSA), and olive oil media plates. Relatively higher P(3HB) hydrolytic activity was observed in the culture supernatant under the co-existence of P(3HB) and the cheese, suggesting that a component of the cheese plays an important role in the hydrolytic activity. These results indicate that microbiota in fermented foods such as cheese can degrade biodegradable plastics, thereby reducing their quality. Therefore, because P(3HB) may be degraded by the microbiota present in cheese, these findings may affect the application of P(3HB) in cheese packaging.

Keywords

P(3HB) Biodegradation Washed rind cheese Cellulosimicrobium cellulans Food packaging Microbiota 

Introduction

Currently, environmental pollution caused by plastic wastes has become a globally important issue, owing to which, biodegradable plastics that are degraded by microorganisms are gaining attention [1, 2]. Because biodegradable plastics have physical and chemical properties similar to those of commodity plastics, various biodegradable plastics have been synthesized and are commercially available in the form of packaging containers, agricultural materials, and general-purpose durable goods. In particular, their use in food containers has been widespread because they are easily degraded in composts [3, 4]. For example, because poly(lactic acid)(PLA), a plastic made from sugar or starch, is easily degradable, it is used in fresh food packaging and tableware for food services. PLA is superior in mechanical strength, transparency, and processability as compared to other biodegradable plastics. However, it is inferior in its water vapor permeability and oxygen barrier property; therefore, several studies to improve its crystallinity have been performed [5].

Currently, companies all over the world are engaged in the development of biodegradable plastics, including Natureworks LLC that produces PLA as a commercial product under the tradename, Ingeo [3, 6]. Starch is used in biodegradable plastic, which is blended with plasticizer, derivatized, or graft polymerized to overcome its low sensitivity to water and poor mechanical properties. Novamont sells modified starch as Mater-Bi, which is blended with cellulose acetate (CA), and Mater-Bi is used in tableware at McDonald’s and at international events such as the Olympic Games [6]. Poly(butylene adipate-co-butylene terephthalate) (PBAT) blended with PLA is sold as Ecovio by BASF [4]. It is used in the manufacture of disposable containers such as espresso capsules that are compostable; however, price fluctuation in crude petroleum, one of the raw materials, influences the price of the product [7]. Poly(3-hydroxybutyrate) (P(3HB)) is also a potential candidate for use in food containers [4] because of its good biodegradability and thermal property. Microbes that degrade P(3HB) and it copolymers have been so far isolated from more kinds of natural environments such as soil, compost, sludge [8], freshwater [9], seawater [10, 11] and deep sea [12] compared to those that do other polymers. This lead to remarkably environmental degradabilities of P(3HB) and its copolymers.

To apply biodegradable plastics to food containers, good strength, flexibility, gas barrier properties, and hydrolysis properties are required. Therefore, there have been constant attempts to improve these properties in biodegradable plastics. However, these plastics can be degraded by microbes from food, and the degradation can cause a decline in the quality of the biodegradable plastic. It is therefore necessary to study the influence of microbes contained in foods on the quality of plastics. Thus far, it has been reported that Bacillus species degrade P(3HB), modified starch, poly(butylene succinate) (PBSu), and PLA, and the fungal strain Penicillium roqueforti degrades PLA [13, 14, 15]. These microbes are known to be present in fermented foods [16, 17]; however, to our knowledge, the effect of such microbes on biodegradable plastics in contact with the fermented food has never been reported.

Fermented foods vary among regions of the world, and are prepared using traditional manufacturing methods with roots in history and culture; they have enriched the dietary life of people worldwide. Microbial metabolism improves the preservation of foods, forms flavors and colors peculiar to fermented foods, and increases nutritive values. Additionally, fermented foods function as probiotics that provide nutritional benefits to the human body. For example, dairy products such as yogurt and cheese [18], pickles such as sauerkraut [19], seasonings such as soy sauce or fish sauce [20, 21], and alcohol such as sake or wine [20, 22] are generated by the action of microbes. Because most of these fermented foods are not completely sterilized, a great variety of microbes abundantly exists in these foods, which might degrade biodegradable plastics.

In this study, we focused on washed rind cheeses, which are produced all over the world, and we expected that microbes specific to the specific production area would be present. Washed rind cheeses are inoculated with Brevibacterium linens early in the ripening stage, by using a product or old-young smearing method, and further fermented by washing the surface with brine [23]. We examined the degradability of biodegradable plastics by bacteria from a type of washed rind cheese, Pont-l’évêque lait cru. The aim of this study was to characterize bacteria that degrade biodegradable plastics present in this cheese and to consider the possibility of using biodegradable plastics as packaging containers for fermented foods.

Experimental

Chemicals

Poly(3-hydroxybutyrate) (P(3HB)), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(ethylene succinate) (PESu), poly(butylene succinate) (PBSu), poly(ε-caprolactone) (PCL), poly(lactic acid) (PLA), poly(52 mol% butylene adipate-co-48 mol% butylene terephthalate) (PBAT) and poly(butylene succinate-co-butylene adipate) (PBSA) were kind gifts from Mitsubishi Gas Chem. Co., Inc., Nippon Shokubai Co. Ltd., Showa Denko K.K., Daicel Chemical Industries Co., Shimadzu Co., BASF AG (Germany), and Showa Denko K.K., respectively. TaKaRa Ex Taq®, plasmid pMD20 and T4 DNA ligase were purchased from TAKARA BIO Inc. Oligonucleotides, GoTaq® Long PCR Master Mix, and Nuclease P1 were purchased from Eurofins Genomics Co., Ltd., Promega KK, and Yamasa Corporation, respectively. Other chemicals were purchased from WAKO Pure Chemical Industries Co., Ltd. and Roche Diagnostics K.K.

To purify polymers, they were dissolved in chloroform, and then were reprecipitated by methanol. The films were prepared by solvent-cast technique from chloroform solutions of polyesters with Petri dishes as casting surfaces. Furthermore, to prepare melt-crystallized film, solvent-casting films were inserted between two Teflon sheets, and were compression-molded on a Toyoseki Mini Test Press by heating at 200 °C for 1 min. After melting, it was kept at 85 °C and isothermally crystallized for 3 days. Unlike other polymers, P(3HB) after reprecipitation was collected and dried in vacuo for 1 day. Purified P(3HB) was put in the metal mold and covered with Kapton polyimide film (DU PONT-TORAY Co., Ltd.). Then, it was pressed with a mini Test Press-10 (Toyo Seiki Seisaku-Sho, Ltd.) for 1 min at 190 °C under the pressure of 15 MPa. The film was annealed at room temperature for 1 day. The film was cut off ca. 2 × 2 × 0.1 cm3. Before use, the film was washed with methanol and distilled water, and dried in vacuo for 1 day.

Media and culture conditions

The microorganisms were cultured on a Luria-Bertani (LB) medium (pH 7.5; components (g L−1):polypepton, 10; yeast extract, 5; NaCl, 5) or mineral media (pH 7.0; components (g L−1): KH2PO4, 4.6; Na2HPO4·12H2O, 11.6; MgSO4·7H2O, 0.5,; NH4Cl, 1.0; FeCl3·6H2O, 0.1; yeast extract, 0.5; agar, 20) supplemented with 0.2% (w/v) emulsified polymer or olive oil, which were prepared as described previously [24, 25]. Unlike other polymers, P(3HB) granules were directly added to the mineral medium.

Isolation of P(3HB)-degrading bacteria

Pont-l’évêque lait cru (washed rind cheese, France) was used as a source of P(3HB)-degrading bacteria. A piece of the cheese (1.0 g) was dispersed in 10 mL of ultrapure water, and 100 μL of suspension was plated onto a medium containing P(3HB). After incubation at 30 °C for 10 days, the colony that formed clearing zone at the periphery was selected and further purified on 2 kinds of plates: LB medium or P(3HB) containing mineral medium, by performing a streak culture.

Phylogenetic analysis

Genomic DNA (gDNA)s were prepared from the isolates as described in reference [25]. 16S rRNA genes were amplified by PCR method. The forward amplification primer was 16Sf, 5′-GTTTGATCATGGCTCAG-3′ (corresponding to positions 36–53 in the 16S rDNA nucleotide sequence of E. coli) and the reverse amplification primer was 16Sr, 5′-TACCTTGTTACGACTTCA-3′ (positions 1517–1533). Reactions were carried out in 50 μL volumes containing 1.25 U ExTaq DNA polymerase (TaKaRa, Japan), 100 nM each primer (16Sf and 16Sr), 25 mM each dNTP (dATP, dTTP, dCTP, and dGTP), 5 μL ExTaq 10 × buffer (TaKaRa, Japan), and 100 ng of gDNA. The PCR cycle parameters were as follows: preheating for 5 min at 94 °C, 25 cycles of denaturation at 94 °C for 20 s, annealing at 55 °C for 30 s, and extension for 2 min at 72 °C, and 1 cycle at 72 °C for 10 min. The PCR products were analyzed in 1.5% (wt. /vol.) agarose to confirm their size and purity, and then ligated to T-Vector pMD20 (TaKaRa Bio Inc., Japan) with T4 ligase (TaKaRa Bio Inc., Japan) at 16 °C. Sequencing of the 16S rDNA sequence was performed by Eurofins Genomics Co. Inc. (Tokyo, Japan), and then was compared with Genebank data using the program blast via NCBI site (http://www.ncbi.nlm.nih.gov/BLAST/). The 16S rDNA sequences were aligned by the program ClustalW via DDBJ site (http://www.ddbj.nig.ac.jp/). A phylogenetic tree was constructed according to the neighbor-joining method with the program MEGA6 [26].

Degradation abilities of isolates for P(3HB)

The hydrolytic abilities of the P(3HB)-degrading bacteria isolates for 8 polyesters (P(3HB), LB with (PHB), PHBV, PESu, PBSu, PCL, PLA, PBAT, and PBSA) and olive oil and chitin were examined by the clear-zone method, as described previously [24]. The hydrolytic abilities were evaluated by the magnitude of clear zone formed surrounding the colony on medium containing each substrate after incubation at 37 °C for 14 days. Further, the degradation abilities of the strains for solid P(3HB), PHBV, PLA and PBAT films (1 cm × 1 cm × 0.1 cm) were evaluated by the weight-loss method [25]. Each strain was incubated at 30 °C for 15 days in medium containing each film. After recovered films were washed with methanol and ultra-pure water, they was dried in vacuo and weighed. Weight loss was calculated by subtracting the weight of degraded film from the initial weight of the film.

Electron microscopy

The isolate was gold-coated by an ion coater (JEOL JFC-1500) after fixation as previously described [12], in order to observe it by a scanning electron microscope (SEM) (Tabletop microscope TM3030, Hitachi High-Technologies Corporation, Japan).

GC content

The DNA guanine + cytosine (GC) content of the isolates and reference strain (Cellulosimicrobium cellulans NBRC15516T) were determined as previously described [27, 28] by high-performance liquid chromatography using a standard mixture for the analysis of DNA GC content with nuclease P1 (Yamasa Corporation, Japan).

Cellular fatty acid composition

Cells of the strain and reference strain (C. cellulans NBRC15516T) were cultivated in LB medium at 37 °C for overnight with shaking. Preparation and identification of cellular fatty acids were carried out according to the MIDI method as described in Ref. [29, 30]. Bacteria standard methyl ester (BAME) mix (Sigma-Aldrich Japan K.K.,Tokyo) was used as standards of fatty acid methyl esters.

Phenotypic analysis

API 20NE and API ZYM (bioMérieux) index systems were used for analysis of physiological and biochemical properties of the isolate and reference strain (C. cellulans NBRC15516 T). In the API 20NE assay, the strains were inoculated into the assay kit at 30 °C for 24 h and 48 h. In the API ZYM assay, the strains, which were suspended in sterile saline, were inoculated into the assay plate and incubated at 37 °C for 4 h. Then a color coupler was added to each cup on the plate and strong light was irradiated there. Thereafter, the test result was evaluated according the color chart supplemented by the manufacturer.

Effect of temperature on growth and P(3HB) degradation ability

The effect of the temperature on the growth and P(3HB)-degrading activity of the isolate was evaluated, respectively. Growth levels were determined by the sizes of colonies on a LB plate at 8 different temperatures (4, 15, 25, 30, 37, 40, 45, and 50 °C), and P(3HB) degradation abilities ware determined by those of clear zones formed on a LB plate containing P(3HB) at 6 different temperatures (4, 15, 25, 30, 37, and 50 °C).

Effect of carbon sources on the growth and P(3HB) depolymerase production

The isolate was aerobically cultivated for 20 h at 37 °C in 3 mL of LB medium and then 10 mL/L of culture was inoculated onto mineral medium supplemented with 0.2% (w/v) various carbon sources (P(3HB), (R)-(−)-3-hydrobutyric acid, (S)-(−)-3-hydrobutyric acid, D-(+)-glucose, succinic acid, lactic acid, D-(+)-sorbitol, adipic acid, D-(+)-mannose, sucrose, LB, LB with P(3HB), and cheese with P(3HB)). The growth levels of isolate were evaluated by optical density at 600 nm(OD600), and the activity of P(3HB) depolymerase was determined as previously described [31]. The standard assay mixture contained 400 mg/L of P(3HB) granules (Monsanto, USA), 0.5 M NaCl and 1 mM CaCl2 in 50 mM Tris-HCl buffer (pH 7.5). A stable suspension of purified P(3HB) granules was prepared by using a sonic oscillator (20 kHz, 250 W) for 10 min. The reaction was started by addition of enzyme and followed by a decrease in turbidity (OD650) of P(3HB) granules, which was measured at 650 nm and 37 °C, using 1 cm light-path cuvettes. One unit of the enzyme was defined as the amount of enzyme required to decrease the value of OD650 by 1 per min.

Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis

Microbiota from Pont-l’évêque lait cru was analyzed using the PCR-DGGE method. Metagenomic DNA was extracted from cheese sample (0.5 g) with a DNA Isolation kit ISOIL for Beads Beating (WAKO Pure Chemical Industries Co., Ltd.). The DNA was preserved at −80 °C until further use. 16S rDNA was amplified by PCR using an iCycler™ Thermal Cycler (Bio-Rad laboratories, Inc.). The PCR reaction mixture was as follows: metagenomic DNA (2.0 μL), GoTaq® Long PCR Master Mix 2X (10 μL), primers 341F–GC (5′-CGCCCGCCGCGCCCCGCGCCCGTCCCGCCGCCCCCGCCCGCCTACGGGAGGCAGCAG-3′) and 907R (5′-CCGTCAATTCCTTT RAGTTT-3′) for amplification of 16S rDNA region (10 pmol each) [25] as well as double distilled (dd)H2O (up to 20 μL). Metagenomic DNA was denatured for 10 min at 98 °C and was quenched in ice-cold water to prevent annealing. Thermal cycles for the amplification of 16S rDNA region were as follows: 1 cycle of 95 °C for 3 min, 14 cycles of 94 °C for 30 s, 64 °C for 10 s (temperature was decreased at 0.5 °C per 1 cycle), 72 °C for 45 s, 16 cycles of 94 °C for 30 s, 57 °C for 10 s, 72 °C for 45 s, and 1 cycle of 72 °C for 10 min. The amplification of target DNA was confirmed using agarose gel electrophoresis.

DGGE was performed using the DCode™ Universal Mutation Detection System (Bio-Rad laboratories, Inc.) to analyze the cheese microbiota. Preparation of DGGE gels and DNA recovery buffer was performed according to the manufacturer’s instruction (Bio-Rad laboratories, Inc.). The reagents of 100% denaturing solution/6% acrylamide gel were as follows: 40% acrylamide/bis solution (5.48 M of acrylamide and 69 mM of N,N′-methylene-bis-acrylamide), 50× TAE buffer [2.0 M of trizma base, 0.25 M of sodium acetate, and 63 mM of ethylenediaminetetraacetic acid (EDTA)], formamide, urea, and ddH2O (up to 50 mL). 10% ammonium persulfate solution and N,N,N′,N′-tetramethylenediamine were added for gelation. Denaturing gradient gels (with 30–55% of denaturant for analysis of microbiota) were prepared using a model 475 gradient delivery system (Bio-Rad laboratories, Inc.). 19 μL of PCR products and 2 μL of 10× loading buffer were loaded on to the gel, and the electrophoresis was performed in 0.5× TAE buffer at 58 °C for 4 h under the conditions of 200 V supplied by an electrophoresis power supply, EPS 601 (GE healthcare life science). The gel was stained with SYBR Green I Nucleic Acid Gel Stain (Roche Diagnostics K.K.), and DNA bands were then visualized with a high performance UV trans-illuminator (Funakoshi Co., Ltd.). Excised DNA bands were incubated in 600 μL of DNA recovery buffer (5 M of ammonium acetate and 2.0 μM of EDTA) at 37 °C overnight, to extract DNA from gels. Extracted DNA was purified with ethanol precipitation and then precipitated DNA was dissolved in 10 μL of ddH2O.

DNA analysis of DGGE bands

DNA was amplified with a semi-nested PCR using DNA extracted from DGGE gels as a template. The PCR reaction mixture was as follows: extracted DNA (2.5 μL), GoTaq® Long PCR Master Mix 2X (5 μL), primers 341F (5′-CCTACGGGAGGCAGCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′) (10 pmol each) for amplification of 16S rDNA [32], and ddH2O (up to 10 μL). Thermal cycles of PCR for the amplification of 16S rDNA were as follows: 1 cycle of 94 °C for 2 min, 25 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 90 s and 1 cycle of 72 °C for 10 min. Amplified DNA were sequenced using the dideoxy method (Eurofins Genomics Co., Ltd.). The sequences were compared with GenBank data using the nucleotide BLAST program at NCBI (http://www.ncbi.nlm.nih.gov) to search the related species.

Accession number of nucleotide sequence

16S rDNA sequences of an isolate, strain PONα was deposited in the DNA Databank of Japan (DDBJ) under the accession number LC186053.

Results and discussion

Characterization of the P(3HB)-degrading bacterial isolate, PONα

From Pont-l’évêque lait cru (washed rind cheese), we isolated a strain, designated as PONα, which formed a clear zone surrounding its colony on a plate containing P(3HB) as the sole carbon source. This strain was gram-positive and filamentous-shaped (approximately 0.5 μm in diameter) (Fig. 1). The GC content of its genomic DNA was 71.6 mol%, whereas that of the reference strain (C. cellulans NBRC15516 T) was 73.8 mol%. The strain exhibited positive results in esculin hydrolysis, gelatin hydrolysis, and β-galactosidase tests, whereas the reference strain showed a negative result for gelatin hydrolysis (Table 1). Other enzyme activities were assayed using the API ZYM kit. Consequently, the following enzyme activities were detected in the strain: alkaline phosphatase, esterase (C4), esterase lipase (C8), leucine arylamidase, trypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, β-galactosidase, α-glucosidase, β-glucosidase, and N-acetyl-β-glucosamidase. Thus, this strain showed similarity to the reference strain (Table 2). The fatty acid profile of strain PONα consisted of 3-OH C-12:0, C14:0, iso-C15:0, anteiso-C15:0, C15:0, iso-C16:0, C16:0, iso-C17:0, and C17:0D. The main fatty acid was found to be anteiso-C15:0, followed by iso-C15:0, similar to the results obtained for the reference strain. The optimum temperature range for the growth on LB medium was found to be 37 °C–40 °C, and the strain was unable to grow at temperatures of 4 °C and 50 °C. The strain PONα formed a clear zone at temperatures higher than 15 °C on P(3HB) medium, whereas the reference strain did not (Table 3). The strain did not form clear zones on PESu-, PBSu-, PCL-, PLA-, PBAT-, or olive oil-containing media, but clear zones were observed on P(3HB), LB with P(3HB), PHBV, and chitin-containing media. The reference strain did not show clear zones on any of the media tested in this study, except for chitin (Table 4). There have been no reports of Cellulosimicrobium species that degraded P(3HB) thus far, although we found a putative ORF (KON75583.1, 64–262) that encodes a P(3HB) depolymerase in the genomic DNA sequence of Cellulosimicrobium cellulans F16 in the NCBI database. Therefore, to the best of our knowledge, this is the first report of a Cellulosimicrobium species that degrades P(3HB).
Fig. 1

Scanning electron micrograph of strain PONα. The white bar indicates 5 μm in length

Table 1

Biochemical and physiological properties of an isolate, PONα and the reference strain

 

PONα

NBRC 15516T

Characteristics

 Gram strain

Positive

Positive

 Oxidase

 Nitrate reduction

+

 Indole production

 Oxidation of glucose to gluconate

 Arginine dihydrolase

 Urease

 Hydrolysis of esculin

+

+

 Hydrolysis of gelatin

+

 β-Galactosidase

+

+

Utilization of carbon source

 Glucose

+

+

 L-Arabinose

+

+

 D-Mannose

+

+

 D-Mannitol

 N-Acetyl-D-glucosamine

+

+

 Maltose

+

+

 Gluconate

+

+

n-Capric acid

 Adipic acid

 DL-Maleic acid

 Citrate

 Phenyl acetate

DNA G + C content (mol %)

71.6

73.8

+: means positive

-: means negative

Table 2

Enzyme production profile of an isolate, PONα and the reference strain

Enzyme

PONα

NBRC 15516T

Alkaline phosphatase

+

+

Esterase (C4)

+

+

Esterase lipase (C8)

+

(+)

Lipase (C14)

Leucine arylamidase

+

+

Valine arylamidase

(+)

(+)

Cystine arylamidase

(+)

(+)

Trypsin

(+)

(+)

Chymotrypsin

Acid phosphatase

+

+

Naphthol AS-BI phosphohydrolase

+

+

α-Galactosidase

β-Galactosidase

+

+

β-Glucuronidase

α-Glucosidase

+

+

β-Glucosidase

+

+

N-Acetyl-β-glucosamidase

+

+

α-Mannosidase

α-Fucosidase

+: means positive

(+): means weakly positive

-: means negative

Table 3

Effect of temperature of the growth on LB medium and clearing zone formation on P(3HB)-containing medium by an isolate, PONα and the reference strain

Temperature (°C)

PONα

NBRC 15516T

Growth

Clearing zone formation

Growth

Clearing zone formation

4

15

+

+

+

25

++

++

++

30

++

++

++

37

+++

+++

+++

40

+++

ND

+++

ND

45

++

ND

ND

50

+++: means growth was excellent, or a large clearing zone was formed

++: means growth was good, or a medium clearing zone was formed

+: means growth was poor, or a small clearing zone was formed

-: means growth was not observed, or a clearing zone was not formed

ND: means no data

Table 4

The ability of clearing zone formation on the plates containing polymers and olive oil by an isolate, PONα and the reference strain

Substrate

PONα

NBRC 15516T

P(3HB)

++

LB with P(3HB)

++

PHBV

++

PESu

PBSu

PCL

PLA

PBAT

PBSA

Olive oil

Chitin

+++

+++

+++: means growth was excellent, or a large clearing zone was formed

++: means growth was good, or a medium clearing zone was formed

-: means growth was not observed, or a clearing zone was not formed

The nucleotide sequence of the 16S rRNA gene of strain PONα was used to search for homologous 16S rDNA sequences in the NCBI database. The results showed that strain PONα belonged to the phylum Actinobacteria and genus Cellulosimicrobium (Fig. 2). The strain showed the highest 16S rDNA sequence similarity with Cellulosimicrobium cellulans (identity level 99%).
Fig. 2

Phylogenetic tree of the P(3HB)-degrading isolate, designated as PONα, 5 bacterial strains detected by PCR-DGGE analysis, and the related strains on the basis of 16S rDNA sequences. The sequences were aligned using the program ClustalW and the tree topology was constructed applying the neighbour-joining method. Accession numbers are given in parentheses. The 16S rDNA sequence of strain PONα (accession no. LC186053) showed the highest similarity with that of Cellulosimicrobium cellulans (AY665978, identity level 99%). The bar indicates 2% estimated sequence divergence

Microbiota in the cheese

Microbiota in the washed rind cheese Pont-l’évêque lait cru were analyzed using the PCR-DGGE method. Five bands were observed in the electropherogram and were numbered in order from the top (well side) to bottom of the gel. Table 5 lists the bacterial taxa corresponding to each DGGE band. PCR-DGGE analysis of 16S rDNA from the metagenome of the washed rind cheese sample revealed that the sample contained bacteria belonging to the phyla Firmicutes and Actinobacteria. These bacterial phyla are commonly observed in other cheeses [33, 34, 35, 36]. However, the genera in the microbiota in this cheese were different from those of other washed rind cheeses. According to Brennan et al. [23], Corynebacterium casei, Corynebacterium mooreparkense, Microbacterium gubbeenense are mainly detected in mature washed rind cheeses, which are made from pasteurized milk. These bacteria were not detected in our study. It is considered that the microbiota of Pont-l’évêque lait cru are influenced by the bacteria in raw milk because this cheese is made from non-pasteurized milk and contaminated with bacteria from the environment during manufacturing processes.
Table 5

Identification of DGGE bands from Pont-l’évêque lait cru based on 16S rDNA

Band

Closely related species

Accession number

DGGE band 1

Streptococcus thermophilus

LC192945

DGGE band 2

Staphylococcus succinus

LC192946

DGGE band 3

Lactococcus lactis

LC192947

DGGE band 4

Brevibacterium linens

LC192948

DGGE band 5

Brevibacterium sp.

LC192949

Effect of carbon source on bacterial growth and P(3HB) depolymerase production

The effects of various carbon sources on the growth of strain PONα were evaluated by measuring the turbidity of the culture media at 650 nm. As shown in Table 6, the growth level of the bacterium depended on the carbon source added to the culture media. The strain expressed relatively high P(3HB) depolymerase activity when D-(+)-glucose or R-(−)-3-hydrobutyric acid was added as the carbon source. Although the strain grew in the presence of lactic acid, no P(3HB) depolymerase activity was detected in the culture supernatants. Although it grew well in the presence of succinic acid, D-(+)-mannose, LB, and LB with P(3HB), only low P(3HB) depolymerase activity was detected. When P(3HB) was used as the sole carbon source, both the growth and the enzyme activity were low. In contrast, when the cheese was added along with P(3HB), the growth and enzyme activity increased, indicating that the cheese probably includes a compound that aids in the expression of the enzyme. However, the strain never expressed P(3HB) depolymerase activity in the presence of cheese alone. This might affect the applicability of P(3HB) in the packaging of cheese because it would be degraded in such environments. We therefore concluded that biodegradable materials containing P(3HB) as constituents are not suitable for packaging of this cheese.
Table 6

Growth levels and P(3HB) depolymerase activity by PONα grown on various media

Carbon source

Growth level

P(3HB) depolymerase activity (U/L)

P(3HB)

+

4.0

R-(−)-3-Hydrobutyric acid

++

42

S-(+)-3-Hydrobutyric acid

++

15

D-(+)-Glucose

++

35

Succinic acid

++

7.0

Lactic acid

+

0.0

D-(+)-Sorbitol

+

9.0

Adipic acid

+

8.0

D-(+)-Mannose

++

6.0

Sucrose

+

4.0

LB

++

8.0

LB with P(3HB)

+++

11

Cheese

++

0.0

Cheese with P(3HB)

++

21

None

+

0.0

+++: means growth was excellent

++: means growth was good

+: means growth was poor

Degradation of P(3HB) film by strain PONα

P(3HB), PHBV, PLA, and PBAT films were aerobically incubated with strain PONα. Weight loss of the films was observed in P(3HB) (0.03 mg/cm2/day) and PHBV (0.05 mg/cm2/day), whereas the weight of PLA and PBAT films did not change after incubation at 30 °C for 10 days. This suggests that strain PONα is able to degrade not only emulsified P(3HB), but also solid P(3HB) and PHBV, which is a P(3HB) co-polymer.

Conclusion

From a type of washed rind cheese, Pont-l’évêque lait cru we isolated a bacterium, designated as PONα that degraded P(3HB) and further discussed the possibility of application for the cheese packaging of biodegradable plastics made of P(3HB). Strain PONα formed a clear zone on P(3HB)-containing mineral plates, although its growth level was poor. In contrast, it grew well in the presence of cheese together with P(3HB). In addition, higher P(3HB) depolymerase activity was observed in the co-presence of P(3HB) and cheese compared with presence of only P(3HB), even though P(3HB) depolymerase activity was never detected in the mineral medium containing only cheese. Therefore, an unknown substance in the cheese appears to facilitate the expression of P(3HB) depolymerase in this strain. This suggests that P(3HB) is not suitable as packaging material for this washed rind cheese because it could be degraded in such environments. In future, it will be necessary to identify an inducer for P(3HB) degradation that is contained in the cheese to increase the practicality of biodegradable materials made of P(3HB) for food packaging.

References

  1. 1.
    Vandewijngaarden J, Wauters R, Murariu M, Dubois P, Carleer R, Yperman J, D’Haen J, Ruttens B, Schreurs S, Lepot N, Peeters R, Buntinx M (2016) Poly(3-hydroxybutyrate-co −3-hydroxyhexanoate)/organomodified montmorillonite nanocomposites for potential food packaging applications. J Polym Environ 24:104–118CrossRefGoogle Scholar
  2. 2.
    Yu H, Yan C, Yao J (2014) Fully biodegradable food packaging materials based on functionalized cellulose nanocrystals/poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nanocomposites. RSC Adv 4:59792–59802CrossRefGoogle Scholar
  3. 3.
    Siracusa V, Rocculi P, Romani S, Rosa MD (2008) Biodegradable polymers for food packaging: a review. Trends Food Sci Technol 19:634–643CrossRefGoogle Scholar
  4. 4.
    Vroman I, Tighzert L (2009) Biodegradable polymers. Materials (Basel) 2:307–344CrossRefGoogle Scholar
  5. 5.
    Arrieta MP, López J, Hernández A, Rayón E (2014) Ternary PLA–PHB–Limonene blends intended for biodegradable food packaging applications. Eur Polym J 50:255–270CrossRefGoogle Scholar
  6. 6.
    Johansson C, Bras J, Mondragon I, Nechita P, Plackett D, Simon P, Svetec DG, Virtanen S, Baschetti MG, Breen C, Clegg F, Aucejo S (2012) Renewable fibers and bio-based materials for packaging applications – a review of recent denelopments. Bioresources 7:2506–2552CrossRefGoogle Scholar
  7. 7.
    Li W, Coffin DR, Jin TZ, Latona N, Liu C-K, Liu B, Zhang J, Liu L (2012) Biodegradable composites from polyester and sugar beet pulp with antimicrobial coating for food packaging. J Appl Polym Sci 126:E362–E373CrossRefGoogle Scholar
  8. 8.
    Mergaert J, Swings J (1995) Biodivesity of microorganisms that degrade bacterial and synthetic polyesters. J Ind Microbiol 17:461–469Google Scholar
  9. 9.
    Mukai K, Yamada K, Doi T (1994) Efficient hydrolysis of polyhydroxyalkanoates by Pseudomonas stutzeri YM1414 isolated from lake water. Polym Degrad Stab 43:319–327CrossRefGoogle Scholar
  10. 10.
    Kita K, Ishimaru K, Teraoka M, Yanase H, Kato N (1995) Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl Environ Microbiol 61:1727–1730Google Scholar
  11. 11.
    Sung C-C, Tachibana Y, Suzuki M, Hsieh W-C (2016) Identification of a poly(3-hydroxybutyrate)-degrading bacterium isolated from coastal seawater in Japan as Shewanella sp. Polym Degrad Stab 129:268–274CrossRefGoogle Scholar
  12. 12.
    Kasuya K, Mitomo H, Nakahara M, Akiba A, Kudo T, Doi Y (2000) Identification of a marine benthic P(3HB)-degrading bacterium isolate and characterization of its P(3HB) depolymerase. Biomacromolecules 1:194–201CrossRefGoogle Scholar
  13. 13.
    Ghosh SK, Pal S, Ray S (2013) Study of microbes having potentiality for biodegradation of plastics. Environ Sci Pollut R 20:4339–4355CrossRefGoogle Scholar
  14. 14.
    Tokiwa Y, Calabia BP (2004) Degradation of microbial polyesters. Biotechnol Lett 26:1181–1189CrossRefGoogle Scholar
  15. 15.
    Yun Y-H, Wee Y-J, Byun H-S, Yoon S-D (2008) Biodegradability of chemically modified starch (RS4)/PVA blend films: part 2. J Polym Environ 16:12–18CrossRefGoogle Scholar
  16. 16.
    McSweeney PLH (2004) Biochemistry of cheese ripening. Int J Dairy Technol 57:127–144CrossRefGoogle Scholar
  17. 17.
    Murooka Y, Yamshita M (2008) Traditional healthful fermented products of Japan. J Ind Microbiol Biotechnol 35:791–798CrossRefGoogle Scholar
  18. 18.
    Plessas S, Bosnea L, Alexopoulos A, Bezirtzoglou E (2012) Potential effects of probiotics in cheese and yogurt production: a review. Eng Life Sci 12:433–440CrossRefGoogle Scholar
  19. 19.
    M’hir S, Minervini F, Di Cagno R, Chammem N, Hamdi M (2012) Technological, functional and safety aspects of enterococci in fermented vegetable products: a mini-review. Ann Microbiol 62:469–481CrossRefGoogle Scholar
  20. 20.
    Machida M, Yamada O, Gomi K (2008) Genomics of Aspergillus oryzae: learning from the history of Koji mold and exploration of its future. DNA Res 15:173–183CrossRefGoogle Scholar
  21. 21.
    Namwong S, Hiraga K, Takada K, Tsunemi M, Tanasupawat S, Oda K (2006) A halophilic serine proteinase from Halobacillus sp. SR5-3 isolated from fish sauce: purification and characterization. Biosci Biotechnol Biochem 70:1395–1401CrossRefGoogle Scholar
  22. 22.
    Versari A, Parpinello GP, Cattaneo M (1999) Leuconostoc oenos and malolactic fermentation in wine: a review. J Ind Microbiol Biotechnol 23:447–455CrossRefGoogle Scholar
  23. 23.
    Brennan NM, Ward AC, Beresford TP, Fox PF, Goodfellow M, Cogan TM (2002) Biodiversity of the bacterial flora on the surface of a smear cheese. Appl Environ Microbiol 68:820–830CrossRefGoogle Scholar
  24. 24.
    Ishii N, Inoue Y, Tagaya T, Mitomo H, Nagai D, Kasuya K (2008) Isolation and characterization of poly(butylene succinate)-degrading fungi. Polym Degrad Stab 93:883–888CrossRefGoogle Scholar
  25. 25.
    Tezuka Y, Ishii N, Kasuya K, Mitomo H (2004) Degradation of poly(ethylene succinate) by mesophilic bacteria. Polym Degrad Stab 84:115–121CrossRefGoogle Scholar
  26. 26.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725–2729CrossRefGoogle Scholar
  27. 27.
    Katayama-Fujimura Y, Komatsu Y, Kuraishi H, Kaneko T (1984) Estimation of DNA base composition by high performance liquid chromatography of its nuclease PI hydrolysate. Agr Biol Chem Tokyo 48:3169–3172Google Scholar
  28. 28.
    Mesbah M, Premachandran U, Whitman BW (1989) Precise measurement of the G + C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39:159–167CrossRefGoogle Scholar
  29. 29.
    Dees SB, Moss CW (1975) Cellular fatty acids of Alcaligenes and Pseudomonas species isolated from clinical specimens. J Clin Microbiol 1:414–419Google Scholar
  30. 30.
    Miller LT (1982) Single derivatization method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxyl acids. J Clin Microbiol 16:584–586Google Scholar
  31. 31.
    Ohura T, Aoyagi Y, Takagi K, Yoshida Y, Kasuya K, Doi Y (1999) Biodegradation of poly(3-hydroxyalkanoic acids) fibers and isolation of poly(3-hydroxybutyric acid)-degrading microorganisms under aquatic environments. Polym Degrad Stab 63:23–29CrossRefGoogle Scholar
  32. 32.
    Nakatsu CH, Torsvik V, Øvreås L (2000) Soil community analysis using DGGE of 16S rDNA polymerase chain reaction products. Soil Sci Soc Am J 64:1382–1388CrossRefGoogle Scholar
  33. 33.
    Delcenserie V, Taminiau B, Delhalle L, Nezer C, Doyen P, Crevecoeur S, Roussey D, Korsak N, Daube G (2014) Microbiota characterization of a Belgian protected designation of origin cheese, Herve cheese, using metagenomic analysis. J Dairy Sci 97:6046–6056CrossRefGoogle Scholar
  34. 34.
    Place RB, Hiestand D, Burri S, Teuber M (2002) Staphylococcus succinus subsp. casei subsp. nov., a dominant isolate from a surface ripened cheese. Syst Appl Microbiol 25:353–359CrossRefGoogle Scholar
  35. 35.
    Randazzo CL, Torriani S, Akkermans ADL, de Vos WM, Vaughan EE (2002) Diversity, dynamics, and activity of bacterial communities during production of an artisanal Sicilian cheese as evaluated by 16S rRNA analysis. Appl Environ Microbiol 68:1882–1892CrossRefGoogle Scholar
  36. 36.
    Rattray FP, Fox PF (1999) Aspects of enzymology and biochemical properties of Brevibacterium linens relevant to cheese ripening: a review. J Dairy Sci 82:891–909CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of Nutrition, Faculty of HealthcareKiryu UniversityMidoriJapan
  2. 2.Division of Molecular Science, Graduate School of Science and TechnologyGunma UniversityKiryuJapan

Personalised recommendations