Abstract
Pluralibacter gergoviae TYB1 is a facultative anaerobic bacterium with a high efficiency of low-density polyethylene (LDPE) degradation using self-producing surface-active compounds (SACs). This bacterium was used for the degradation of LDPE. In this study, analysis was performed to check the SAC production efficiency of P. gergoviae TYB1 by drop collapse assay, oil displacement test, emulsification index, and foamability testing. RSM was performed to scale up the production of SAC for its further utilization for experimental purposes to improve the biofilm formation of P. gergoviae TYB1 on LDPE. SAC extracted from the broth supernatant was subjected to FT-IR, TLC, and GC-MS analyses. Within 30 days of LDPE treatment along with bacteria and SAC, bacterial SAC improved biofilm formation and biodegradation of the LDPE film by P. gergoviae TYB1. The results were showed decreased hydrophobicity and improved biofilm formation on the LDPE films. LDPE degradation was measured using weight loss analysis, FTIR, and SEM. The results were indicated that LDPE degradation was enhanced by up to 17.5% without any pre-treatments for 30 days of treatment with P. gergoviae TYB1 and SAC. SAC was enhanced biofilm production on LDPE and improved biodegradation of LDPE by P. gergoviae TYB1 was confirmed.
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Introduction
The widespread use of low-density polyethylene (LDPE) endangers both terrestrial and marine ecosystems, as evidenced by issues like polyethylene (PE) obstructions in fish, bird, and marine animal intestines. Also, hundreds of species from different habitats are at risk of extinction due to the mistaken consumption of this plastic waste1. The biodegradation of PE involves use of microbes or microbial communities that modify and consume the polymer as a source of energy leading to changes in its physio-chemical properties such as weight loss, structural deterioration, and eventually carbon fixation as a biomass. The demand and consumption of plastic polymers have been increasing continuously for decades due to day-to-day customer demands for maintaining a modern and luxurious lifestyle2. In 2017, the plastic production reached 348 million tons and every year 4% increases have been observed due to wide usage of plastic products3. Plastics have been incorporated into the food chain from various sources. The plastic waste has been contaminated the entire earth4. Among all type of plastics, PE in the form of LDPE is about 64% used widely as a single used polymer in food packaging, detergents, clothing, shelter, transportation, and chemical substances5. Microplastics contaminate aquatic and terrestrial environments and are now abundant in air6. Microorganism-mediated degradation is an ecofriendly method for reducing pollution7. Different forms of PE have been demonstrated to be degraded by over 20 bacterial species. Gram-negative and Gram-positive species from the genera Rhodococcus, Staphylococcus, Streptococcus, Streptomyces, Bacillus, and Pseudomonas, Ralstonia, Stenotrophomonas, Klebsiella, and Acinetobactor are among them8,9,10,11. Fungi such as Aspergillus, Acremonium, Fusarium, Penicillium, Phanerochaete, Trichoderma, and Paecilomyces have been identified as degrading PE12. A few fungal species, such as Rhodotorula mucilaginosa, Alternaria alternata FB1, and Zalerion maritimum have been found to degrade PE in the maritime environment13.
Plastics are novel materials for microorganisms because of their high molecular weight and crystallinity, and because they cannot be taken up into the cell for degradation. They do not have evolved enzymatic machinery for the complete degradation of PE and not fully understood. PE is a synthetic new polymer to the microbial world. Microbes are trying to adopt to utilize PE as a new sole carbon source for their energy production. Hence, the enzyme system of PE degradation is important to understand completely. The absence of functional groups and the hydrophobic nature of polymers are strong barriers that allow microbes to form a biofilm over the surface14. Microorganisms prepare a multilayer biofilm on the surface to stabilize them over the long term15. Microbial coverage on the PE surface plays a significant role in the biodegradation process. A biofilm is a type of covering over the surface that facilitates the colonization of bacteria16. Few bacterial species have been explored for their biodegradation activity, but no effort has been made to enhance biofilm formation to adapt microorganisms to high-level degradation17,18,19,20. Enhanced high-density polyethylene (HDPE) degradation was observed in vitro by physical, chemical, and biological treatments with Aspergillus terreus21. Two isolated PE degraders, Bacillus and Enterobacter genera, were isolated, and their degradation of PE showed up to 6.1% and 10.7% weight loss within 60 d of treatment22. Pseudomonas species showed a degrading efficiency of 5% for untreated PE within 45 days23. PE biodegradation was examined using bacteria isolated from the marine ecosystem of the Gulf of Mannar, India24. Immediate focus on ecofriendly biodegradation is needed because of the plastic toxicity and biohazardous nature of the environment and their habitats. Surface-active compounds (SACs) are amphiphilic in nature25 and it is having potential to enhance the biofilm formation through reduction of surface tension and conversion of hydrophobicity to hydrophilicity of the LDPE. It showed the novelty of the work with distinct types of approaches compared to previous findings. The main advantages of SAC are its nontoxicity, biodegradability, good compatibility, high selectivity, and increased specificity under extreme environmental conditions (i.e., temperature, pH, and salinity), and it can be reproduced from renewable sources. However, the lack of knowledge regarding its structure and molecules has prevented its commercialization in food and medicinal industries. Even though research has reported significant LDPE degradation with bacteria and fungi, more attention has been paid to the environmental degradation of waste LDPE26. The present research has focused on the extraction and production of biosurfactants from Pluralibacter gergoviae TYB1 and its application for the enhancement of biofilm formation to facilitate a prominent level of biodegradation within a brief period. To the best of our knowledge, this is the first study to use a combination methodology involving SAC production and biofilm formation on LDPE. The aim of this research was to confirm SAC production by P. gergoviae TYB1 and analyse it using different methods and then production in an optimized culture condition verified by RSM. In this study, biofilm formation ability was checked using the test tube method, and hydrophobicity was evaluated using the BATH assay. SAC is a vital compound produced by bacteria that able to degrade the LDPE. SAC enhances biofilm formation by changing functional groups through the addition or deletion of other functional groups, which was characterized by FT-IR analysis. Biofilm formation and biodegradation were enhanced by SAC treatment. SAC was characterized by FT-IR, TLC, and GC-MS analyses. These research findings can be straightforwardly implemented at a commercial or industrial level.
Results
SAC production and characterization
P. gergoviae TYB1 (Fig. 1b) was employed in the current investigation to produce SAC and to treat the SAC to increase the biodegradation activity by enhancing the biofilm production on LDPE film (Fig. 1a). Centrifugation was performed to separate bacterial cells from NB culture media and then transferred into MSM media to determine SAC production. SAC production was confirmed by the following tests drop collapse, oil spreading, emulsification, and foamability assays. P. gergoviae TYB1 culture supernatant (CS) was utilized for the drop collapse experiment, and after the addition of CS, the oil drops flattened from their initial state (Fig. 1c and d). According to these findings, the oil drop collapsed because of the lowering of the surface tension by the SAC. It was found that the SAC present in the bacterial suspension may cause an oil drop to collapse on a hydrophobic surface with low interfacial tension, or it depends on the destabilization of liquid droplets by surface-active agents. An oil spreading assay was performed, and satisfactory results were obtained for the confirmation of SAC production by P. gergoviae TYB1 (Fig. 1e&f). CS of P. gergoviae TYB1 created a sizable halo in the middle of the thin oil layer in the oil displacement assay. A wide displacement of oil was observed after a minute of CS application due to the presence of SAC in the CS, which was measured as 3 cm on both kerosene and crude mustard oil. P. gergoviae TYB1’s CS was subjected to foamability analysis. Kerosene oil was added to the CS and vertex for five minutes the foam was formed due to the presence of the SAC when kerosene was mixed with CS vigorously for 5 min. The foam was left for 24 h, and its height was measured. Foaming stability was assessed as 75% (Fig. 1h). P. gergoviae TYB1’s CS were treated with kerosene oil to create an emulsifying layer, the emulsification index (EI24%) was measured (Fig. 1g). The CS EI24% value for the kerosene oil was 18.03 ± 0.97%. For the emulsification test, SDS and Tween 20 were used as positive controls (18.73 ± 0.81 and 16.97 ± 0.97, respectively), and MSM without bacterial inoculation was used as a negative control. The emulsification layer remained stable for up to 24 h. Hydrocarbon emulsion is an appropriate technique to measure surface activity27. The bacteria streaked on blood agar plate showed partial haemolysis around the colonies. Thus, P. gergoviae TYB1 was found to possess α-haemolytic activity (Fig. 1i). After the haemolytic assay SAC production were confirmed. Optimization of bacterial growth and SAC production was performed in a lab-scale by RSM analysis. In a lab-scale experiment, a suitable temperature of 36 ± 1 °C and a pH of 5.8 ± 0.15 were determined to be ideal for bacterial growth and high amount of production of SAC. The findings demonstrated that among all other carbon sources, starch at 3.16 ± 0.01 g/l was observed better growth, and for nitrogen sources, ammonium nitrate at 3.5 ± 0.1 g/l, beef extract at 4.1 ± 0.05 g/l demonstrated the maximum growth of P. gergoviae TYB1 (1.24 × 109±0.24 CFU/ml) and SAC production under ideal conditions (Fig. 2). Following optimization of the conditions for high production of SAC, the EI24% also increased from 18.03 ± 0.97% to 81.43 ± 1.19% (Fig. 1g). A Box-Behnken experimental design was used to optimize SAC synthesis by P. gergoviae TYB1 (Figs. 3 and 4). The response variables listed in Tables 1 and 2 were correctly evaluated. Design Expert version-7.1 was employed for the optimization of the production of SAC under the circumstances, as per the ANOVA results that described the RSM model equation (Figs. 3 and 4). The quadrating condition did not exist, nor was there a lack of fit. For bacterial growth and EI24, the model suitability was explained with a coefficient of determination R2 0.963211 (bacterial growth) and R2 0.948202 (SAC, EI24), demonstrating 96.32% and 94.82% variability in the response. The adjusted R2 and predicted R2 values were 0.933 and 0.906 for bacterial growth, and 0.852 and 0.792 for (EI24) SAC production, respectively. The cell viability was 16.00401% for bacterial growth and 30.38915 for (EI24) SAC production. The 3D surface plots of these factors are shown in Figs. 3 and 4. The quadratic model in Table 3, ANOVA findings showed that the final model equation obtained by RSM with Design Expert Version-7.1 could be used to adequately characterize the generation of SAC production under a variety of operational situations. The optimized culture conditions showed effective bacterial growth and SAC production (Figs. 3 and 4; Tables 1, 2, 3 and 4). The experimental data were maintained as follows: coefficient of variables (A-pH, B-Temperature, C-Starch conc., D-Ammonium nitrate, E-Beef extract), mean value, standard errors, and F value using the following equations:
(a) LDPE film (3 cm x 3 cm); (b) P. gergoviae TYB1 in LB agar; (c, d) analysis of SAC production by Drop collapse assay performed with mustard oil and kerosene oil respectively; (e, f) analysis of SAC production by Oil displacement test - oil in the centre of water surface without SAC and oil displacement from centre to outer surface after adding SAC respectively; (g) Analysis of SAC production by Emulsification test (positive control − 1% SDS; negative control (without bacterial inoculation)). (h) Emulsification index was analysed after optimization of SAC production; (i) haemolytic assay by growing P. gergoviae TYB1 on blood agar.
Production and extraction of SAC from P. gergoviae TYB1: (1) Foam formation during incubation period was indicating SAC production; (2) Solvent extraction for separation of organic layer from solvent layer; (3) solvent evaporation; (4) Crude SAC processed for further characterization.
Optimization of bacterial growth compared with various factors such as temperature, pH, carbon and nitrogen sources (a-j) by Response Surface Methodology (RSM).
Optimization of SAC production in the parameter of EI24 (%) compared with various factors such as temperature, pH, carbon and nitrogen sources (a-j) by Response Surface Methodology (RSM).
The fitted RSM equation for bacterial growth and emulsification index were:
(Here, Y is the bacterial growth response and A, B, C, D, A, B, C, D, and E are the factors affecting bacterial growth).
(Here, EI24% is the response variable and A, B, C, D, A, B, C, D, and E are the factors affecting EI24%)
Presently, the carbon and nitrogen sources were optimized for the maximum production of SAC as starch and beef extract respectively. The optimized conditions were found to be significantly increased the SAC activity of EI24% was observed after 24 h incubation was 81.43 ± 1.19% (Figs. 3 and 4). Under ideal growth conditions, SAC production was measured at 2.7 ± 0.1 g/l SAC with starch as a sole carbon source in the media (Table 4). Dried SAC isolated from P. gergoviae TYB1 was soluble in methanol, ethyl acetate, chloroform, and hexane, and had an off-white powder appearance28. In the current work P. gergoviae TYB1 was observed that production of lipopeptide type SAC analysed and confirmed by TLC, FTIR and GC-MS analyses. In RSM analysis, the bacterial growth was positively correlated with medium pH (r2-0.3279), temperature (r2-0.890), starch (r2-0.633), ammonium concentration (r2-0.5027) and beef extract (r2-0.4079) at the significant level of P < 0.0001. Similarly, EI24% of SAC during the media optimization for the highest growth of selected bacteria and high-level production of SAC was positively correlated with pH (r2-0.0438), temperature (r2-0.8547), starch (r2-0.2133), ammonium concentration (r2-0.059) and beef extract (r2-0.2397) at the significant level of P < 0.0001 (Tables 1, 2 and 3).
Characterization of SAC
Biochemical analysis revealed that the SAC produced by the P. gergoviae TYB1 was mainly composed of lipid and protein. The protein content was found to be 288 ± 0.57 µg/ml whereas the lipid content was 332 ± 0.25 mg/g. A minimal fraction of carbohydrate (72.75 ± 0.3 µg/ml) detected in the SAC sample. The FTIR spectrum of the SAC revealed few peaks that provide specific information about functional groups that potentially involved to characterise the SAC i.e., lipoprotein or glycolipids. The characteristic peaks observed from the spectrum with their corresponding functional groups (Fig. 5a). The distinctive peaks measured from the spectrum with respect to the functional groups of O-H stretch, H-bonded; alcohols, phenols, N-H stretching mode; and aliphatic primary amine were at 3334.32 cm− 1. The existence of O-H stretching was indicated the presence of fatty acid in the sample. At the wavelength 2348.87 cm− 1 (O = C = O stretch; CO2), and 1640.16 cm− 1 (C = N, imine/oxime) range was showed that the presence of amide II bands. Correspondingly, peaks at 1151.29 cm− 1 (C-N stretch, aliphatic amines), 1076.08 cm− 1 (C-O stretch) confirmed the presence of ester, fatty acid, and 1003.77 cm− 1 (O-H, stretch; carboxylic acid). Existence of aliphatic amine and O = C = O stretch was showed that the presence of lipid moiety in the SAC. FT-IR results were indicated the presence of lipopeptide type biosurfactant (Fig. 5a).
FTIR analysis (a), GC-MS analysis (b) and TLC analysis of SAC extracted from P. gergoviae TYB1, (c) Iodine and (d) Ninhydrin sprayed on silica for finding the presence of lipids and amino acids.
GC-MS were used to characterize the bioactive compounds present in the crude CS. Using the Standard WILEY library, Butanal, Methyl (2E)-9-(Dimrthylamino)-7-hydroxy-2-methyl-9-oxo-2-nonenoate, 3-Penten-2-one, Dimethylsulfoxonium formylmethylide, Dimethyl Sulfoxide, Acetic acid, Methane, Dimethyl sulfone, Decan, Cyclohexene, 1-Methyl-4-1-Methylethenyl, 1,3,5-Triazine-2,4-Diamine, N,N’-bis(1-Methylethyl-6-(Methyl sulfonyl, S-Methyl methanethiosulfinate, L-Methionine, N-trifluoroacetyl-N-propyl-, methyl ester 1-Ethanol, 2-(ethylsulfinyl), Methoxyacetic acid, 6-ethyl-3-octyl ester, Acetic acid, 2-[4-(1 H-1,2,3,4-tetrazol-1-yl)phenoxy]− 2-Ethyl-1-Butene, methane, bis(methylsulfonyl)- 3,5,5-Trimethyl-2-cyclohexen-1-one, S-(2-Fluoroethyl) methanesulfonothioate, Cycloheptasiloxane, tetradecamethyl, 1- Decanethiol, Pregn-5-en-20-one, Trimethylsiloxy, O-Methyloxime Cyclooctasiloxane, Hexadecamethyl, Methyl 7-(3-(3-Hydroxyocta-1(E)-enyl)-1-Hydroxy-4-Oxocyclopenta-2-yl) heptanoate, Sulfurous acid, hexyl octyl ester, 5,5-Diethylheptadecane 3-Fluoro-phenyl)-2-Methoxy-6,6-Dimethyl-9-thioxo-8,9-dihydro-6 h-7-thia-5 A, 8-diaz-Cyclopenta[E]acenaphthylene-4,5-DioneI, 1 H-Purin-6-amine, (2-fluorophenyl) methyl, 5,5-Diethylheptadecane 2-Penten-4-yn-1-ol, 5-(methylthio)-, (E)- Cyclododecasiloxane, and Tetracosame. In Fig. 5b, the gas chromatogram of the SAC was shown. Dimethylsulfoxonium formyl methylide, dimethyl sulfoxide, acetic acid, and methane sulfinyl were observed in the CS and all the compounds found to be of industrial value. These compounds indicate a complex mixture of organic molecules, which is nature of lipoprotein type of SAC. Each of these compounds can contribute to overall surface-active properties of the SAC, making it useful in various applications such as bioremediation, pharmaceuticals, cosmetic and food industries. SAC was purified and characterized by B. subtilis YPS-32, B velezensis KLP2016O, B. subtilis HSO 121 (Zhou et al., 2023; Meena et al., 2021; Zhao et al., 2012). Compared to the previous reports with present studies, it was showed similarity of presence of cyclic lipoprotein type of SAC on the basis of GC-MS analysis (Fig. 5c&d).
TLC analysis of partially purified SAC was showed a colour spot in plates with Rf value of 0.862 for lipid (Fig. 5c) and 0.650 for protein and free amino acid contents (Fig. 5d). The SAC fraction was showed negative reaction with Molish reagent, no spots were detected indicating that the absence of carbohydrate whereas it was showed positive reaction with iodine vapour (a yellow band was developed) and ninhydrin reagent (a purple colour spot) indicating the presence of lipid moieties and free amino acids in the SAC. The above results of TLC analysis were demonstrated that the lipoprotein nature of SAC. Glucose, olive oil and leucine were used as standard components in the TLC experiment and Rf values of the standard compounds were 0.960, 0.896 and 0.642, respectively.
Enhancement of biofilm formation on LDPE and confirmation of biodegradation by P. gergoviae TYB1 by addition of SAC
Analysis of a very dense covering of microbial colonization explained that the existence of SAC caused by bacteria to bind on LDPE. For the successful binding of a bacterial enzyme to the polymer chain, SAC is facilitating an effective functional group and surface. The results of our research demonstrated that the efficiency of bacteria to make colonization on LDPE surface and the thick layer of bacterial consortia that covered on it (Fig. 6a-c & g-i). LDPE are hydrophobic, which prevents the adhesion and colonization of bacteria on the polymer surface. Because the polymer chain lacks a functional group, biofilm development and enzymatic activity were both reduced. However, the creation of biofilms is a crucial step that triggers deterioration. SAC is a surface-active agents have the potential to lower the polymer’s surface tension and change its nature to one that is hydrophilic, which enables bacteria to adhere on it. In the current study, P. gergoviae TYB1 was used to produce lipopeptide type of SAC, which could improve biofilm formation by lowering the hydrophobicity of the polymers (Fig. 6a-c & g-i).
Biofilm formation by SEM analysis (a-c) a-LDPE film without bacteria (Control), b- LDPE film with bacteria and c- LDPE film with bacteria and SAC. Surface modification of LDPE was observed by SEM analysis for biodegradation of LDPE by P. gergoviae TYB1 (d-f) d - LDPE film without bacteria (Control), e - LDPE film with bacteria and f - LDPE film with bacteria and SAC. Inverted microscopic analysis of biofilm on LDPE after a month of incubation (g-i) g -LDPE film without bacteria (Control), h - LDPE film with bacteria and i- LDPE film with bacteria and SAC.
BATH assay is a secondary examination technique to check the efficiency of bacteria for SAC production and biofilm formation on the LDPE surface. The results indicated a hydrophobicity as 107 ± 7%, which indicates the potential of the bacteria P. gergoviae TYB1 to produce enormous amounts of SAC by selected bacterial strain. The outcomes of the secondary screening revealed that the levels of biosurfactants produced by the bacterial isolates varied from one another. Among the finest makers of biosurfactants were isolates with high EI24%, fat drops, an extended clear zone with low surface tension, and a decent BATH percentage. In comparison to the others, isolate Z47 demonstrated strong significance (p < 0.0001) based on the lowest surface tension and the highest EI24%. As a result, it was chosen as a potential producer of biosurfactants to be molecularly identified and utilized. In current research work P. gergoviae TYB1 showed remarkable results as compared to earlier findings. In the correlation co-efficient analysis, the bacterial growth was correlated with pH (r2: 0.982), foamability (r2: 0.915), and negatively correlated with oil displacement properties (r2: -0.866) at the significant level of p < 0.001). SAC production was directly propositional to the bacterial growth (r2: 1.000), biomass (r2: 0.380), EI24% (r2: 0.669), foamability (r2: 0.915) and indirectly propositional to the oil displacement property (r2: -0.866), beef extract (r2: -1.00) and starch (r2: -0.50) (Table 5). The viability of the bacterial biofilm over the LDPE was observed by applying of Coomassie brilliant blue staining. The viable bacterial cells were noticed, and the multilayered biofilm was observed on the LDPE (Fig. 6d-f). Bacteria may be able to bind more quickly than the untreated because of changes in the hydrophobicity to hydrophilicity of LDPE by SAC application. These research outcomes suggested that carbon scarcity may enhance the growth of biofilms on hydrophobic polymers like LDPE.
After viability testing, the SEM analysis indicated that, after 30 days of incubation, the biofilm growth was higher in LDPE film treated with SAC (Fig. 6a-c & d-f). Over the surface of the LDPE, there was a mass generation of bacterial biofilm analysed by SEM (Fig. 6a-c & g-i). The results indicated that the production of microbial depolymerizing enzymes and other catabolic activities leads to the surface alteration of LDPE, including the creation of holes (bacterial action following biofilm formation), corroded by microbial enzymes, adhesion, and cleavage (crack region) of LDPE film (Fig. 6d-f). Untreated LDPE films did not modify in a 30-days of incubation without bacteria, giving clear view at 2.35 KX. Biofilm over the LDPE by P. gergoviae TYB1 was indicated that the microbial adherence, but the biofilm formation was not efficient due to hydrophobic nature of polyethylene, 4.15 KX and 586 X. SAC added LDPE film along with bacteria was visualized that the highest microbial adherence, multilayered biofilm on the surface of LDPE, erosion and cracks made by attachment by P. gergoviae TYB1 observed at 1.40 KX and 23.3 KX (Fig. 6d-f). The SEM images showed that the biodegradation efficiency of P. gergoviae TYB1 for LDPE film within a fleeting time as compared to previous findings by other microbes. The SEM micrographs and these outcomes agreed (Fig. 6d-f), It showed varied levels of sample surface degradation at different points. The FT-IR results demonstrated that the degradation of LDPE with SAC was more intense than that of LDPE without treatment (Fig. 6d-f).
After 30 days of treatment of bacteria with LDPE, the dry weight loss of LDPE film was initially 4.8% without treatment of SAC, and then it was increased up to 17.5% after the SAC treatment (Fig. 7). The results were showed that bacterial degradation increased analysed by the weight loss of LDPE after 30 days of treatment with SAC without any physical and chemical pretreatments. Because of the carbon from the LDPE film was added in the medium being consumed during the SAC treatment, the weight loss of the LDPE was significantly increased. The bacterial colonization was enhanced by the SAC which leads to conversion of hydrophobic to hydrophilic surface of LDPE. This conversion significantly increased the bacterial degradation of LDPE by their depolymerising enzymes.
Weight loss of LDPE analysed after 30 days of incubation; the corrosion of LDPE occurred by the enzymes released by the P. gergoviae TYB1.
FT-IR analysis of LDPE film treated by P. gergoviae TYB1 without SAC and with SAC indicated changes in functional groups as compared to control (Fig. 8a-c). LDPE films treated with P. gergoviae TYB1 displayed variation in the intensity of bands in different regions for both conditions (Fig. 8b & c). The absorption bands were detected in the control spectra at 722.21 cm− 1 (C-H bend), 1463.71 cm− 1 (C-C stretch), 1739.48 cm− 1 (CHO stretch), and 2913.91 cm− 1 and 2847.38 cm− 1 (both resulting from C-H stretch). In the spectra of the LDPE sample treated with P. gergoviae TYB1, a drop indicated the cleavage of C–H bonds peak at 2369.44 cm− 1 and 2341.16 cm− 1 (both attributable to C = NH+ charged amines). A rise in the already-existing peaks and the development of a few new ones at 1078.98 cm− 1 and 400–1200 cm− 1 (C-O stretch 1˚ aldehyde), 918.91 cm− 1 (O-H bend Carboxylic acid), 669.17 cm− 1 (C-N stretch amide bond) region of FT-IR spectra, indicated the formation of new intermediate products (Fig. 7b). The presence of new functional groups at the region of 3846.33 cm− 1, 3790.4 cm− 1, 3715.19 cm− 1 (both due to -OH alcohol), 2347.91 cm− 1 (C = NH+ charged amines), 2175.31 cm− 1 (C ≡ N stretch isonitriles), 1709.59 cm− 1 (-CO-NH-CO- stretch amide), 1585.2 cm− 1 (C = N stretch), 1530.24 cm− 1 (C = C stretch ) and 1461.78 cm− 1 (O = C-O stretch carboxylic acid) was observed in SAC treated LDPE film along with bacteria (Fig. 8c).
FTIR analysis: (a) LDPE film without bacteria (Control), (b) LDPE film with bacteria and (c) LDPE film with bacteria and SAC.
In the current experiment with P. gergoviae TYB1, the FT-IR spectra clearly demonstrated that the included and removal of alcohol, carboxylic, aldehyde, amines groups, and internal double bond index, which aids in the confirmation of LDPE biodegradation. Both the untreated and treated conditions’ bacteria initially started the biodegradation of LDPE.
Discussion
Given their widespread presence in the earth’s biosphere, microbes are thought of as biological molecules’ “mines.” A biomolecule derived from microorganisms; SAC has ability to function as surface-active agent. SAC distinctive qualities—such as their relative ease of manufacture, low toxicity, and specificity have drawn the interest of various researchers recently. Chemical structural classification of the molecules released by various bacteria results in a diversity of structures among the molecules categorized as surface active compounds originating from microbes29. Microbial SAC have unique biological benefits and can conduct a variety of physiological tasks. For instance, they promote the biodegradation of environmental contaminants and improve the availability of water immiscible substrates through the emulsification process30 During the haemolytic assay, the bacterium P. gergoviae TYB1 produced α- haemolysis, by showing a partial haemolysis around the colony on blood agar plate, which reasserts SAC production. The haemolytic activity is a one of the initial indicative parameter for the screening of bacterial SAC production31. SAC enhances the biofilm formation because it is the initial step to start biodegradation process by improving enzymatic activity on the surface. Enzymes are integral to the formation and function of biofilms on PE surfaces. They are not only aid in the initial colonization and maturation of the biofilm but also enhance the biodegradation process by breaking down complex polymers into simpler, and more manageable molecules. It reduces the surface tension of PE by allowing the bacteria to make colonization on LDPE and help to bind the bacterial enzymes on the LDPE to react on LDPE for its breakdown. This enzymatic activity is vital for the sustainable and efficient degradation of PE waste32. Biosurfactants are essential for improving enzyme binding and activity on plastic surfaces. Biosurfactants play a major role in the biodegradation of plastics by lowering surface tension, enhancing biofilm development, boosting enzyme activity, and altering the plastic surface. Addressing plastic pollution may be greatly aided by the synergistic action of enzymes and surfactants33. Drop collapse34, oil displacement35, emulsification index are the techniques that have been developed to screen the SAC from the microorganisms. Although, they are referred to as cell-bound SAC in most research articles, these SAC may be secreted in bacterial cell suspension, or they may stay attached to the bacterial cell wall. There are very few reports on SAC supporting biofilm formation on LDPE and its biodegradation. In the previous study of confirmed that a clean zone was formed after adding the CS on the oil layer. In the reaction formation of clean zone may be due the of the SAC in the CS36. P. gergoviae TYB1’s CS was observed as a good emulsifier, the emulsification index (EI24%) was measured with kerosene oil was 17% and it remained stable, this work process was compared with previous techniques27. According to the kind of solvents employed, the stable interaction of the hydrophobic and hydrophilic phases can be reason for the formation of the emulsifying layer37. In a recent investigation, the SACS produced by Brevibacterium 7G and Ochrobactrum 1C were not found to emulsify against diesel oil or kerosene, but both were effective emulsifiers for motor oil, with emulsification ranges of 45% and 90%, respectively38. In this experiment, P. gergoviae TYB1 revealed SAC production with high emulsification index in kerosene oil, it will be effective in emulsifying hydrocarbons, which could increase the availability of the resistant hydrocarbons, according to the current emulsification data39.
As per recent research 2.6 g/l SAC production was reported in Bacillus licheniformis (ON795916) and it was characterized as lipopeptide by FTIR and TLC analysis. In the current research P. gergoviae TYB1 was observed with 2.7 ± 0.1 g/l was showing slightly high production of SAC with similar type40. Spent engine oil observed as a carbon and energy source for SAC production41, bacterial growth and observed that Enterobacter cloacae B14 having SAC activity reported as 42.2%. SAC production was observed by using E. cloacae B14 with the specific pH range from 2 to 10 and a temperature range from 30 to 37 °C42. Additionally, SAC had a stronger emulsification activity than other Enterobacter species, such as strain SF-4 (40%)43, strain LS1 (44%), strain CG101 (15%), and LS8 (50%) observed by previous researchers44. Enterobacter sp. MS16 was identified from soil contaminated by diesel oil, biosurfactant production was observed 1.5 g/l by sunflower oil added in culture media as a single carbon source reduction in surface tension 68%28. It was examined that the production of 0.05 and 0.01 g/l SAC by the two bacterial strains of Pseudomonas sp. BP10 and Rhodococcus sp. NJ2 with emulsification activity of 75 and 35%, respectiely25. Production of 2.0 and 2.5 g/l SAC was reported based on dry weight from Brevibacterium 7G and Ochrobactrum 1 C strains, respectively45. Pseudomonas guguanensis strain was observed for biosurfactant production with 3.01 g/l. The biosurfactant effective to reduce the surface tension from 72 to 38 ± 0.33 mN/m and had 52 ± 0.33% EI24%. This biosurfactant was preliminarily specified to be a glycolipid and mono rhamnolipid as evident from TLC, FTIR, and GC-MS analyses46.
In the case of EI24%, E. cloacae BAGM01 was showed activity from 29.4 to 47.7%; 60–80% of EI24% by P. aeruginosa strain but both values, are lesser than current finding by P. gergoviae TYB147. In this work P. gergoviae TYB1 was showed 81.43 ± 1.19% emulsification index with kerosene oil and 75% foamability. Extracted SAC was showed high stability even after 24 h observation. It was observed that Enterobacter cloacae SJ2 produce rhamnolipid (conformed by FTIR and MALDI-TOF analysis) type biosurfactant with 68.14% emulsification index percentage (EI24%) and the bacterial hydrophobicity was observed 79.2%. It was showed that optimum production of biosurfactant 2.61 g/l in 36 h of incubation period, 150 rpm agitation, pH 7.5, 37 °C temperature, 2% glucose as carbon source and with 1% yeast extract as sole nitrogen source48. In current research work SAC production by P. gergoviae TYB1 was observed as 2.7 ± 0.1 g/l with 81.43 ± 1.19% emulsification index percentage (EI24%) and 107% bacterial hydrophobicity, 72 h of incubation period, 120 rpm agitation, pH 5.8, 36 °C temperature, 3.16% starch as carbon source and with 4.1% beef extract as sole nitrogen source.
FT-IR data has been confirmed by the correlation of previous work related to SAC production in Enterobacter sp41,42. By using FT-IR spectra, Jha and his colleagues determined the cyclic lipopeptides generated by the B. subtilis R1 strain, fengycin and surfactin49. Sharma and coworkers identified the polysaccharide and lipid components in the SAC produced from Enterococcus faecium by FT-IR spectrum50. Monorhamnolipids was obtained by P. guguanensis as commercial eco-friendly hydrocarbon dispersants46. Similarity has been found in Infrared spectrum of the SAC obtained from Bacillus mycoides, a broad stretching peak was observed at 3161 cm− 1, characteristic of the hydroxyl and amine groups. 2967 cm− 1 is assigned to the (-C–H) aliphatic chains. The CO–N bond’s stretching mode was demonstrated by an absorption band at 1691.65 cm− 1, while the presence of ester carbonyl groups (C = O in COOH) in the SAC is indicated by a weakly symmetric stretching peak at 1455 cm− 151. From FTIR, it was presumed to be lipoprotein with the characteristic peaks. According to the information that compiled from e-sources, the identified chemicals are beneficial in a variety of applications with high demand. Through, GC-MS investigation of the SAC generated by Salmonella bongori, the six industrially significant chemicals have been discovered31. GC-MS result explained about lipopeptide production from Enterobacter sp. strain N18 isolated from soil samples52. Related results found in Citrobacter and Enterobacter53.
A purple colour spot was observed in the ninhydrin spray revealed the presence of amino acids and proteins in SAC. Lipoprotein biosurfactants typically exhibit Rf values in the range of 0.2 to 0.8. The test surfactant’s components show a broader range of Rf values, with one component being highly polar (Rf = 0.0) and others being less polar (Rf = 0.862 and 0.650). This suggests that the test SAC was a complex mixture with varying polarities with high contents of lipid and protein so that these findings suggested that the examined sample contained SAC of the lipoprotein or lipopeptide type. This finding was corelated with the findings of54,55,56,57. Similarly, Bacillus subtilis R1 isolated from an oil contaminated desert site in India and produced SAC observed that as efficient biocontrol agent and useful for oil recovery49. The SAC produced by Enterobacter MS16 was reported as glycolipid by TLC analysis having efficient emulsification activity against various hydrocarbons28. Lipopeptide type SAC production was observed in Enterobacter sp. isolated from contaminant soil area58. Similar report was found in Enterobacter N18 to a surfactin-producers and explained about lipopeptide producing Enterobacter family52. In previous studies of lipopeptides production by E. cloacae C3 strain was proved by TLC analysis. The findings of our study revealed that modifications on the functional group as well as enhancing a thick layer of bacterial consortia59. Involvement of the Bacillus SecYEG Pathway and biosurfactant production was explored to understand the enhancement of biofilm Formation60. It was demonstrated that the attachment of various functional groups caused the biofilm formation. Similar work was explained by Tavasi and his colleagues, found that Pseudomonas strains adhered to crude oil more than other strains, with P. aeruginosa demonstrating the highest cell attachment (95.15 ± 0.21%), indicating its greater capacity to create biosurfactants61. However, Bacillus had the highest cell adhesion (94.23 ± 0.71%), whereas E. coli had the lowest adherence (60.15 ± 1.42%). Similar changes on surface hydrophobicity of bacteria cell in response to carbon deprivation was reported previously62. Previous research demonstrated that Rhodococcus ruber made biofilm on polyethylene showed survivability even after 60 days of incubation and stayed attached to the substrate without any external carbon supplementation63.
Recent research explained that bacterial communities obtained from Manila Bay (Philippines) was showed degradation efficiency against LDPE. But they did not explain about weight loss as well as incubation period was 91 days64. In current research with P. gergoviae TYB1 weight loss was observed 4.8% without any treatment and 17.5% with SAC treatment in 30 days (Fig. 7). Few bacterial isolates belong to the genera Halomonas, Bacillus, Alteromonas, Photobacterium, and Aliishimia was subjected to a 60-day co-incubation period with LDPE sheets to assess their capacity to colonize PE. Analysis was done by FTIR showed a significant change in the surface of LDPE film and changes on function groups. Bioaugmentation of Enterobacter cloacae AKS7 caused an improved degradation of LDPE in soil. It was reported that UV-treated LDPE degradation was more efficient than UV-untreated LDPE. E. cloacae AKS7 exhibited ~ 30% higher functional richness and ~ 30% enhanced functional uniformity than the other non-bioaugmented microcosms that was indicating the formation of an enriched ecosystem that could offer various functions including polymer degradation65. In previous research, HDPE-degrading bacterial strain Bacillus cereus CGK5 was identified from the faecal matter of the cow, weight loss of HDPE was observed 1.83% after 90 days incubation. Recent research was conducted in same area, results indicated that 43% weight loss of LDPE films within 120 days of incubation with Bacillus cereus strain NJD166. Current results showed that the significance of bacterial strain P. gergoviae TYB1 for the LDPE degradation. The emergence of new functional groups within the region of 3846.33 cm− 1, 3790.4 cm− 1, 3715.19 cm− 1 ((both due to -OH alcohol)), 2347.91 cm− 1 (C = NH+ charged amines), 2175.31 cm− 1 (C ≡ N stretch isonitriles), 1709.59 cm− 1 (-CO-NH-CO- stretch amide), 1585.2 cm− 1 (C = N stretch), 1530.24 cm− 1 (C = C stretch ) and 1461.78 cm− 1 (O = C-O stretch carboxylic acid) was observed in BS treated LDPE film. Formation of carbonyl (keto and ester), double bonds (-CH = CH-) and terminal ⁄ vinyl double bond (-CH = CH2) reported with Pseudomonas sp. showed high degradation efficiency to HDPE67. PE film exposed to P. aeruginosa ISJ14 revealed alterations in functional groups. In contrast, the PE films that was exposed to ISJ14 showed variance in the band’s intensity across various locations. The typical absorption bands were allocated for the control spectrum at 729.01 cm− 1 for the C-H bend, 1,465.59 cm− 1 for the C14C stretch, 1713.55 cm− 1 for the CHO stretch, and 2916 and 2855 cm− 1 for the C-H stretch. In the spectra of LDPE samples treated with P. aeruginosa ISJ14, a peak at 2913.50 and 2853.50 cm− 1 for MSM and 2912.00 and 2854.00 cm− 1 for BHM showed a reduction that denoted the cleavage of C-H bonds. In the 1200–1400 cm− 1 range of FTIR spectra, there has been increased in previously observed peaks as well as the emergence of few new peaks, which suggests the development of novel intermediate products68. Related results found in LDPE degradation with consortium of bacteria and fungi identified from different environmental samples2.
Dissolved oxygen or oxygen existing in the broth caused the polymer chain to oxidise through free radicals, resulting in the production of carbonyl groups. But the process is too slow, in the case of SAC treated LDPE oxidation is speedup due to the reduction of hydrophobicity of LDPE. SAC facilitates addition or deletion of functional group to enhance enzyme binding on the LDPE surface to increase the degradation process. Carbonyl groups was produced rapidly in SAC treated LDPE than untreated LDPE. After that carbonyl groups easily transform into carboxylic groups and begin the β-oxidation process and then bacteria complete the citric acid cycle, results in the formation of CO2 and H2O. After incubated with bacterial strains, there was an improvement in the amount of the carbonyl residues in the polyethylene, which was primarily attributed to enzymatic activity. The SAC-treated LDPE film exhibited bond cleavage and the creation of a new functional group, both of which increased the degree of deterioration. The appearance of a terminal double bond in response to introduction to a biotic environment, and the same phenomenon has been noted for terminal double bonds following incubation with both conditions. Esters may have formed, or the Norrish mechanism of degradation may have caused the additions of terminal double bond. Currently, the addition of carboxylic, amide and hydroxyl groups were observed because of high enzymatic activity. Isonitriles, amide and carbonyl ions have been reported as major products in the presence of SAC. The formation of double bonds in the polymer chain occurred by the Norrish type II reaction69. The incredibly low intensity of the bands around 1739 cm− 1 in untreated and 1709 cm− 1 SAC treated LDPE confirmed the results of moderate surface degradation and negligible oxidative degradation under given condition. At the same time, the results suggested that the variable intensity of the bands around the surface deterioration of the samples varied based on the local circumstances, the local contact with the medium, and the local concentration of SAC, as confirmed by 3662 cm− 1. At the same time, it proved that the high efficiency of P. gergoviae TYB1 for biodegradation of LDPE compared to previously reported microorganisms. Hydrophobic nature of LDPE is one of the major barriers for microbial enzymatic reaction so as per the current research outcome SAC could be an effective compound which act as an enhancer of LDPE biodegradation through enhancing the biofilm formation in future research for the biodegradation of different type of polymers.
Recent researchers and technologists are taking interest on the development of bioproduct which could be a potential bioremediatory, to manage the negative impacts of pollutants on humans and wildlife. The use of biological molecules is a promising way to address this serious social problem. In previous studies, researchers were focusing to search a potential microorganism either bacteria or fungi for plastic degradation. Few of them got positive results with few microorganisms but the efficiency of degradation was extremely low and not sufficient to solve this significant issue because of the accumulation of tons and tons of plastic wastes in the world. However, few bacteria were showed considerable efficiency against polyethylene degradation, but time and degradation percentage are extremely low. As per our research, if any enhancer would be used to enhance the activity of bacteria so the degradation efficiency could be increased manyfold to be an effective tool for managing the PE wastes as much as possible. Researchers have been attempted with chemical treatment, but no report has been documented with the biological treatment. In this research work SAC were produced by P. gergoviae TYB1 then it was utilized as an enhancer for the biodegradation of LDPE. SAC has a wide variety of applications on different industrial purpose due to its high emulsification activity, foamability, antimicrobial activity, and antifouling. It can be applicable for the bioremediation of other toxic contaminants in the environment. In extreme conditions, microorganisms contribute to plastic degradation by enzymatic activity for softening and disruption of plastics with the help of SAC. Biodegradation under unique ecofriendly conditions opens a path for reducing disposed plastic waste. Few approaches had been developed for the screening of SAC such as drop collapse method, oil displacement method, emulsification index, and foamability test. Only limited number of research have been performed available on SAC production from culture media. The present research reveals the SAC secretion by P. gergoviae TYB1. Present studies on preliminary screening methods for SAC production by P. gergoviae TYB1 than extracted as a powder form. Till now no one has utilized SAC as a treatment material for the enhancement of LDPE degradation.
In the current investigation, research study explained that the biodegradation efficiency of P. gergoviae TYB1 isolated from wastes dumping site. P. gergoviae TYB1 was found to be an effective LDPE degrader and could produce SAC having high emulsification activity. The strain exhibited high degradation capacity compared to other bacteria. The SAC possesses high surface activity and exhibits high emulsification activities. Therefore, based on current findings, lipopeptide SAC could be applied as a compound enhancing the LDPE biodegradation and all the properties of SAC which facilitates the P. gergoviae TYB1 as a potent degrader of LDPE. SAC is having a similar efficiency as commercially importance and with a lower production cost, it could be an effective product for various environmental applications (cleaning and detergent, cosmetics, pharmaceuticals, and agriculture), particularly in the bioremediation of LDPE contamination sites. Moreover, further studies need to be conducted to explore the lipopeptide SAC application and strain improvement for the LDPE degradation.
Materials and methods
Materials and bacterial culture
A commercially available Low-Density Polyethylene (LDPE) film with a thickness of 50 μm thickness and 0.95 g/cm3 density were used (3 × 3 cm) was sterilized with 70% ethanol and dried under sterile conditions before the experiments (Fig. 1a). P. gergoviae TYB1 was isolated from the waste dumping place of Tiruchirappalli, Tamil Nadu, India by using PE degrading media (PDM). In 100 ml of PDM containing (g/l): NH4NO3, 1.0; MgSO4·7H2O, 0.2; K2HPO4, 1.0; CaCl2·2H2O, 0.1; KCl, 0.15; yeast extract, 0.1; and microelements for 1.0 mg/l of each of the following (FeSO4·6H2O, MnSO4, and ZnSO4·7H2O, ). P. gergoviae TYB1 (109 CFU/ml, OD600) seed culture was kept at 4 °C and produced in nutrient broth media (NB) for healthy growth, and subsequently made an inoculum for using the culture to conduct an experiment later in both NB and nutrient agar medium (NAM) were utilized for keeping bacterial culture alive (Fig. 1b).
Blood agar test
By using blood agar, the haemolysis assay was performed to check the haemolytic activity of P. gergoviae TYB1. Bacteria were inoculated in the LB agar (Luria Bertani media, Hi-media) plate prepared with 5% human blood and incubated for 24 h at 37 °C. The haemolysis was observed after incubation31 .
Confirmation of production of SAC by P. gergoviae TYB1
P. gergoviae TYB1 was grown in NB media for 24 h at 37 °C in 120 rpm in a shaking incubator. The incubation was continued till the bacterial growth was reached 108 CFU/ml measured by UV spectrophotometer. The SAC formation was initially confirmed in mineral salt medium (MSM). MSM contains (g/l): Na2HPO4, 33.9; KH2PO4, 15.0; NaCl, 2.5; Starch, 5; Beef extract, 5. 5 ml of mother culture inoculum of P. gergoviae TYB1 was inoculated in the MSM media, prepared for the production of SAC and incubated for 3–5 days51.
Drop collapse assay
All the glass slides were cleaned with distilled water, ethanol, and then rinsed again before use. After that, 1.8 µl of mineral oil was applied on the slides, and they were allowed to equilibrate for a while to create a homogenous oil covering. Using a micropipette, a 5 µl aliquot of culture supernatant was applied on the slide. Results were observed visually, and a beaded or entire drop indicated that absence of SAC and oil collapsing due to presence of SAC70.
Oil spreading assay
The diameter of the clean zone, which appears after dropping a surfactant-containing solution on an oil-water interface, was measured using the oil displacement test. The studied SAC’s effectiveness at reducing surface tension can be assessed using the binomial diameter. A 15 cm diameter beaker was filled with 40 ml of distilled water. The surface of the distilled water was covered with 50 µl of mineral oil to make a thin covering, and 10 µl of test supernatant solution was added to the centre of the oil surface35.
Emulsification index
Utilizing a hydrocarbon (Kerosene) and the same volume of culture supernatant used to test the presence of SAC, the emulsification index (EI24%) was calculated. The mixture was then vortexed for two minutes at a high speed and allowed to stand for 24 h71. The emulsification index was calculated by EI24(%) = (Total height of the emulsified layer /Total height of the liquid layer) x 100.
Foamability assay
The bacterial culture was grown in the mineral salt medium (MSM) and centrifuged to take culture supernatant31. The CS was vigorously shake and check the foaming capability due to the presence of SAC produced by the P. gergoviae TYB1. The following formulae was applied to calculate foamability (%), Foaming Capacity (%) = (Foam height/Total height of the solution) × 100.
Bacterial adhesion to hydrocarbons (BATH) assay
The BATH test is a technique for measuring the bacterial adhesion capability on hydrophobic surfaces. The optical density (OD) was measured at 600 nm at about 0.5, after the cell pellet had been collected, twice washed with phosphate buffer salt solution (g/l, 16.9 K2HPO4, and 7.3 KH2PO4), and suspended with the buffer solution in an equal amount. 2 ml of cell suspension and 100 l of crude oil (n-hexane) were combined in an eppendorf tube, and the vortex was shaken for three minutes. The solution was maintained one hour for separation after mixing well. Finally, the OD of the aqueous phase was detected at 600 nm following the separation of the two layers (oil and aqueous). To check cell adherence to the crude oil, the percentage was taken for the calculation from the OD values.
% of bacterial cell adherence = [ 1 – (OD shaken with oil /OD original)] × 100.
Optimization of culture conditions for the SAC production by RSM method
P. gergoviae TYB1 was grown in the mineral salt medium (MSM) and centrifuged to take culture supernatant. SAC production was confirmed by standard methods such as the drop collapse assay, oil spreading assay, emulsification index, and foaming assay31. Bacterial growth was optimized to produce SAC using the one factor at a one-factor-at-a-time technique. The maximum production capacity may be achieved by a few variables, including temperature (from 25 °C to 40 °C), pH (from 2 to 10), incubation period (from 24, 48, 72, 116, and 140 h), carbon (starch, glucose, sucrose, lactose, maltose, and fructose) and nitrogen sources (yeast extract, beef extract, urea, NH4Cl, and (NH4)2SO4 ranging from 1 to 6% (w/v). All the factors were examined effectively using substrates that varied in range and concentration. The efficiency of SAC production was ascertained by examining the emulsification activity in each set of experiments with the highest degree of SAC production. Graphical and statistical analyses were used to determine accuracy42. All samples were processed for growth measurement (OD600) using spectrophotometric analysis, and the emulsification layer/activity (EI24%) was calculated according to72. The RSM approach was used after a lab-scale test to optimize the production of SAC by examining the concentrations of various physical and chemical parameters51. ANOVA was used to evaluate the statistical significance of RSM products.
Extraction and characterization of extracted SAC from P. gergoviae TYB1
Extraction of SAC
To collect crude SAC, incubated culture was centrifuged at 8,000 rpm for 10 min to collect cell free supernatant then acidification was done with 6 N HCl to maintain pH 2.0. The mixture was kept for overnight at 4˚C. Precipitate SAC was collected by centrifugation at 10,000 rpm for 20 min at 4˚C. Followed by centrifugation, then pellet was dissolved in 10 ml of distilled water. Purification was performed by vigorously mixing with chloroform and methanol (2:1 v/v) twice, after that solvent phase was separated by using separating funnel, then solvents were evaporated, and SAC was weighted. Partially purified SAC was kept at 4 °C.
Compositional analysis of SAC
The phenol sulfuric acid method73 was used to measure the total carbohydrate content of SAC, with D-glucose used as a standard. The colorimetric method74 was used to determine the lipid content, oleic acid was used as a standard. The Lowry’s method75 was used to measure the total protein content by using bovine serum albumin as a standard.
Thin layer chromatography (TLC)
After being dissolved in methanol at a concentration of 100 g/ml, a sample of partially purified SAC (0.1 g) was pulled through a glass capillary and spotted onto a silica gel (Merck) plate at a depth of roughly 1 cm. The tank containing the plate was then filled with the mobile phase, which was a mixture of chloroform, methanol, and acetic acid (65:15:2, v/v/v). The TLC plate was taken out and allowed to air dry once the solvent had reached the top of the plate. The retardation factor (Rf) value of the SAC bands was determined by the following formulae: Rf = The distance travelled by the compounds/ The solvent’s travel distance. Chemical visualization techniques such as 1% ninhydrin solution (1 g ninhydrin in 100 ml distilled water after treatment, then the plates were incubated at 100 °C for 15 min until the appearance of definite spots), UV exposure (10 min) and iodine test (10 min) for detection of lipids, sugars, and free amino groups, respectively, were used to check partially purified SAC main ingredients76.
FT-IR spectra of the dried biosurfactants
The SAC was examined by FT-IR analysis using a Bruker 113 V FT-IR spectrometer with a liquid N2 cooled mercury-cadmium-telluride (MCT) detector. KBr (200 mg) and SAC (2 mg) were combined to create powder. Then the powder was compacted into a tiny pellet that could be easily measured using an FT-IR spectrum with a wave number range of 4000 –400 cm− 1. OPUS 3.1 (Bruker Optics) software was used to analyse IR spectra.
GC-MS analysis
The sorts of components contained in the retrieved SAC were examined using GC-MS analysis along with their molecular weight. The recovered SAC sample was injected in one millilitre on a 5MS column of a GC-MS device (Perkin Elmer, Massachusetts, USA) with helium serving as the mobile phase. Using a CP 3800 Saturn 2200 GC-MS, the qualitative and quantitative analyses were conducted. The set temperature ranged from 80 to 350 °C at a rate of 3 °C per minute. 200 degrees Celsius was the ion temperature, and the scan range was 20 to 500 atomic mass units. Based on a comparison of their mass spectra with the Wiley library, the identification of chemical components was done77.
Investigation of LDPE biodegradation by P. gergoviae TYB1 based on enhancement of biofilm formation and level of biodegradation by SAC treatment
Biofilm formation analysis of P. gergoviae TYB1 was performed using the test tube method to determine the efficiency of bacteria for surface adhesion78. Subsequently, experiments were performed using LDPE films. The LDPE film was sterilized with 70% ethanol and dried overnight at 45 °C79. Weight loss of the LDPE films was determined using a weighing balance. To check biofilm formation and biodegradation efficiency of P. gergoviae TYB1, LDPE was inoculated in Minimal Medium (MIM) containing (g/l): KH2PO4, 0.2; CaCl2, 0.1; MgSO4.7H2O, 0.1; NH4H2PO4, 0.05; FeSO4, 0.035; Urea, 1.0 with P. gergoviae TYB1 and incubated for 30 days at 37 °C in 120 rpm in the rotatory shaker. As a control, LDPE film alone was introduced into the optimized medium. Biofilm formation on the surface of the LDPE film was observed in the following experimental setups: a- LDPE + medium (control), b- LDPE + medium + P. gergoviae TYB1, and c- LDPE + medium + P. gergoviae TYB1 + SAC (1 mg/50 ml).
Characterization of LDPE film for biodegradation
Weight loss analysis of LDPE
P. gergoviae TYB1 colonised the surface and created a biofilm over the LDPE. 2% SDS (v/v) (aqueous sodium dodecyl sulphate) and 70% ethanol solutions were applied to the LDPE surface twice, followed by a final wash with distilled water80. The washed LDPE samples were gathered on filter paper and then allowed to dry for a whole night at 60 °C before being weighed81. The equation below was used to compute the weight loss.
Percentage of weight loss (%) = [(Final Weight-Initial weight)/ Initial weight] × 100.
Viability of surface attached bacteria on LDPE
After the incubation period, the LDPE film was taken out of the culture medium and carefully rinsed with water to get rid of any medium residue or loosely adherent cells. Using 70% ethanol as a fixative, the bacterial biomass on the film was dried for 24 h at room temperature. An inverted microscope was used to study the bacterial biofilm biomass after it was stained with Coomassie brilliant blue staining. Viable cells were expressed as colour cells and dead cells were stained by Coomassie brilliant blue stain and expressed as blue colour due to the loss of cell membrane integrity and dead cells allows the stain to enter the cells.
FT-IR analysis of LDPE film
In the frequency range of 4000 –400 cm− 1, a Perkin Elemer, USA, Model: Spectrum RX1 was employed with a resolution of 2 cm− 1. Relative absorbance intensities were determined to be 1740 cm− 1 for the ester carbonyl bond, 1715 cm− 1 for the keto carbonyl bond, 1650 cm− 1 for the terminal double bond made of vinyl, 908 cm− 1 for the internal double bond, and 1465 cm− 1 for the methylene bond. Keto carbonyl bond index (KCBI) = I1715 / I1465; Ester carbonyl bond index (ECBI) = I1740 / I1465; Vinyl bond index (VBI) = I1650 / I1465; Internal double bond index (IDBI) = I908 / I1465; crystallinity (%) = 100 – [(1 - (Ia/1.233Ib)/1 + (Ia/Ib)) × 100]67.
Field-emission scanning Electron microscopy (FE-SEM) analysis for confirmation of biofilm formation on the surface of LDPE
LDPE films were exposed to P. gergoviae TYB1 culture for 30 days were examined using FE-SEM to determine the formation of biofilm and erosion of LDPE surface. After removing of media adhering with bacterial colonies by washing LDPE films in 0.01 M phosphate buffer for 2 min, the bacterial morphology of the biofilm on the PE surface was investigated. LDPE films were taken out of the media and washed with 2% SDS and warm distilled water for 10 to 20 min to get rid of the bacterial biomass before being checked for surface modification. LDPE films were then fixed in 4% glutaraldehyde at 4˚C for 2 h and dehydrated in 50% ethanol for 30 min. Overnight, 70% ethanol was used to incubate the LDPE films at room temperature, dried, mounted and sputter coated with gold for 40 s and scanned through FE-SEM77.
Statistical analysis
All the experiments were repeated three times, and all the data was represented with mean value ± standard deviation at the significant level of P < 0.001.
Data availability
All supporting data and protocols have been provided within the article. P. gergoviae TYB1 genome was submitted to NCBI GenBank using the following accession number: JF738074 and on the following link: https://www.ncbi.nlm.nih.gov/nuccore/JF738074.1/.
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This research work was funded by the Department of Science and Technology (DST), Government of India, New Delhi, India under Women Scientist-B scheme [File no.: DST/WOS-B/WWM-6/2021(G)].
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Conceptualisation was performed by NS and BV: methodology was provided by BV, PG and SDKS: data curation was done by NS and BV: formal analyses, investigation and editing were conducted by CK, STS, and RK: writing—original draft and editing were prepared by NS, BV, VK, and VS: approved funding and supervision. All authors reviewed the manuscript.
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Soni, N., Kumarasamy, V., Gupta, P. et al. Enhancement of low-density polyethylene biodegradation through the production of surface-active compounds by Pluralibacter gergoviae TYB1. Sci Rep 15, 23270 (2025). https://doi.org/10.1038/s41598-025-04163-5
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DOI: https://doi.org/10.1038/s41598-025-04163-5
