Introduction

The production and enormous use of polymers like polyurethanes (PU) is a growing threat to the environment due to unabated dumping. The environmental pollution and xenobiotic nature of these polymers is always a worldwide problem (Pathirana and Seal 1984; Bentham et al. 1987; Kay et al. 1991). Apart from waste treatment, PU-degrading bacteria are important in maintenance of PU-based paint formulations and in evolving protective technologies (Halim-El-Sayed et al. 1996). Therefore, exploration of environmental isolates for solid waste treatment and analysis of their behavior towards PU-based paints and coatings may prove to be useful in issues related to environmental management and material maintenance. In view of the requirement for a defined PU-degrading community, it appears desirable to isolate microorganisms from appropriate ecological niches.

Polyester polyurethane is more susceptible to microbial attack as compared to polyether types of polyurethanes. Several reports have appeared in the literature on the susceptibility of polyurethanes to fungal attack (Ossefort and Testroet 1966; Darby and Kaplan 1968; Kaplan et al. 1968). Certain types of polyurethanes are reportedly susceptible to degradation by soil fungi such as Curvularia sengalensis, Fusarium solanii, Aureobasidium pullulans and Cladosporium sp. (Crabbe et al. 1994). The bacterial species known to attack polyurethanes include Comamonas acidovorans, Pseudomonas fluorescens, P. chlororaphis, and Bacillus subtilis, (Nakajima-Kambe et al. 1995, 1997; Howard and Blake 1998; Ruiz et al. 1999; Howard et al. 1999, 2001b; Rowe & Howard 2002). The management of PU solid waste requires efficient bacteria from various ecological niches having PU-utilizing ability which may be explored effectively in monoculture or consortia for solid waste treatment.

Therefore, this investigation describes isolation of polyurethane-utilizing B. pumilus, from a river receiving industrial waste. The isolate has been characterized by conventional biochemical and advanced molecular biology techniques. The polyurethane-degrading ability has been confirmed using a sensitive Rhodamine-B plate assay incorporating water-dispersible polyurethane (Impranil DLN) in the growth medium. The isolate was also screened for lipase and esterase activity in an Olive oil-Rhodamine-B plate assay.

Materials and methods

Isolation of PU-utilizing bacteria

Various PU contaminated water and soil samples were collected from sites receiving industrial wastes. Samples were separately inoculated into nutrient broth and incubated on a shaker incubator at 30°C for 6–8 h and cultures showing growth were plated on nutrient agar after serial dilution in sterile saline (0.89%, w/v NaCl) to obtain isolated colonies (Seeley and Wandermark 1962). Later each colony type was marked and carefully subcultured initially on medium containing 0.05% (w/v) water-dispersible polyurethane in yeast extract salts (YES) media (Crabbe et al. 1994) followed by serial dilution and plating on Luria-Bertani (LB) agar plates. These plates were incubated at 30°C for 18 h and later observed for growth to screen PU-utilizing isolates on LB-PU agar plates.

Identification of bacteria

All isolates growing on nutrient agar plates after preliminary isolation were identified by conventional microbiological and biochemical techniques as described in Bergey’s Manual of Systematic Bacteriology. The isolated bacterial cultures were gram stained and observed under the light microscope (Carl Zeiss).

Growth on Yeast extract-supplemented medium

Bacterial cultures of environmental isolates and a type culture, B. subtilis (MTCC 2423, Microbial Type Collection Centre, Chandigarh India) were grown in minimal media containing 0.3% (w/v) water-dispersible PU Impranil DLN, supplemented with yeast extract (Crabbe et al. 1994) as well as minimal media devoid of yeast extract. In brief, a 500-ml Erlenmeyer flask containing 100 ml minimal media was inoculated with bacterial culture and incubated at 30°C on a rotary (150 rev min−1) shaker incubator (Orbitek, India). The growth of isolates NMSN-1a, NMSN-1d and type culture B. subtilis (MTCC 2423) was determined on intervals of 0, 1, 15 and 45 days, by surface spreading of an aliquot of appropriately diluted culture on nutrient agar plate and monitoring colony forming units (c.f.u.) per ml.

Growth kinetics of NMSN-1d in Water-dispersible PU Impranil DLN

The growth kinetics of NMSN-1d in minimal media was determined using varying concentration of water-dispersible PU Impranil DLN (1–4 g l−1) with and without yeast extract separately. Minimal media was prepared as mentioned above (Crabbe et al. 1994) and supplemented using yeast extract (0.02 g l−1). The growth of NMSN-1d was monitored at 30°C on a shaker incubator (150 rev min−1). The growth was observed on regular intervals of 0, 4, 8, 12, 24, 36, 48, 60 and 72 h. The data was expressed as mean c.f.u. ml−1 ± standard error (\(\bar{\rm X}\) ± SE).

Plate assay for PU utilization and enzyme production

Both NMSN-1a and NMSN-1d isolates were streaked on LB -PU (0.3% w/v) agar plates and differentiated for PU utilization by formation of zones of clearance along the streak. The plates were incubated at 37°C for 18–20 h. Cultures showing zones of clearance around the colonies were considered positive for PU utilization (Vega et al. 1999). The environmental isolates were grown in PU-YES medium and the cultures were assessed for their ability to produce polyurethanase in a separate plate assay (Howard et al. 2001a). The plates were incubated 18–20 h at 37°C and considered positive for polyurethanase activity if formation of an orange fluorescent halo around the wells was observed on u.v. irradiation (Howard et al. 2001a).

For lipase assay, 2 × YT plates containing olive oil (1% v/v) and Rhodamine B (0.001% w/v) were prepared and bacterial isolates were streaked and incubated at 37°C for up to 72 h. Formation of an orange fluorescent halo around the colonies under UV irradiation was considered positive for enzyme activity (Kouker and Jaeger 1987). For esterase activity, Tween 20-Tributyrin agar plates were prepared (Tween 20, 1% v/v). Production of esterase was checked by formation of powdery deposits around the colonies (Sommer et al. 1997).

PCR amplification of 16S rRNA gene

Genomic DNA from all isolates was extracted using phenol:chloroform extraction procedure (Marmur 1961). The 16S rRNA gene was PCR amplified using Universal primers (Weisberg et al. 1991; Pavitran et al. 2004) on a thermal cycler (UNO II Biometra, Germany). The PCR mixture contained 5 μl of 10× PCR buffer, 4 μl of 25 mM MgCl2, 1 μl of 10 mM dNTP’s, 100 pmol of each primer (0.5 μl), 1 μg of template DNA and the reaction volume was made up to 50 μl with sterile distilled water (Sambrook et al. 1989). The hot start PCR was performed at initial denaturation of 5 min at 94°C followed by addition of 1U, Taq DNA polymerase (Cat #EP0402, MBI Fermentas, USA). Subsequently, 30 cycles had thermal profile of denaturation at 94°C for 60 s, annealing at 52°C for 1.5 min and polymerization at 72°C for 1.5 min. The final extension was at 72°C for 7 min. The PCR products were resolved on agarose gel electrophoresis (1% w/v, Sigma Cat #A9539) and visualized after staining with ethidium bromide on a UV transilluminator (Biometra, Germany).

16S rRNA gene - HaeIII restriction profile

Amplified products were gel eluted using a MinElute gel Extraction kit (Cat.No.28604, Qiagen, Germany). The concentration of purified PCR product was determined spectrophotometrically at 260 nm (Model UV-1201 Shimadzu, Japan). The amplified 16S rRNA genes were digested with HaeIII (MBI Fermentas, USA) at 37°C for overnight. Type cultures B. subtilis (MTCC 2423), and B. pumilus (NCIM 2327, National Collection of Industrial Microorganisms, India), were also included for comparison. The restriction profile of these isolates along with type cultures has been generated on agarose gel and their phylogenetic relatedness has been analysed using PD Quest software (Discovery Series, BioRad, USA).

Dendrogram construction

The RFLP (Restriction Fragment Length Polymorphism) pattern obtained from restriction digestion of 16S rRNA gene using HaeIII, has been used to deduce the genetic relatedness of the environmental isolates as determined by comparison of the banding pattern of the digested 16S rRNA gene. A matrix containing similarity values was obtained with the Dice coefficient. The phylogenetic tree was constructed using a Diversity Database Software (Bio-Rad, USA). A dendrogram was constructed according to the unweighted-pair group method, using arithmetic average (UPGMA) cluster analysis.

Sequencing of the 16S rRNA gene

The purified 16S rRNA gene of four isolates (1a, 3a, 3c & 1d,) was sequenced using ABI prism dye terminator cycle sequencing ready reaction kit and ABI cycle sequencer (Applied Biosystems /Perkin Elmer). DNA sequencing was performed by using M13 forward and reverse primers. The sequences obtained were analysed on NCBI, BLAST database to determine relatedness of each bacterial isolate (Altschul et al. 1990).

Results

Isolation of PU-degrading bacteria

Three types of samples (soil, water and buried PU foam) were collected from polluted sites for successful isolation of polyurethane-utilizing bacteria. All these samples when inoculated on conventional nutrient media indicated substantial growth and nine different types of colonies were observed on solid medium. However, only two colony types were found growing on minimal broth containing 0.05% PU without yeast extract, whereas PU-minimal media supplemented with yeast extract supported growth of seven isolates. For comparison, a type culture of B. subtilis (MTCC 2423) was also included in the study. Environmental isolates NMSN-1d & NMSN 3c, as well as type culture of B. subtilis (MTCC 2423) showed growth with or without yeast extract in minimal broth containing 0.3 %(w/v), PU as sole source of carbon and energy. The bacterial growth of NMSN-1a, NMSN-1d and type culture B.subtilis (MTCC 2423) in YES-PU media for 45 days was observed (Table 1).

Table 1 Growth of NMSN-1a & NMSN-1d environmental isolates and B. subtilis (MTCC2423) type culture after enrichment for 45 days in YES-PU media
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Characterization of bacterial isolates

All nine cultures growing on nutrient broth media were obtained as single colonies on fresh nutrient agar plates and subjected to biochemical characterization according to Bergey’s Manual of Systematic Bacteriology. In biochemical tests, five bacterial isolates NMSN-1d, NMSN-2a, NMSN-3a, NMSN-3b, NMSN-3c were found resembling the genus Bacillus. Isolate NMSN-1b belonged to the genus Pseudomonas, isolate NMSN-1c to the genus Acinetobacter and isolate NMSN-2b was determined as E. coli. Biochemical tests in the case of NMSN-1a isolate remained inconclusive. Catalase and oxidase were positive for all isolates, whereas gelatinase, urease, amylase, esterase and lipase production indicated variation. Isolates NMSN-1d, NMSN-2a & NMSN-3c showed lipase activity. Isolates NMSN-1b, NMSN-1c, NMSN-2b, NMSN-3a & NMSN-3b were positive for esterase (data not shown) as evidenced by plate assays, however NMSN-1a was negative for both the enzymes. Isolates (NMSN- 1a, NMSN-1d, NMSN-3b & NMSN-3c) were found to grow in high salt concentration (10% w/v), of these three represented Bacillus sp.

Growth on PU plates and polyurethane degradation

Bacillus pumilus (NMSN-1d) showed good growth on LB agar plates containing water-dispersible PU as well on YES-PU agar plates. This isolate was found to show clearing zone around colonies (Fig. 1b) indicating PU utilization within 12 h, as compared to an unrelated environmental isolate NMSN-1a, obtained from the same environment (polluted water sample, Fig. 1a). The presence of a polyurethanolytic enzyme in PU degrader (NMSN-1d) has been checked by incubating PU-grown culture lysate and culture supernatant in punched holes of PU-Rhodamine-B agar plates. NMSN-1d indicated production of extracellular enzyme, as indicated by formation of an orange fluorescent halo around the well charged with culture supernatant (Fig. 2).

Fig. 1
figure 1

Bacteria utilizing water-dispersible polyurethane showing zone of clearance around culture streak or colonies on LB-PU plate (Impranil DLN 0.3% w/v) (a) B. pumilus NMSN-1d culture showing zone of clearance around streak and Exiguobacterium sp. NMSN-1a no zone of clearance. (b) Isolated colonies of B. pumilus NMSN-1d showing zone of clearance

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Fig. 2
figure 2

Bacillus pumilus NMSN-1d culture filtrate showing a fluorescent halo around wells indicating extracellular polyurethanase activity (e) as compared to Exiguobacterium sp. NMSN-1a showing no activity (e, i)

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Effect of yeast extract on growth kinetics of NMSN-1d in minimal medium containing PU-Impranil DLN

The effect of yeast extract has been assessed on the growth kinetics of NMSN-1d in the presence of different concentrations of PU. The lag phase appeared to be very short in presence of yeast extract as compared to that of without yeast extract (<4 h). The log phase was found to be 24 h in the presence or absence of yeast extract. After 24 h the growth came to the stationary phase both in the presence or absence of yeast extract. However the total viable count remained almost 1 log higher in presence of yeast extract than in its absence (Tables 2, 3).

Table 2 Growth of Bacillus pumilus NMSN-1d in varying concentration of PU (Impranil DLN) in the presence of yeast extract at different time intervals
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Table 3 Growth of NMSN-1d in Minimal medium-PU in the absence of yeast extract with varying concentration of Impranil DLN
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Lipase and esterase activity

Bacillus pumilus (NMSN-1d), showed lipase activity on 2×YT-olive oil plate as orange fluorescent halo around the colonies under UV illumination (Fig. 3) indicating the production of a true lipase. The type culture B. subtilis (MTCC 2423) was found to produce esterase, as indicated by a powdered zone around the colonies in Tween 20-agar plates, whereas isolate NMSN-1d and NMSN-1a showed no esterase activity (Fig. 4).

Fig. 3
figure 3

Lipase activity shown by (a) NMSN-1d as indicated by formation of orange fluorescence halo around the wells charged with culture, no orange fluorescence halo around (b) B.subtilis (MTCC 2423) and (c) Exiguobacterium sp. NMSN-1a on 2×YT-Olive oil—Rhodamine B agar plates

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Fig. 4
figure 4

Esterase activity shown by B. subtilis (MTCC 2423) as indicated by a powdery zone around the colonies (b), whereas no powdery zone around B. pumilus NMSN-1d (a) & Exiguobacterium sp. NMSN- 1a (c) on Tween 20-Tributyrin agar plates

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PCR amplification of 16S rRNA gene and HaeIII restriction profile

A 1.5 kb of 16S rRNA gene was amplified by PCR for all nine bacterial isolates (Fig 5a). The restriction profile for all these isolates was obtained using HaeIII (5′GG↓CC3′), a blunt cutter, to determine its close relationship with other members of genus Bacillus (Fig. 5b). Type cultures of B. subtilis (MTCC 2423) and B. pumilus (NCIM 2327), were found to give four restriction fragments (600, 450, 300 and 120 bps). Therefore, environmental isolates NMSN- 1d, NMSN-2a, NMSN-3a & NMSN-3c, were considered closely related strains of B. subtilis and B. pumilus in view of the similar pattern of banding, the RE profile of the non-PU degrader NMSN-3b also resembled Bacillus species (Fig. 5b, lanes 1–6 & lanes 7–8; 5c, lane 3). Relatedness of the other isolates could not be ascertained in the present study (Fig. 5c, lane 2, 3, 5, & 6).

Fig. 5
figure 5

PCR amplification of 1.5 kb 16S rRNA gene as observed in (a) lane 1 to 9 for various isolates NMSN-1a, 1b, 1c, 1d, 2a, 2b, 3a, 3b, & 3c. (b) RFLP pattern of amplified 16SrRNA gene of B. subtilis strain 168 (lane1), PU enriched B. subtilis 2423 (lane2), NMSN-2a (lane3), B. subtilis MTCC 2423 (lane 4), B. pumilus, NCIM 2327 (lane 5), NMSN-1d (lane 6), Unknown isolate (lane U), NMSN-3a (lane7), NMSN-3c (lane8), NMSN-1a (lane9). (c) RFLP pattern of NMSN-1b (lane1), NMSN-1a (lane2), NMSN-3b (lane3), NMSN-2b (lane4), NMSN-1c (lane5), Pseudomonas putida, MTCC 2492 (lane6). Lane M, is 100 bp DNA ladder

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16S rRNA gene sequencing

16S rRNA gene of NMSN-1a, NMSN-1d, NMSN-3a and NMSN-3c isolates were sequenced and BLAST analysis was done through Genbank (http://www.ncbi.nlm.nih.gov/ BLAST). BLAST analysis indicated NMSN-1d (GenBank: EF070205) to be closely related to B. pumilus c-10 strain (99% homology). Similarly isolate NMSN-3c, was also found to be closely related to B. pumilus. The-non PU degrader isolate NMSN-3a was considered to be a B. cereus and NMSN-1a (GenBank:EF070206) was found to be an extremophile Exiguobacterium sp.

Discussion

The aim of this study was to isolate potential PU-utilizing bacteria by exploring ecological niches receiving industrial waste. We describe here isolation of nine environmental bacteria, of which two B. pumilus strains (NMSN-1d & 3c) were found to grow in presence of water-dispersible PU. The strain NMSN-1d was found to show poly-urethanolytic activity within 12–14 h, the other strain NMSN-3c showed a delayed effect in a PU plate assay. In view of its relative superiority, isolate NMSN-1d was considered a promising strain and characterized further.

It is considered that PU-attacking bacteria first bind to PU surfaces in the same way as biofilm-forming bacteria, thereby facilitating its bioavailability for further bacterial attack (Blake et al. 1998; Ribeiro et al. 2005). In this study a B. pumilus (NMSN-1d) was isolated from PU foam floating on water. Potential PU degraders were isolated in minimal media without yeast extract where B. pumilus NMSN-1d showed optimum growth, whereas yeast extract supplementation showed an inducing effect on PU-degrading bacteria. Further, the same has been substantiated in growth kinetics experiments where presence of yeast extract resulted in a shorter lag phase and an earlier log phase as compared to cultures without yeast extract supplementation. The overall effect as observed was found to give a relatively higher bacterial count in presence of yeast extract. In view of these observations, yeast extract supplementation is considered to have inducing effect on PU-degrading bacteria. Similar observations have been reported by other investigators with Corynebacterium sp. and P. aeruginosa (Kay et al. 1991, 1993). The growth-enhancing effect of yeast extract is considered to be due to its surface-active and nutritive properties that facilitate bioavailability of nutrients and target substrate (Kay et al. 1991).

All the isolates were characterized first by conventional biochemical techniques and were further characterized by PCR amplification of 16S rRNA gene followed by its RFLP analysis using a blunt cutter restriction endonuclease HaeIII (BsuRI). The dendrogram based on RFLP data has been generated to ensure genetic relatedness of the isolates. The UPGMA-based dendrogram (Fig. 6), indicated two major clusters, one of B. cereus NMSN-3a, the other of B. subtilis (MTCC 2423). The B. cereus NMSN-3a cluster showed distant genetic relatedness (dice coefficient 0.48) to the B. subtilis cluster. The potential PU degrader isolate NMSN-1d clustered with type culture of B. pumilus (NCIM 2327), within the B. subtilis cluster, showing its close genetic relationship to B. pumilus (dice coefficient 0.9). The other isolate NMSN-3c, which showed slow growth and delayed enzyme production, also clustered with B. subtilis (MTCC 2423) indicating genetic relatedness (dice coefficient 0.85) however, both are closely related to B. pumilus (dice coefficient 0.85). It is considered that many strains of B. pumilus and B. subtilis cannot be differentiated in single restriction enzyme RFLP analysis. In RFLP analysis, the NMSN-3c isolate could not be differentiated clearly, therefore, the 16S rRNA gene of this culture along with other isolates has been subjected to sequencing. The sequence data confirmed that the promising PU-utilizing isolate NMSN-1d is closely related to B. pumilus strain c10 (gb/AY373359.1), whereas the other culture of Bacillus sp. (NMSN-3c) is considered to be related to B. species (strains 41KF2b and ROO40B) which appear to be closely related to B. pumilus.

Fig. 6
figure 6

Phylogenetic tree showing the genetic relatedness of NMSN-1d and NMSN-3c with type cultures

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In this study degradation of PU has been confirmed in a sensitive plate assay. Degradation of PU and olive oil was evident in the Rhodamine-B plate assay, where utilization of these substrates is considered to be an effect of bacterial lipases (Kouker and Jaegar 1987). Susceptibility of polyurethane to microbial attack appears to be the effect of lipase- and esterase-like enzymes, as has been reported elsewhere (Howard 2002). In a separate study PU degradation is reported to be a synergistic effect of both exo- and endo-polyurethanases (Wales and Sagar 1988). B. pumilus NMSN-1d, found to degrade Impranil DLN at a concentration of 3 g−l proves to be a very active polyurethanase-like enzyme-producing strain that may find application in polyurethane waste treatment.

Utilization of PU by bacteria is considered to be mediated either by lipases, esterases or proteases as reported in various gram positive and gram-negative bacteria (Akutsu et al. 1998; Howard and Blake 1998; Ruiz et al. 1999; Allen et al. 1999; Stern and Howard 2000; Rowe and Howard 2002; Howard et al. 2002). In agreement with these investigators our results indicated that PU degradation in B. pumilus (NMSN-1d), appears to be mediated by true lipases.

Attempts are being made to ascertain the extent of degradation by FTIR and GC-MS analysis of exposed PU and characterization of enzymes involved in degradation.