Introduction

Plastics, a synthetic polymer, are consumed in trillions of amount (about 140 million tons) annually as packaging materials (Caruso 2015; Roy et al. 2008) which are increasing continuously day by day Sekhar et al. 2016). Among plastics, high-density polyethylene (HDPE) is widely used in packaging industry due to its effectiveness and versatility (such as light weight, inexpensiveness, durability, easy processing, etc.) (Rivard et al. 1995; Begum et al. 2015; Witt et al. 2001; Muller et al. 2001). It shows less branching and more linearity than low-density polyethylene (exhibiting random branching resulting in low packing of chains), which provides it a high packing density (Arutchelvi et al. 2008). Polyethylene is xenobiotic in origin and resistant to degradation in nature. Moreover, its hydrophobic character leads to a very slow degradation (Orhan and Büyükgüngör 2000; Hadad et al. 2005). Studies indicate towards no clue of deterioration in polyethylene sheet incubated in moist soil for 12 years (Otake et al. 1995). Polyethylene wastes are normally discarded as landfill or thrown in water bodies to decompose/degrade (Priyanka and Archana 2011). Its accumulation rate is 25 million tons per annum, so its degradation is great challenge to the scientists (Sangale et al. 2012; Orhan and Büyükgüngör 2000).

Recycling of polyethylene is one of the environmentally attractive solutions. Currently, a very small part of the plastics are recycled and the remaining goes to the burial sites (Bhardwaj et al. 2012). Landfills are less in number and are rarely satisfactory, whereas incineration creates highly toxic fumes, causing air pollution (Bhatia et al. 2013). The potential hazardous emissions from incinerating polyethylene include hydrogen chloride, dioxin, cadmium, and fine particulate matter. Moreover, chlorinated plastics can release harmful chemicals into the surrounding soil, which harms organisms by reaching ground water or other surrounding water. However, biodegradation could be the best alternative to combat this environmental pollution by polyethylene (Satlewal et al. 2008; Gu 2003). Biodegradation is bio-chemical in nature in which enzymes released from microorganisms act as a catalyst. So far, the richness of microorganisms able to degrade polyethylene is limited to 17 genera of bacteria and 9 genera of fungi (Restrepo-Flórez et al. 2014). Aerobic metabolism results in carbon dioxide and water (Starnecker and Menner 1996), whereas anaerobic metabolism results in carbon dioxide, water, and methane as the end products, respectively (Gu et al. 2000). The metabolites released after biodegradation are non-toxic to the environment and redistributed through the carbon, nitrogen, and sulfur cycles. Thus, biodegradation does not create environmental pollution.

The microbial degradation of plastics leads to a cleavage of the polymer chain into oligomers and monomers, which is promoted by certain enzymatic activities. Microbial cells absorb these water soluble degradation products for use in their metabolism. Biodegradability is defined as the propensity of a material to breakdown into its constituent molecules by natural biological processes (often microbial digestion). The vulnerability of the polymers to microbial attack generally depends on enzyme availability, availability of a site in the polymers for enzyme attack, enzyme specificity for that polymer, and the presence of coenzyme if required (Reich and Stivala 1971). According to some reports, partial degradation of polyethylene could be achieved after UV irradiation (Cornell et al. 1984) thermal treatment (Awasthi et al. 2017; Albertsson et al.1998) and oxidation with nitric acid (Brown et al. 1974). Thermal or radiation treatments on polyethylene reduce the polymeric chain size and form oxidized groups such as carboxyl, carbonyl, and hydroxyl which are more easily degraded by microorganisms (Albertsson et al. 1995). Oxidized groups modulate the microbial attachment by increasing the surface hydrophilicity (Tribedi and Sil 2013). Therefore, polyethylene degradation will be boosted if a more oxidized surface is used as a substrate (Awasthi et al. 2017). These treatments modify the properties such as crystallinity level and morphological changes of the original polymer, and facilitate the polymer biodegradation (Lee et al. 1997).

Klebsiella pneumonia, a Gm-negative bacterium, has shown importance in waste water treatment (Maal et al. 2014), citrate decomposition (Brynhildsen and Rosswall 1989), nitrogen fixation (Iniguez et al. 2004), and hydrogen production from biodiesel waste containing glycerol (Liu and Fang 2006). Moreover, it qualifies as a suitable microorganism for LDPE degradation (Anbuselvi and Pandey 2015). It has been stated that Klebsiella pneumoniae secretes lipase (Peil et al. 2016), tyrosinase, laccase, and peroxidase enzymes (Dhanve et al. 2008), which are capable of degrading polyethylene via groove formation. In addition, the extracellular polymers released by this microorganism act as surfactants which facilitate the exchanges between hydrophilic and hydrophobic phases. Such exchanges favor the penetration of microbial species into the polyethylene. However, studies on biodegradation of polyethylene, particularly HDPE, by Klebsiella bacterium are scarce. In the present paper, biodegradation study of thermally treated HDPE is described, using an indigenous bacterial stain K. pneumoniae CH001.

Materials and methods

Pretreatment of high-density polyethylene

High-density Polyethylene (HDPE) carry bags, (40 μm in thickness) which are used in cloth packaging, were procured from local market for use in the present study. HDPE strips of 10 × 4.5 cm size were treated thermally in a preheated hot air oven at 70 °C for 10 days to enhance the biodegradation. Thermally pretreated HDPE biodegrades easily as oxidized polyethylene chain leads to the carbonyl group formation. These strips were washed and disinfected with a solution containing (7 ml Tween-80, 10 ml bleach, and 983 ml sterile water). These films were transferred aseptically into 70% (v/v) ethanol solution for 30 min incubation, and dried overnight at 45–50 °C, and weighed before biodegradation.

Screening of bacteria for the biodegradation of HDPE

Isolation and screening of bacteria were done from a plastic waste dumpsite, Diesel Locomotive Works (DLW), Varanasi, India. Bacterial culture was isolated by serial dilution method and all the assays for studying colonization and degradation of HDPE were carried out in Nutrient Broth. The isolates were further screened for their ability to utilize HDPE as a primary C source against polyethylene films of different weight. Furthermore, nutrient agar plates treated separately with increasing concentration of polyethylene were inoculated with loopful culture of overnight grown bacterial strain and were incubated at 30 °C for a week. Bacterial isolate showing the clear zone around their colonies was further screened for their degradation ability in broth (Nutrient broth, NB). Liquid cultures (100 ml) were incubated in flasks (250 ml) on a rotary shaker (120 rpm) at 30 °C for 60 days. Thermally oxidized, sterile polyethylene films incubated in culture medium containing 0.05% sodium azide (to check microbial contamination) were used as control in the present study. The experiment was carried out in triplicates, and mean and standard deviation are reported here.

Biodegradation assay

The biodegradation assay was performed in 250-ml flasks by adding 1000 µl of active bacterium into 100 ml of NB containing HDPE film (NB was prepared by dissolving 1.3-g nutrient broth in 100-ml double distilled water). The assay was performed using (1) NB + bacterium + HDPE as treatment taking NB + HDPE as controls. The flasks were incubated at 30 + 2 °C with continuous shaking (120 rpm) for 2 months. Degraded samples were recovered from the nutrient broth for characterization only after the bacterium had attained its stationary growth phase.

Evaluation of biodegradation

The extent of Biodegradation of HDPE film was done using weight loss, change in pH, mechanical strength, and spectroscopic and microscopic analyses.

Weight loss measurement

The reduction of polymer integrity due to the microbial attack results into weight loss. As biodegradation is a surface phenomenon, so the weight loss is proportional to surface area. To estimate the actual weight loss, the bacteria-laden HDPE was washed with 2% (v/v) aqueous sodium dodecyl sulfate (SDS) solution for 4 h and further washed with distilled water to remove the bacterial biofilm from the HDPE surface (Gilan et al. 2004). Multiple samples were weighed with an accurate five-digit balance (Elico) and average values are reported here. The washed HDPE was placed on a filter paper and dried overnight at 60 °C before weighing.

The weight loss (cf. Fig. 1) was calculated by the formula written below:

Fig. 1
figure 1

Change in residual weight of HDPE with incubation time. The graph shows that there is regular decrease in weight during incubation of HDPE film with K. Pneumoniae. Values represent the average of three independent experiments. Error bars indicate standard deviation (± SD). Statistical significance between the groups was evaluated at p = 0.05 significance level

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$${\text{Percentage of weight loss}} = \frac{{\left( {{\text{Initial weight}} - {\text{Final weight}}} \right)}}{\text{Initial weight}} \times 100.$$

Reduction in mechanical strength

The material testing machine (Model INSTRON 4206) with a crosshead speed of 10 mm/min was used to estimate the mechanical strength of the treated and control HDPE strips at room temperature. Tensile strength of the incubated HDPE films was measured in 10-day interval. A regular pattern of reduction in comparison to control polyethylene films was investigated in 60 days (cf. Fig. 2). The result was reported as average value of three observations. The testing conditions for both treatment and control were maintained at room temperature (35–37 °C) with a relative humidity of 50%.

Fig. 2
figure 2

Change in tensile strength of HDPE with incubation time. Tensile strength of control film remains unchanged in the 60 days, while there is a regular pattern of decrease in tensile strength of incubated films. Values represent the average of three independent experiments. Error bars indicate standard deviation (± SD). Statistical significance between the groups was evaluated at p = 0.05 significance level

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Change in pH

Change in pH of NB was measured by Elico LI614 pH Analyzer at every 15th day for biodegradation study of HDPE. It was observed to change from 7 to 5.7 in 60-day duration (Fig. 3) which confirms biological degradation.

Fig. 3
figure 3

Change in pH of media with incubation time. There was decrease during incubation: at day 0, pH was 7.0, and after 60 days, it was reported 5.7. Values represent the average of three independent experiments. Error bars indicate standard deviation (± SD). Statistical significance between the groups was evaluated at p = 0.05 significance level

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Microscopic analysis of HDPE degradation

Microscopic analysis was performed to examine the degradation of HDPE film by the bacterium K. pneumoniae. HDPE films were recovered from the conditioned media and washed with 2% SDS to remove the adhered organisms, if any. Thereafter, the films were air-dried overnight, and the surface morphology of the microbe-treated or untreated films was examined by scanning electron microscopy (SEM) (ZEISS, EVO18) (Fig. 4a, b) and atomic force microscopy (AFM) (Fig. 5a, b) Oxford INSTRUMENT X-act NT-MDT). The polythene films were cut into small strips, coated with gold, and examined under SEM. In AFM analysis, all images were obtained with a scan speed of 1.0 Hz and a resolution of 512 × 512 pixels.

Fig. 4
figure 4

SEM image of HDPE after a 15 days, b 30 days, c 45 days, and d 60 days. Note It is clear from the SEM images that formation of biofilm on the film surface within 15 days, film gets cracks and holes with the passage of time when incubated with K. Pneumoniae

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Fig. 5
figure 5

AFM of HDPE a Control (untreated) and b after 60-day treatment. Note We can see that the control film is smooth, while after 60 days’ incubation roughness of film increases which confirms the biodegradation of film by K. pneumoniae

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Fourier transform infrared (FTIR) analysis

The Shimadzu 8400S was used to determine the formation of new or disappearance of any functional groups from the sample. FTIR is a decisive and precise method to detect structural changes occurring in polymers due to thermal and biological treatments. Three types of polyethylene samples were analyzed: (i) untreated, (ii) thermally treated, and (iii) thermally treated and then incubated with bacterial strain. It was done using the frequency range of (4000–400 cm−1). HDPE films were affixed directly to the standard infrared sample plates. Spectra of untreated sample were recorded as control and spectra of treated sample were taken after 60 days (Fig. 6).

Fig. 6
figure 6

FTIR of HDPE a control, b thermally treated HDPE, and c thermally treated HDPE after bacterial degradation. Note It is clear in Fig. 6a that there are various groups in the control film in Fig. 6b; carbonyl group appears at 1765 cm−1 which indicates oxidation of film after abiotic treatment, and in Fig. 6c, formation of carboxylic group (3423 cm−1) denotes further oxidation of abiotically treated film after incubation with K. Pneumoniae

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GC–MS

Incubated NB medium of control and treated HDPE film was subjected to GC–MS analysis to analyze the degradation intermediates (Fig. 7) and understand the mechanism of HDPE biodegradation using gas chromatography–mass spectrometer (JEOL GCMATE II GC–MASS SPECTROMETER, JNU, New Delhi). For this, the bacterial pellets were removed by filtration after 2 months of incubation period, and the filtrates were extracted with diethyl ether.

Fig. 7
figure 7

Note It is clear in Fig. 7a that GC–MS of control HDPE film is different from the treated HDPE film which has various monomers, acidic compounds, and oxidized compounds which are present at less retention time after biodegradation

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Statistical analysis

Data were subjected to one-way ANOVA to observe the variation in weight loss, tensile strength, and media pH with incubation time. Post-hoc (Tukey) test (P < 0.05) was performed to observe the significance of difference between control and treated HDPE films in varying days of incubation. Statistical analysis was carried out using the software SPSS-16.

Results and discussion

Characterization of potential strains and phylogenetic analysis

The selection criteria of the bacterial culture were based on effectiveness of polyethylene degradation in nutrient broth media. The isolated bacterial strains were morphologically and biochemically characterized and identified on the basis of 16S rRNA gene sequence analysis. A phylogenetic tree was constructed by software version 8 of Molecular Evolutionary Genetics Analysis (MEGA). The phylogenetic analysis was grounded on BLAST search applying 16S rDNA gene sequence, which demonstrated its maximum homology (100%) with bacterium K. pneumoniae strain AANP1 with gene bank Accession Number: KY494861.1. Based on the cladistic analysis as well as homology valuation, it was concluded that the selected bacterial isolate could be regarded as K. pneumoniae CH001. The sequence of K. pneumoniae strain CH001 has been deposited in NCBI with accession no. MF399051 (http://blast.ncbi.ntm.nih.gov).

Weight loss

ANOVA result indicates that weight loss with incubation time was significant (F = 2152, P < 0.001). Klebsiella pneumoniae CH001 was found to significantly degrade (18.4%) of the thermally pretreated HDPE after 60 days of incubation (Fig. 1). Similar results were also reported by Sudhakar et al. (2008) where thermal pretreatment was found to increase HDPE degradation when incubated with B. sphericus (i.e., 9% against the 3.5% in case of untreated HDPE). Thus, increased weight loss was observed after thermal treatment as compared to untreated HDPE. Similarly, Balasubramanian et al. (2010) also observed a respective loss of 12 and 15% in HDPE weight after 30 days of incubation with Arthrobacter sp. and Pseudomonas sp. Moreover, the HDPE films were found to develop a biofilm on the surface within 15 days of incubation with K. pneumoniae CH001. It might be attributed to the fact that thermal pretreatment oxidizes polyethylene chain which leads to the carbonyl group formation supporting biofilm development on HDPE. The biofilm reduces the hydrophobicity of the polymer, which resulted in the enhancement of the degradation rate in the present study. However, strong adhesion and relatively high survival rate of K. pneumoniae biofilm to the polyethylene surface (unpublished data) might be attributed to the production of protein which enables the formation of a stable biofilm. Such attributes, in spite of the low carbon availability, confirm its efficiency in utilizing polyethylene as a carbon and energy source.

Change in tensile strength

ANOVA result indicates that tensile strength was found to significantly reduce with incubation time (F = 204, P < 0.001). In the present study, a significant reduction in tensile strength (approximately 50%) was observed after 60-day incubation (Fig. 2). Similar results were also reported by Hanaa et al. (1998) and Lee et al. (1991). Nowak et al. (2011) reported that biodegradation reduces the percentage elongation of polyethylene films. Similarly, Jakubowicz et al. (2006) showed a reduced elongation of thermally treated LDPE film after incubation with compost microorganisms.

Change in pH of nutrient media

ANOVA result indicates that pH of the media showed a significant decrease with incubation time (F = 89.5, P < 0.001). We observed a significant reduction in pH of media containing thermally treated HDPE incubated with Klebsiella as compared to control (media not containing Klebsiella) in the present study (Fig. 3). The reduction in pH from 7 to 5.7 proves that the culture is metabolically active in media containing thermally treated HDPE incubated with Klebsiella and is utilizing HDPE as source of carbon for its growth. However, it also indicates towards the production of several monomers post-degradation (Awasthi et al. 2017; Arutchelvi et al. 2008). It has been further confirmed by FTIR and GC–MS analysis (Figs. 6 and 7).

Surface morphology

SEM and AFM

As evident in the SEM micrographs (Fig), we observed some localized degradation of the polyethylene around the bacterial cells in the biofilm in the present study. The bacterial biofilm showed a cell-like molded pattern in the polyethylene. Such shapes have previously been noticed for biodegradable polymers, e.g., poly-β-hydroxybutyrate (Otake et al. 1995). Skariyachan et al. (2015, 2017) observed similar alteration in surface topology for HDPE films that were treated with bacterium via SEM image. The treated HDPE film showed rough surface with a number of cracks and grooves after 60 days of incubation (cf. Fig. 4d). On the contrary, the untreated film retained a smooth surface under the same condition. Similar alterations in the surface topology have also been observed from AFM image analysis (cf. Fig. 5b). It indicates that bacterium K. pneumoniae CH001 secretes enzymes capable of degrading polyethylene, resulting in grooves formation.

FTIR

The changes in bond scission, chemical transformation, and formation and disappearance of new functional groups to determine any changes in the chemical structure of the polyethylene were determined with the help of FTIR (Usha et al. 2011; Suresh et al. 2011). Control spectra of HDPE film, non-treated with bacterium, displayed a number of peaks reflecting the complex nature of the HDPE (Fig. 6a). In the present study, an increase in wavenumber at 1765 was observed for all samples during the thermal pretreatment at 70 °C for 10 days (Fig. 6b). It is consistent with some previous studies (Albertsson et al. 1998; Karlsson and Albertsson 1998; Khabbaz et al. 1998, 1999). These studies reported that ester- and keto-carbonyls are the major products formed during abiotic oxidation of polymer under thermal oxidation or after oxidoreductase activity. Polyethylene samples oxidized when kept in oven for 10 days as indicated by the absorption band around 1765 cm−1.(Figure 6b) It indicates toward Norrish-type II reaction, which lead to the formation of double bonds in the polymer chain. It is assigned to the C=O stretching vibration of a ketone group, which grows in intensity with prolonged aging. However, the FTIR of the thermal-treated HDPE incubated with K. pneumoniae showed considerable changes in the absorption intensities, as compared to the control. The disappearance/or utilization of carbonyl groups (i.e. 1765 cm−1) might be due to the enzymatic attack of K. pneumoniae through Norrish-type mechanism. It is consistent with Dolezel (1967) who observed a decrease in the amount of carbonyl groups with prolonged exposure to a microbial environment. Awasthi et al. (2017); Albertsson et al. (1998); Weiland et al. (1995) also observed a reduction in the carbonyl group after 150 days of incubation with a mixed fungal culture.

The intensity of 1765 cm−1, which determines the extent of degradation, decreased with increase in incubation time in the present study. The decrease in weight complemented the decrease in carbonyl index. Gajendiran et al. 2016; Das and Kumar 2015; Balasubramanian et al. 2010 reported that carbonyl groups once formed (after heat treatment) can further be attacked by microorganisms. It is consistent with observed higher carbonyl and double-bond groups in thermal-pretreated than untreated HDPE samples after exposure to K. pneumoniae. Oxidized polyethylene molecules are hydrolyzed by the extracellular enzymes into fatty acids which are further metabolized by ß-oxidation (Albertsson et al. 1998). It is also consistent with the new absorption bands at 3423 cm−1 (Fig. 6c) observed in the present study due to the formation of carboxylated compound. Bhatia et al. have carried out a similar work with a bacterial consortium, and observed a relative shift in the peaks. These peaks were observed due to the vibrations in the stretching of the O–H bond in alcohols and phenols. An absorbance range of 3500–3200 cm−1 corresponds to the presence of alcohols and phenols (Bhatia et al. 2014).

GC–MS

Incubated NB medium of control and treated HDPE film was subjected to GC–MS analysis to identify the degradation intermediates of HDPE biodegradation. The organic compounds were found as carboxylic acids and alkanes. It is consistent with (Pramila and Ramesh 2015; Mahalakshmi et al. 2012). The carboxylic acids were n-deconic acid, docosanoic acid, undeconic acids, n-deconoic acid, hexadeconoic acid, propanoic acids, oleic acids, oxalic acids, cyclopropanetetradeconic acid, benzene dicarboxylic acid, phthylic acid, octadecatrienoic acid, acetic acid, hexanoic acid, octadeconoic acid, butanoic acid, oxamimidic acid, undecanonic acid, docosanoic acid, tetradecanoic acid, and I propyldodecanoic acid (cf. Fig. 7). Thus, it confirms the production of carboxylic acids as observed in FTIR analysis (cf. Fig. 6a–c). We observed a further degradation of carboxylic acids by K. pneumoniae sp. which produced alkane compounds such as ethane, pentane, decane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, undecane, dodecane, and non-adecane. It indicates towards the cleavage of polymer chain yielding carbonyl radical, which can react with an alkoxy radical on the polyethylene chain (Norrish I-type reaction). It is consistent with the findings of Albertsson et al. (1998) who observed that the long chain of HDPE was cut into small pieces as alkanes and carboxylic acids.

As already noted, a significant amount of low-molecular-weight compounds was released to aqueous media from oxidized polyethylene film. We observed 2-Butene, 2-methyl at 7.2 retention time, Ethene-1,2-dichloro-(2)-1,1 di at 7.6 retention time, acetic acid ethyl ester at 12.71, and Methane, trichloro- at 13.22 retention time. In a similar study, Kounty et al. (2006) observed a similar release of low molecular compounds to water media by Rhodococcus rhodochrous. Konduri et al. (2010) stated that alkanes and carboxylic acids (i.e., ethane, pentane, decane, acetic acid, n-deconic acid, propanoic acids, oleic acids, and oxalic acids) were degraded by A. terreus MF12 by β-oxidation after consumption.

Conclusion

Biodegradation of HDPE has received great deal of attention in recent years, which has limited research so far. Despite use in LDPE degradation, exploration of Klebsiella in HDPE degradation has been little explored. We observed a tremendous efficiency of a novel K. pneumoniae CH001, isolated from a landfill site, as compared to the other microbial species reported for HDPE degradation in the literature. The relatively significant degradation ability of our isolated strain in the present study indicates that microbes might be adapting towards the HDPE use as C and energy source by natural evolution. It suggests that microbes seem to be able to remodel their enzyme-based metabolic pathway for the biodegradation of synthetic HDPE polymers for carbon quenching in the absence of other carbon sources. The biofilm formation on the HDPE surface in the present study was viable and a high number in population strengthens this fact. The limited studies on HDPE biodegradation indicate that this strain could be of potential use in management of plastic waste.