Adaptation of Pseudomonas sp. AKS2 in biofilm on low-density polyethylene surface: an effective strategy for efficient survival and polymer degradation
© Tribedi et al.; licensee Springer. 2015
Received: 14 January 2015
Accepted: 10 March 2015
Published: 25 March 2015
Pseudomonas sp. AKS2 can efficiently degrade low-density polyethylene (LDPE). It has been shown that this degradation of LDPE by AKS2 is correlated to its ability to form biofilm on the polymer surface. However, the underlying mechanism of this biofilm-mediated degradation remains unclear. Since bioremediation potential of an organism is related to its adaptability in a given environment, we hypothesized that AKS2 cells undergo successful adaptation in biofilm on LDPE, which leads to higher level of LDPE degradation. To verify this, the current study investigated a number of parameters of AKS2 cells in biofilm that are known to be involved in adaptation process.
Successful adaptation always develops a viable microbial population. So we examined the viability of AKS2 cells in biofilm. We observed the presence of viable population in the biofilm. To gain an insight, the growth of AKS2 cells in biofilm on LDPE at different time points was examined. Results showed a better reproductive competence and more colonization for AKS2 biofilm cells than planktonic cells, indicating the increased fitness of AKS2 biofilm cells than their planktonic counterpart. Towards understanding fitness, we determined the hydrolytic activity, different carbon source utilization potentials, functional diversity and homogeneity of AKS2 biofilm cells. Results showed increased hydrolytic activity (approximately 31%), higher metabolic potential, higher functional diversity (approximately 27%) and homogeneity for biofilm-harvested cells than planktonic cells. We also examined cellular surface hydrophobicity, which is important for cellular attachment to LDPE surface. Consistent with the above results, the cell surface hydrophobicity of biofilm-harvested AKS2 cells was found to be higher (approximately 26%) compared to that of their planktonic counterpart. All these results demonstrated the occurrence of physiological as well as structural adaptations of AKS2 cells in biofilm on LDPE surface that resulted in better attachment, better utilization of polymer and better growth of AKS2 cells, leading to the development of a stable colony on LDPE surface.
The present study shows that AKS2 cells in biofilm on LDPE surface undergo successful adaptation that leads to enhanced LDPE degradation, and thus, it helps us to understand the underlying mechanism of biofilm-mediated polymer degradation process by AKS2 cells.
KeywordsPolyethylene-based plastic material Bioremediation Pseudomonas Biofilm Adaptation
In the modern era, plastic-based materials have a variety of domestic and industrial applications. However, the widespread use of this non-biodegradable material poses a major threat to the environment. An example of a widely used non-biodegradable polymer is polyethylene. Although there are reports of microbial degradation of polyethylene, the rate is very slow [1,2]. Moreover, this microbial degradation requires the pre-oxidation of polyethylene, either by physical or chemical treatment [1-4]. Previously, we reported that Pseudomonas sp. AKS2 can degrade 5% ± 1% of low-density polyethylene (LDPE) in just 45 days, without any prior oxidation of polyethylene . This report also documented that AKS2 developed biofilm on polyethylene surface efficiently, and there was a linear correlation between this biofilm formation and the ability to degrade polyethylene . However, the underlying mechanism of this biofilm-mediated LDPE degradation by AKS2 remains unclear.
Biofilm represents a complex association of microorganisms in a given habitat , and its formation is a bacterial survival response to hostile environment . Microorganisms are known to be capable of altering their structural and physiological activities through biofilm formation as this allows survival under varied environmental conditions. Such alteration in activities for better survival in a given habitat is known as adaptability.
In general, adaptation is a biological process by which an organism becomes more competent to live in a given habitat . Following adaptation, microorganisms exhibit different physiological and structural activities compared to their non-adapted counterparts . Thus, adaptation provides a kind of biological insurance for an organism to encounter varying environments in a given ecological niche. The adaptive traits may be structural, behavioural or physiological. Structural adaptation includes variations in shape and size of the organism. The alteration of membrane fluidity, by both psychrophilic and thermophilic bacteria, is an example of structural adaptation. This type of modulation in membrane fluidity serves as a protection against harsh temperatures. While behavioural adaptations are composed of inherited behaviour chains, physiological adaptations allow the organism to perform special functions for the adjustment of cellular growth and development, regulation of temperature, etc. Bacterial secretion of exo-polysaccharides for their attachment to a solid surface is an example of physiological adaptation.
Existing literature documents that bioremediation potential of an organism is related to its adaptability in a given environment . Adapted Rhodococcus erythropolis DCL14 cells were shown to degrade alkanes and alcohols at higher rate compared to their non-adapted counterparts . Thus, we hypothesized that successful adaptation of AKS2 population in biofilm on LDPE surface resulted in its sustained retention at this site, which resulted in enhanced polymer degradation.
To verify this hypothesis, we investigated the various parameters of AKS2 structure and physiology such as fitness, metabolic potential, functional diversity and homogeneity that are relevant to the adaptation. We observed increased fitness, higher levels of functional diversity and homogeneity, and increased cell surface hydrophobicity for biofilm adapted cells compared to the planktonic cells, indicating the physiological and structural adaptations of AKS2 cells in biofilm. Thus, these results demonstrate that successful adaptation of AKS2 cells in biofilm on LDPE surface resulted in enhanced degradation of LDPE.
Bacterial strain and culture condition
Pseudomonas sp. AKS2 was previously isolated from Kolkata municipal solid waste dumping ground soil (Kolkata, India) . It is a potential degrader of polyethylene succinate (PES)  and LDPE . This isolate was grown in 100 ml of sterile basal media containing 300 mg of sterile LDPE films at 30°C for different time periods as per the requirement of the experiments. Basal media were prepared as described previously . Commercially available LDPE was used in all the experiments. LDPE films were made additive free by washing with 70% ethanol. The surface area of each polyethylene film used was 5 × 4 cm.
Dual staining for the assessment of AKS2 viability in biofilm
To determine the viability of AKS2 cells in biofilm on polyethylene surface after the incubation, LDPE films were removed from the conditioned medium and the adhered bacterial population, if any, were stained with 4 μg ml−1 acridine orange for 15 min . Thereafter, LDPE films were washed with sterile Milli-Q water (Millipore Corporation, Billerica, MA, USA) and further treated with 4 μg ml−1 ethidium bromide for another 15 min. LDPE films were again washed with sterile Milli-Q water. Thereafter, dried films were observed under a fluorescence microscope (Olympus IX 71, Olympus Corporation, Tokyo, Japan).
Measurement of AKS2 fitness
Microbial fitness represents the ease of reproduction of an organism in a given environment. In order to examine the fitness of AKS2 biofilm cells, we compared the colonization and reproduction potential of biofilm-harvested cells with that of planktonic AKS2 cells. For this experiment, AKS2 cells were grown for 30 days in media containing LDPE films as sole C-source. After the incubation, cells that adhered to LDPE films were extracted. These cells represent biofilm-harvested cells. To compare the colonization and reproduction potential, biofilm-harvested and planktonic AKS2 cells were inoculated separately in equal numbers (approximately 104 cells) into 100 ml of basal media containing 300 mg of LDPE film as sole C-source and incubated at 30°C for different lengths of time. After incubation for 5 and 10 days, LDPE films were taken out from the growth media and examined under a fluorescence microscope after staining with acridine orange as described in the previous section. All experiments were performed in triplicate.
Fluorescein diacetate hydrolysis assay
To examine the hydrolytic activity of bacterial cells either harvested from biofilm or planktonic condition, fluorescein diacetate (FDA) hydrolysis assay was performed . Briefly, equal numbers of cells (approximately 106 cells) taken from the respective origin were separately added to 1.5 ml of 60 mM sodium phosphate buffer, pH 7.6. FDA solution was added to it to attain a final concentration of 10 μg ml−1. The flask was then shaken at 30°C for 30 min. These samples were then centrifuged at 6,000 rpm for 5 min, and the absorbance of the supernatant was measured by a spectrophotometer (V-630, Jasco, Tokyo, Japan) at 494 nm. Samples without FDA served as a control.
Evaluation of bacterial cell surface hydrophobicity
Physiological profiles of AKS2 population
where L is the Lorenz curve and F is the standardized cumulative distribution of the standardized population.
For cluster analysis, data from the richness tests using BiOLOG-ECO plates were collected from either biofilm-harvested cells or planktonic cells of AKS2. The similarity matrix was generated by Euclidean distances, which were used to build a dendrogram with the unweighted pair group mean averages (UPGMA) algorithm wherein the linkage was single. Cluster analysis was performed by using the software Minitab 16.
Experimental results were subjected to statistical analysis of one-way analysis of variance (ANOVA) in order to evaluate statistically significant differences among samples. Mean values were compared at different levels of significance using the software Minitab 16. All experiments were performed in triplicate.
AKS2 cells exhibit increased fitness for their growth on LDPE surface
Biofilm-harvested AKS2 cells exhibit increased functional diversity and metabolic activity
AKS2 cells exhibited increased functional homogeneity in biofilm
Biofilm-harvested AKS2 cells exhibit higher level of cell surface hydrophobicity
Polyethylene surface modulates the adaptation of AKS2
The present study investigated structural and physiological properties of AKS2 cells during LDPE degradation. It has been reported that adapted marine microorganisms can survive in extremely unfavourable environmental conditions containing high concentrations of pollutants and toxic substances like heavy metals, hydrocarbons, xenobiotics and other recalcitrant compounds by forming biofilm . Similarly, LDPE degradation by AKS2 was also found to be increased concomitantly with the increased biofilm formation . Though biofilm was shown to enhance bioremediation, the high cell density inside a biofilm is known to cause a stressful environment [24,25]. The ability of an organism to adapt to the different microenvironments in biofilm is an important survival strategy against this environmental stress [26,27]. In addition, a previous report has documented that under stressful conditions, microorganisms undergo phenotypic diversification to enhance their adaptive potential . Towards understanding the adaptation, we observed that biofilm cells on polyethylene surface exhibited higher metabolic activity, in particular hydrolytic activity, compared to planktonic cells. This increased metabolic activity may help these cells to degrade and utilize the polymer to establish a sustainable population. Biofilm-harvested cells also exhibited increased functional diversity. This indicates that microorganisms are undergoing phenotypic diversifications, which leads to better adaptation. Moreover, biofilm-harvested cells exhibit lower Gini coefficient which is an indicator of increased functional homogeneity. Therefore, majority of the individual AKS2 cells within the population have similar levels of metabolic potential with respect to utilization of a wide range of carbon sources. This enables the individual microorganisms to efficiently grow and establish a stable population in biofilm as the possibility of intra-species competition is greatly reduced. It has been documented that Rhodococcus tolerates extreme conditions by structural adaptations such as the modification of cell membrane and the alteration in cell surface hydrophobicity . We also observed increased cell surface hydrophobicity of biofilm-harvested AKS2 cells compared to their planktonic counterpart. This increased cell surface hydrophobicity enables AKS2 cells to attach to the hydrophobic LDPE surface more efficiently compared to planktonic cells. The emergence of this trait demonstrated the occurrence of structural adaptation in biofilm of AKS2 cells on polyethylene surface. Taken together, the increased metabolic potential, higher level of functional diversity and homogeneity, and the increased surface hydrophobicity resulted in better colonization and higher reproduction potential of biofilm-harvested AKS2 cells.
Phenotypic alteration by an organism in an imposed condition has been considered an important strategy for better adaptation . In our previous study, we observed that AKS2 cells in biofilm on LDPE surface exhibited a significant alteration in their shape and size compared to the planktonic form . In biofilm, AKS2 cells become more round shaped and smaller in size than planktonic cells . This structural adaptation may provide AKS2 cells a better access to the available nutrients in its surroundings. In the same study, we also observed that AKS2 cells adhering to LDPE secreted exo-polysaccharides for their better attachment and formation of biofilm on the polymer . Again, it suggests the occurrence of physiological adaptations of AKS2 cells on LDPE surface. Collectively, these results demonstrated that AKS2 cells have adapted successfully in biofilm on LDPE surface resulting in a viable population and better degradation of the LDPE polymer.
The stability of an ecosystem depends on the balanced interactions between biotic and abiotic components . These biotic and abiotic components are connected together through nutrient cycles and energy flows . Thus, the abiotic surface of an ecosystem is likely to play a significant role towards adaptation of an organism in a given ecological niche. Towards this, our results showed different levels of functional diversity and evenness of the same organism, AKS2 cells, for two different polymers: LDPE and PES. Thus, the result demonstrates the possible involvement of LDPE towards adaptation of AKS2 cells in biofilm on LDPE surface.
Towards understanding biofilm-mediated LDPE degradation by AKS2, we verified the adaptation of AKS2 cells in biofilm by examining their viability and fitness. The results showed a viable population of AKS2 cells in biofilm with increased fitness compared to their planktonic counterpart. Further investigation revealed higher metabolic potential, higher functional diversity and homogeneity, and higher level of surface hydrophobicity for the biofilm-harvested AKS2 cells than planktonic cells. All these physiological and structural properties are known to be connected to the adaptability of an organism, and thus, these observations strongly support the view that AKS2 cells have adapted successfully in biofilm and thus developed a viable and stable population which resulted in enhanced polymer degradation. Thus, the current study deciphers the underlying mechanism of LDPE degradation by AKS2 biofilm cells with an enhanced rate.
In conclusion, the current study demonstrates structural and physiological adaptation of AKS2 cells in biofilm on polyethylene surface wherein the nature of the polymer plays an important role. This adaptation leads to enhanced LDPE degradation through biofilm formation.
Analysis of variance
Average well colour development
Bacterial adhesion to hydrocarbon
- G :
- H :
Shannon diversity index
- L :
Unweighted pair group mean averages
We thank Dr. Srimonti Sarkar for critical reading of the manuscript. This work is supported by a grant in aid from the Department of Biotechnology, Government of West Bengal, India (Sanction no. 555-BT (Estt)/RD-21/11).
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