Isolation and Identification of Pseudomonas sp.
Typical colonies of bacterial isolates were sub-cultured on nutrient agar and incubated at 37°C for 24 h. Isolated identification was performed according to the morphological, staining reaction, cultural and various biochemical characteristics by following the Bergey’sMannual of Systematic Bacteriology. Based on presumptive identification of bacteria by Gram’s staining protocol Pseudomonas sp. was identified as gram negative rod. Depending upon the different biochemical characterization the isolated bacteria was identified as P. aeruginosa strain (Fig. 1). It was further confirmed by streaking the strain onto the Cetrimide agar which is selective media for isolating P.aeruginosa. The isolates were showed green pigmented, circular and opaque colony morphology after incubation. Similar to ours study, the another author reported that the isolated bacteria were also grown in milk agar with cetrimide for the preliminary detection of Pseudomonas spp.(Szitaetal, 1998). Based on the results it was reasonable, to identify the isolated bacterium as P. aeruginosa. This identified strain was used for the biodegradation of pre-treated polypropylene mask pieces. The bacterium P.aeruginosa isolated from Sisdol land fill site and Sanothimi household garbge site soil in Nepal has similar biochemical properties(Badahitet al, 2018). It has been reported that P. aeruginosa ISJ14 showed tremendous efficiency to degrade low density polyethylene (LDPE) in BHM(Gupta and Devi, 2020).In contrast, the present study reports for the first time that P.aeruginosastrain is capable of biodegradation of surgical face mask made up of hardy PP like substance.
Cell surface hydrophobicity of bacteria
The capability of bacteria to use any substrate depends upon its development on and adherence to that substrate. The adhesion ability of bacteria to either hydrophilic or hydrophobic surfaces is addressed by a number of physical factors, together with the forces which help the bacterium to adhere to solid substrates, properties of that substrate and the nature of bacteria. For the most part, a hydrophobic bacterium favors a hydrophobic surface for adhesion, though the inverse is substantial for bacterium with hydrophilic properties(Gupta and Devi, 2020). In the current investigation, the hydrophobicity of mid- log phase cells of P.aeruginosa at 0.2ml concentration of xylene, we showed a significance increase in hydrophobicity (30.39%) (Table 1 and Fig. 2). These results are in agreement with Gupta and Devi (2020), who observed that bacterial cells in log phase are more hydrophobic in nature. Previous study also documented similar findings, where, maximum increase in hydrophobicity i.e, the isolates KocuriapalustrisM16 andBacillus subtilis H1584 showed approximately 24% turbidity reduction at 0.25 µl and a maximum reduction of turbidity 32% at 150µl concentration of hydrocarbon like hexadecane(Harshvardhan and Jha, 2013). Another recent research also reported that the hydrophobobicity of L.monocytogenes strain CICC 21332 showed lowest hydrophobicity (12.5%) and the strain FSIS 57034 displayed highest percentage of CSH (74.81%) at 1ml of xylene concentration(Fun et al, 2020).
Table-1. Cell surface Hydrophobicity of P.aeruginosa
S. No | Conc. Of Xylene | OD value | %Hydrophobicity |
01 | 0 | 1.02 | 0 |
02 | 0.05 | 0.89 | 12.74 |
03 | 0.10 | 0.82 | 19.61 |
04 | 0.15 | 0.76 | 25.49 |
05 | 0.20 | 0.71 | 30.39 |
Growth of P.aeruginosa planktonic cells and surface attached cells on PP films
The growth pattern of bacteria attached on the surface of PP film was examined in planktonic cells and viable count of surface attached bacterial species (Arkatkaret al, 2010; Andes et al, 2004). The data illustrated in Figures 3 and 4 show a surface attachment pattern of bacteria on the PP film. The bacterial cell growth was characterized by a precipitous raise in planktonic cells after 15 and 30 days of incubation and is also revealed by a raise in the surface-attached bacterial mass. After 15-20 days of incubation the growth of P.aeruginosa was able to reach a steady, almost 107 CFU/ml in all the liquid media used. In all the three media, biofilm formation patterns exhibited analogous to that of the growth of planktonic cells. The results recommend that P.aeruginosa cells exhibit better colonization, formation of biofilm and fractional biodegradation of PP film in all the three media. These observations not only specify high affinity of P.aeruginosa cells for the PP film but also increase the possibilities of P.aeruginosa cultures to form biofilms by hydrophobic interactions at low levels of carbon availability. In considering of concurrence model, the course of action of biofilm formation by microbes is began when the planktonic growth of cells achieves high density and help for the attachment of bacterial cells to a surface through cell signaling, likewise bringing about the development of microcolonies that will eventually frame the mature biofilms (Costertonet al, 1999). This biofilm population is diverse and comparativelysteadyintendedforextensiveperiod of time (Bodtkeret al, 2008). Conversely, this circumstance is not inevitably familiar to the entire biofilm forming bacterial species. In our investigation, PP films provided as a substrate for the attachment and biofilms formation as well as a source of carbon for P.aeruginosa strain. The extended incubation of P.aeruginosa with PP films resulted in a solid biofilm on PP surface which may lead to the fractional deduction of this polymer. Likewise, the formation and maintenance of active biofilm throughout the 30 days of incubation possibly will perhaps by the utilization of low molecular massmaterials in the polymer. Similar findings were recorded in earlier studies such as biodegradation of untreated films of polyethylene by P.putita IRN22, Micrococcus luteus IRN20, Acinetobacterpittii IRN19 (Montaveret al, 2019) and other bacterial genera including Delftia, Stenotrophomonas and Comamonas(Peixotoet al, 2017) and Galleria melonella isolated from the gut of the wax worm also have been establish the capabilities of PE degradation (Cassoneet al, 2020).
Table-2. Viability of P.aeruginosa on different day interval
S.No | Days | Cfu/ml in NB(10−7dilution) | Cfu/ml in BHM (10−7dilution) | Cfu/ml in MSM (10−7dilution) |
1 | 0 | 1.07×105 | 0.8×105 | 0.92×105 |
2 | 10 | 2.01×106 | 1.9×106 | 1.92×106 |
3 | 20 | 2.02×107 | 1.95×107 | 1.99×107 |
4 | 30 | 1.97×107 | 1.98×107 | 1.98×107 |
Table-3.Monitoring the planktonic growth of P.aeruginosa (log cfu/ml)
Media | 0 DAY | 15TH DAY | 30TH DAY |
NB | 0.9×105 | 2.0×105 | 2.08×105 |
BHM | 1.1×107 | 1.9×107 | 2.07×107 |
MSM | 1.2×107 | 2.0×107 | 2.12×107 |
Dry Weight Determination of recovered Polypropylene Mask pieces
The residual polypropylene mask strips were recovered from the media after one month (30 days) of incubation. The adhered media and bacterial biomass were washed with appropriate solutions and allowed to air dry. The air dried films were weighed and the final weight loss for P.aeruginosa in respective culture media (NA, NB, BHM agar, BHM broth, MSM agar and MSM broth) has been provided in table 4 and Fig. 5a and 5b. The growth kinetics of the P.aeruginosastrain in media demonstrated its colonization on PP mask pieces surface consequently, reduction in weight was observed as a result of utilization of pp mask film as nutrient source. Our study describes the potency of the P.aeruginosastrain was very high because it shows 26 and 33 % of weight reduction in case of PP films placed in NB and NA, 28 and 31% weight reduction observed in BHM agar and BHM broth and 24 and 23% weight reduction was noted in MSM agar and MSM broth after one month duration (30 days). Similar findings were reported by several other researchers on LDPE surface (Kapriet al, 2010; Shah et al, 2015; Sahet al, 2010). Whereas, the LDPE film treated by P.aeruginosastrain showed 20% reduction after 120 days of treatment also has been reported (Kyawet al, 2012). Several other researchers also reported the same findings on LDPE surface (Kapriet al, 2010; Shah et al, 2015; Sahet al, 2010). Whereas, the LDPE film treated by P.aeruginosastrain showed 20% reduction after 120 days of treatment also has been reported (Kyawet al, 2012). However, the recent study provide strong evidence for PP microplastic degradation by Rhodococcus sp. strain 36 with 6.4% degradation and Bacillus sp. strain 27 with 4% also depicted the good degradation capacity of bacterial strains (Autaet al, 2018).
Table-4. Weight reduction of PP mask pieces after 30 days treatment with P.aeruginosa on different media sources
Media sources | Initial weight (g) | Final weight(g) | % Weight reduction |
Control | Treated | Control | Treated | Control | Treated |
Nutrient agar | 0.1 | 0.1 | 0.1 | 0.074 | 0.0 | 26 |
Nutrient broth | 0.1 | 0.1 | 0.1 | 0.067 | 0.0 | 33 |
BHM agar | 0.1 | 0.1 | 0.1 | 0.072 | 0.0 | 28 |
BHM broth | 0.1 | 0.1 | 0.1 | 0.069 | 0.0 | 31 |
MSM agar | 0.1 | 0.1 | 0.1 | 0.076 | 0.0 | 24 |
MSM broth | 0.1 | 0.1 | 0.1 | 0.077 | 0.0 | 23 |
Surface Changes Analysis
The surface morphology changes on the PP mask pieces before and after biotic exposure was investigated with the help of Field- Emission Scanning Electron Microscope (FE-SEM). The Fig. 6a, 6b and 6cshowed that the surface changes recorded during FE-SEM analysis both the PP mask films treated with P.aeruginosastrain(T1A, T1B, T2A, T2B, T3A and T3B)and untreated negative control(C1A, C1B, C2A, C2B, C3A and C3B) after 30 days of treatment. It was examined that signs of surface deterioration appeared on the PP mask films treated withP.aeruginosastrain after 30 days of incubation. On the other hand, the control film (un-treated with P.aeruginosastrain) keep hold of a smooth surface under same incubation condition was noted. Similar findings were reported in surface morphology of the LDPE films treated with Pseudomonas spp. by SEM after 40, 80 and 120 days of incubation (Kyawet al, 2012).The another study also reported that the P.aeruginosa ISJ14 used to treat LDPE film showed maximum deterioration after 60 days of treatment when observed under the FE-SEM (Gupta and Devi, 2020). A cross reference to the earlier research studies on LDPE biodegradation, many authors have reported the similar morphological changes on LDPE degradation by Aspergillusspp. (Zahra et al, 2010).A.clavatus JASK1 (Gajendiranetal, 2016).
Structural Analysis using FTIR
Structural changes in biologically treated PP mask films were further analyzed with the help of FTIR.This investigation of the degraded PP films had shown the stretching of numerous functional groups after incubation with the P.aeruginosa strain. This was established by the alterations in the peaks of the FTIR spectra amidst the control and test samples in all the media used. Table 5a, 5b and 5c recapitulate the sort of functional group implicated in stretching by the role of P.aeruginosa, the wave number and IR band position on the PP films.A considerable reduction in the Carbonyl Index was observed on the samples incubated with P.aeruginosastrain for 30 days. The drop in CI with respect to the negative control was showed in Fig. 7a-7l. In our study, we observed maximum reduction in CI on PP mask filmsincubated with P.aeruginosastrain for 30 days. Spectrophotometric variations ofPP mask films and the value of CI which determines the maximumdegradation when compared to un-treated negative control. Thus, our results suggested that theP.aeruginosastrain has great ability to degrade the PP mask films. The LDPE film incubated with P.aeruginosa PAO1 showed maximum reduction in CI was reported by (Kyawet al, 2012; Skariyachanetal, 2015). In the present study, FTIR analysis affords a close view of N-H stretching of aldehydes group at 3190.18 cm-1. The C-C absorption peaks were shifted as evident at 1255.18, 1302.34, 1794.87, 2427.49 and 2617.26 cm-1. A similar observation was reported by several authors (Howard and Hilliard, 1999). Our results were supported by various previous research studies noticed the formation of functional groups and disappearance of these groups in the LDPE degradation using the strainBacillus amyloliquefaciens(Das etal, 2015). In relation to our study, (Gajendiranet al, 2016) have also noticed visible modifications in the synthetic polymers undergo biodegradation, before and after exposure to microbes by FTIR analysis. The conformational changes on PP mask film were supported by the changes in the peak values of almost all functional groups (Fig .7a-7l).
Table-5a. Comparison of IR band position in the PP films after incubation with P.aeruginosa in NB and NA(both control and test)
S.No | Incubation period | IR band position in BHM broth control-test (cm−1) | IR band position in BHM Agar control-test (cm−1) | Functional group involved |
1 | 30 days | 572.18 - 460.23 | 1002.24 – 808.93 | C-X stretching |
2 | 2950.10 – 2838.92 | 2918.97 – 2917.61 | C-H stretching |
3 | 2916.61 – 2722.13 | 2947.73 – 2838.92 | C-H stretching |
4 | 3762.08 - 3189.68 | 3761.52 - 3189.68 | O-H stretching |
Table-5b. Comparison of IR band position in the PPfilms after incubation with P.aeruginosa in BHM broth and BHM agar (both control and test)
S.No | Incubation period | IR band position in BHM broth control-test (cm−1) | IR band position in BHM Agar control-test (cm−1) | Functional group involved |
1 | 30 days | 572.18 - 460.23 | 1002.24 – 808.93 | C-X stretching |
2 | 2950.10 – 2838.92 | 2918.97 – 2917.61 | C-H stretching |
3 | 2916.61 – 2722.13 | 2947.73 – 2838.92 | C-H stretching |
4 | 3762.08 - 3189.68 | 3761.52 - 3189.68 | O-H stretching |
Table-5c. Comparison of IR band position in the PPfilms after incubation with P.aeruginosa in MSM broth and MSM agar (both control and test)
S.No | Incubation period | IR band position in MSM broth control-test (cm−1) | IR band position in MSM Agar control-test (cm−1) | Functional group involved |
1 | 30 days | 562.12 – 458.98 | 460.00 – 459.35 | C-X stretching |
2 | 2839.02 – 2722.15 | 2839.57 – 2838.65 | C-H stretching |
3 | 2916.76 – 2838.83 | 2918.00 – 2917.14 | C-H stretching |
4 | 3760.91 - 3346.85 | 3351.15 - 3190.32 | O-H stretching |