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Navigating novel biological routes to migitate biofouling is of great worth inorder to allow sustainable performance of Membrane Bioreactors (MBRs) in wastewater treatment technology. Recently, it was confirmed that a number of natural compounds in plants, and vegetables have an anti-biofouling effect, reducing the formation of biofilm. This study addressed the Pseudomonas aeruginosa PAO1, a Gram-negative bacterium for anti-biofouling knocks of Piper betle extract (PBE). Various concentrations (1000-5000 µg/mL) of PBE were used to determine the Minimum Inhibitory Concentration (MIC) (3000 µg/mL) that was required not to kill the cells but grow at its minimum. The anti-biofouling effects of PBE were evaluated via a microtiter plate assay; time kills studies (TKS) and changes in the growth (µ) profile. The morphology of biofilm formed on membrane was also studied via SEM analysis. PBE revealed a significant reduction (≥80 %) in biofilm formation and growth rate (87% ± 0.04) by Pseudomonas aeruginosa.
Membrane bioreactors (MBRs) have emerged as one of the innovative technologies in wastewater treatment (Yun et al. 2006). However, high-quality purification systems have faced a major problem due to biofilm formation on the membrane surface, or biofouling. It reduces permeate flux, shortens the membrane life, increases the membrane cost and eventually adds additional capital cost for membrane replacement. The control of membrane irreversible fouling resulting from strongly bound fouling materials is a difficult and challenging task (Yu et al. 2010).
So far, extensive research has been pursued to investigate the possible methods to prevent or reduce membrane biofouling. Many physico-chemical methods have been used, for regular physical and chemical cleaning, etc. (Ramesh et al. 2006), and may not be effective and energy efficient. Sometimes it is hard to reach all the areas that are contaminated with biofilm. Acidic and alkaline solutions are sometimes used to remove biofilm from surfaces by washing, but there is an issue of adverse environmental impact. Thus it appears that biological control of microbial attachment would be a novel promising alternative for mitigating membrane biofouling and would be a new niche that deserves further study (Xiong and Liu 2010). It would be better to prevent biofilm formation rather than killing the cells after it forms. However, killing the cells using antibiotics, as practiced in industry, for example, does not always work, because it is not usually possible to kill all the cells completely for an extended time, and some cells still can attach onto the solid surface to form a biofilm (Costerton 1999).
Many natural products of plants are well known for antimicrobial activities (Kubo et al. 2006), and in this study, it was hypothesized that these may help reduce biofilm formation (Sendamangalam et al. 2011). No information, however, is available on the Piper betle (L.) extract to biofouling control in membranes. Consequently, the present study was undertaken to evaluate the knocks of PBE on biofilm formation and growth by Pseudomonas aeruginosa PAO1.
Material and methods
The materials used in this study were nutrient broth for cell culture; crystal violet dye (MERCK, 101408-0025) was used to stain the biofilm cells. Methanol (PROLABO) was used to fix the attached bacteria. Glacial acetic acid (DMF, Fisher scientific) was used to resolubalize the dye bound to the adherent cells. A sterile 96 well clear flat bottom tissue culture microtiter plate (Corning, SIGMA) with a lid was used for biofilm assay. All materials were autoclaved for 20 min at 1200C before use.
Model Bacterial strain
Natural environment bacterial variance leads to an extremely complex biofilm system that is poorly known. Therefore, for this work, a single representative bacterium, namely Pseudomonas aeruginosa PAO1 was used as a model bacterium because of its ability to foul surfaces rapidly, its rapid reproduction rate, and its significance as a pathogen (Lee et al. 2010). This species is gram negative; rod shaped (1.5-2 µm long and 0.3-0.6 µm wide) and can be cultured from almost all natural waters.
Preparation of bacterial suspension
The stock of the Pseudomonas aeruginosa PA01was kept in glycerol at -70 °C for further use. The bacterial specie was revived in nutrient broth at 37 °C overnight. The cells were harvested by centrifugation at 10000 rpm for 10 min. The cells were then resuspended in nutrient broth, and the concentration was standardized at 106 cells/ml by using a spectrophotometer (Shimadzu) at 550 nm (Nalina and Rahim. 2006). A synthetic wastewater (Yun et al. 2006) (Table 1) was used as the bacterial growth medium to study the biofilm.
Preparation of plant extract
Piper betle L. leaves were obtained from one source in Menkatab, Pahang, Malaysia. Aqueous extract of P. betle was prepared by boiling small pieces of the fresh leaves of the plants in distilled water for several hours until the final volume was one third of the initial volume. Following this, the decoction was centrifuged at 10,000 rpm to eliminate sediments. The supernatant was divided, into one ml aliquots, in micro-fuge tubes. It was concentrated using a speed-vacuum concentrator (HETO/HS-1-110, Denmark). The concentrated extracts were weighed into sterile micro-fuge vials and prepared into stocks of 20 mg/ml using sterile distilled water as diluents (Fathilah and Rahim, 2003). The extracts were dissolved by sonicating the microfuge vials in a sonicator (DAIHAN).
Determination of MIC of Piper betle extract
The MIC of PBE was assessed using a broth dilution method (Smullen et al. 2007). Briefly, Pseudomonas aeruginosa PAO1 was grown overnight in nutrient broth medium. A 0.1 mL sample was taken from the culture when the stationary growth phase was reached after 16 hrs. The sample was transferred to culture tubes that contained 15 mL of the culture medium. The PBE solutions were prepared as follows: The PBE was dissolved in DMSO at different concentrations resulting in a 1% DMSO solution when added to the broth. The solution of 1% DMSO and PBE of various concentrations were tested for their effects on the growth of Pseudomonas aeruginosa. 0.1 mL of the PBE solutions of different concentrations were taken and added to the culture tubes. At a regular interval, 0.1 mL of the solution from each culture tube was serial-diluted and plated on the nutrient agar plates. The plates were incubated at 37°C and colonies were counted after 24 hrs. The MIC assay was done in triplicates, and the averages of the results were taken.
Biofilm susceptibility assay
The Pseudomonas aeruginosa PAO1 was grown over night in synthetic dye wastewater containing glucose, at 37 °C. Aliquots of 100 μL were inoculated in 9 parallel wells of a 96-well microtiter plate. Two microliter of PBE was added to each well. The final concentration of PBE in a well was 3000 µg/mL. Control (without PBE) was also included in the study. The plate was incubated at 37 °C for 12, 24, 36, 48, 60 and 72 h. Then the content of each well was aspirated. The wells were rinsed three times after incubation period with 150 µl of physiological saline. The plate was vigorously shaken so that non-adherent bacteria removed and fixed the remaining bacteria with 100 μL of 99.99 % ethanol for 10 min. The liquid was poured off from the plate, and the plate was dried in the air. The adhered bacterial material was stained adding 100 μL of crystal violet (2 %) for 15 min. The plate was rinsed with tap water to rinse off excess violet and the plate was air dried. The dye bound to the adherent cells was re-dissolved with 100 µl of 33% (v/v) glacial acetic acid per well (Burton et al. 2007) and the adhered cells were quantified via an ELISA reader (TECAN) at 570 nm. The tests were done in triplicates, and the averages were taken.
Effect on growth profiles
The antibacterial activity of PBE was measured via an assay procedure, which involved the alterations in the optical absorbance as an indication of the bacterial profile. A total of 50 μL of this suspension was used to inoculate 20 ml of synthetic dye wastewater with concentration of 3000 μg/ml of PBE. Control (untreated with extract) was also included in the study. The contents of each tube were mixed well using a vortex mixer. The OD readings of each tube were set to zero with a test tube containing everything else except the bacterial cells. This was to accommodate differences in OD of the mixture caused by the varying colour intensities of plant extract. The cultures were incubated at 37 °C in shaking water bath and the growth of Pseudomonas aeruginosa was monitored by measuring the changes in OD of each tube periodically via spectrophotometer (U-1800, HITACHI) and recorded at every 30 min intervals over a period of 8 h. The growth curves of bacterium at various concentrations were plotted and compared with the profile of control. The growth rate (µ) under different growth conditions were then determined using the following equation (Cappuccino and Sherman, 2005). All tests were carried out in triplicate and repeated three times for reproducibility of results.
Where, N is No. of cells at log phase, No is No of cells at zero time and t is time to reach, to is zero time log phase.
Time kill studies
The Pseudomonas aeruginosa was grown in synthetic dye wastewater at 37 °C for 24h. The turbidity of suspension was adjusted to 0.5 McFarland standard in sterile normal saline. A total of 200 μl of this suspension was used to inoculate 20 ml of nutrient broth with increasing concentrations of PBE ranging from 1000 to 5000 μg/ml. Control (untreated with extract) was also included in the study. Suspensions were incubated at 37 °C for 24h, and the number of CFU was determined on nutrient agar using a serial dilution method at various time points (Eliopoulus et al. 1996).
The development of the biofouling (biofilm) layer on membrane with time (12h and 36h) was assisted by SEM. Typical SEM micrographs gathered at different operating times, starting at a virgin membrane. The morphology difference in membrane surface treated with PBE and the control was observed. The voltage used was 15KV with 1.5 nm resolution. Membrane for surface analysis was prepared by cutting hollow fibre membrane with razor blade. The sample was fixed with 2% (v/v) glutaraldehyde in 0.1M phosphate buffer at pH 7.2 for 2h and then washed twice for 10 min and again immersed for 1h in 0.1M phosphate buffer. The fixed sample was dehydrated with ethanol and coated with aurum-platinum alloy (with coating depth 10 nm) for 2 min. The specimens were air dried in a desiccator at 250C for 24h before being delivered for further instrumentation analysis.
All experiments were conducted in triplicates and results obtained in experiments were expressed in terms of means (average) and standard deviation (±) using SPSS 10.0 software. Probability (p-value) less than 0.05 and 0.01 was considered significant and highly significant, respectively.
Results and discussion
Minimum Inhibitory concentration (MIC) of PBE
Various concentrations (1000-5000 µg/mL) of the PBE were used and the minimum concentration was selected that was needed not to kill the cells but grow at its minimum as the MIC. Figure 1 shows the log (CFU/mL) results for the cells measured soon after the addition of PBE. The control in figure 1 shows the number of cells in the growth medium without the PBE. These results demonstrate that the CFU, i.e. cell viability in the presence of the PBE was not significantly affected, when the PBE concentration was at the MIC. The concentration of PBE was selected at 3000 µg/mL. This would ensure that the addition of extract would not kill the bacteria cells but instead would allow the growth of cells to be its minimum.
As seen in Figure 1, it can be clearly observed that the bacterial culture grew exponentially until the beginning of stationary phase and started to die once the PBE were added. The PBE were added after 16 hours of inoculation. The MIC value of PBE against Pseudomonas aeruginosa was close to the finding reported by other researchers (Fathilah et al. 2009).
Biofilm susceptibility assay
The microtiter plate, results are shown in figure 2 and the percentage reduction from the control is shown in table 2. Each bar graph represents the absorbance of crystal violet dye bound to biofilm cells. Therefore, a large absorbance indicates more biofilm formation. The increasing time resulted in a less reduction of biofilm. PBE exhibited an inhibitory effect on the formation of biofilm generated by Pseudomonas aeruginosa, with a 79% minimum biofilm inhibition at concentration of 3000 µg/mL. Biofouling causing bacteria are protected by formation of biofilm. Bacteria in a biofilm are invariably less susceptible to antimicrobial agents than their planktonic counterparts (Wilson 1996). Unlike the effects of PBE on planktonic cells, as determined by MIC, PBE exhibit ≥ 80% reduction of biofilm even at 72h biofilm. However, in terms of 79% minimum biofilm reduction, we found that PBE have significant effect on the formation of biofilm by Pseudomonas aeruginosa.
Time Kill studies
The time kill kinetic studies were specifically performed against Pseudomonas aeruginosa owing to its importance in the initiation of biofilm formation. The results of time kill studies are shown in Fig 3. The MIC of PBE (3000 µg/mL) showed a 3-log reduction in growth in 10h, compared to the control. The kill kinetic study showed that PBE extract exhibited a time dependent killing effect against Pseudomonas aeruginosa.
Growth profile and growth rate
The growth profile in the figure 4 strongly suggested that antimicrobial activity of PBE towards Pseudomonas aeruginosa was bacteriostatic and may have been targeted at the early lag phase of the growth cycle. PBE seemed to have created an environment for the cells to perform their normal biological functions. This explains for the reduction (87% ± 0.04) in growth rate (0.10 µ ± 0.03). The attainment of minimal population size as the bacteria enters the stationary phase indicated the bacteriostatic activities. Under the stressed growth environment the bacteria were unable to perform normal biological function and eventually ceased to propagate. Such growth inhibiting mechanism has also been reported when the requirement for the nutrient was restricted for Streptococcus sanguinis growth (Fathilah et al. 2007).
The SEM profiles could reveal the development of biofilm on the surface of membrane and the treated membrane as clearly shown in figure. Figure shows the view of the virgin membrane, which demonstrates a porous structure of the randomly oriented fibres with variable sub-micron to micron diameter. Figure shows the control membrane without treatment of PBE. The surface of this membrane was comprised of bacteria clusters covered with biopolymers, indicating that biofouling occurs on the membrane surface (An et al. 2009). One could clearly visualize the foulants on this membrane. Although there was a clear interface between membrane and foulant, the foulant might still diffuse into the membrane to some extent. SEM revealed a developed biofilm and the extracellular polymeric substances (EPS) (Figure). The EPS production and presence can be linked to the growth of micro-organisms (Ivnitsky et al. 2005).
These investigations exhibit that PBE is effective in reducing biofilm formation and growth rate caused by Pseudomonas aeruginosa. At MIC level, PBE used in experiments, reduced the bacterial growth rate and biofilm formation without killing all the bacterial cells. How PBE inhibit biofilm formation was not investigated. Further study is necessary to determine the antibiofouling mechanisms and the application of PBE in membrane reactor systems.