Isolation, Identification, and Evaluation of

Philippine Journal of Health Research and Development

Isolation, Identification, and Evaluation of Polyethylene Glycol and Low-Density Polyethylene-Degrading Bacteria from Payatas Dumpsite, Quezon City, Philippines Nicole R. Bolo*1, Ma-an Jane C. Diamos1 Glenn L. Sia Su1, Melody Anne B. Ocampo1, Lani M. Suyom1 *Corresponding author’s e-mail address: [email protected] 1

Department of Biology, University of the Philippines, Manila

R E S E A R C H A R T I C L E Abstract Background: The use of plastics plays a significant role in today's global economy. However, the problem arises when these plastics are not properly managed and they end up in the environment such as in Payatas Dumpsite. The potential of employing microorganisms in the degradation of these plastic wastes is not well-explored, particularly in the Philippines. Objectives: This study aims to isolate and identify the bacteria present in Payatas Dumpsite that have the potential to degrade plastic components such as Polyethylene Glycol (PEG) and Low Density Polyethylene (LDPE) films. Methodology: Grab soil and leachate samples were obtained from Payatas Dumpsite and cultured microorganisms were morphologically and biochemically identified and evaluated for their plastic degrading capabilities. Results: Results of the study showed that the microorganisms, namely Kocuria kristinae, Dermacoccus nishinomiyaensis, Pseudomonas stutzeri, and Acinetobacter haemolyticus were present in the soil samples. These microorganisms' plastic degrading capabilities were proven through their emission levels of carbon dioxide. Scanning Electron Microscope (SEM) microphotographs displayed the plastic degradation of the microorganisms. Conclusion: The microorganisms isolated from Payatas Dumpsite have the potential to degrade plastics, particularly PEG and LDPE. Future studies could be done regarding the extraction of enzymes used by the isolates and the subsequent pathway for the plastic degradation process employed by the four microorganisms. Key words: plastic polymer-degrading bacteria, biodegradation, Payatas dumpsite, PEG, LDPE

Introduction Metro Manila, or the National Capital Region (NCR), is the largest metropolitan center in the Philippines. Although NCR is the smallest region in the country in terms of land area, it has the second highest population figure [1]. The increasing population in the region causes different types of environmental problems, such as accumulation of waste products including proper plastic waste disposal. In the Philippines, domestic usage of 30, 740 metric tons of plastic packaging fills 1M cubic meters of landfill [2]. In clearing waterways and road repairs due to accumulation of plastic wastes, the government spends P791 million [2] 50

which could have been instead redirected to providing basic social services. In spite of this, plastic products have brought tremendous convenience to our lives. Polyethylene (PE) has been utilized in making grocery bags, food packaging films, and toys; polypropylene (PP) for the creation of straws, car seats and container caps; and lastly, polystyrene (PS) for making disposable food trays, and laboratory wares [3]. The exceptional mechanical properties of PE and its relatively low cost led to the abundance and popularity of the mentioned polymer to the general public. It is also known that plastics, specifically PE, are inert materials [4]. This means they require long periods of time to be degraded. Phil J Health Res Dev March 2015 Vol.19 No.1, 50-59

Isolation, Identification and Evaluation of Polyethylene

The extreme durability of plastics has caused their accumulation in the environment at a rate of approximately 25 million tons per year [5]. As a result, plastic wastes have become omnipresent in the environment [6]. Local statistics also support the current and urgent need to address the problem of existing, and accumulating plastic wastes [2]. To address this problem, various disposal methods have been used in Southeast Asia [6]. However, suggested solutions for plastic disposal are certainly not without disadvantages. For example, composting is problematic due to high operating and maintenance costs, poor maintenance of facilities, and more expensive compost costs compared to commercial fertilizers [7]. Incineration is also known to emit greenhouse gases as a by-product. Landfills as well as open dumpsites, if not managed properly, will cause ground water contamination, gas migration, and also fire breakouts because of methane gas generation [7]. In fact, Payatas dumpsite has been reported to release an estimated amount of 2 liters/second or 63 million liters of leachate each year into ground water and river systems for the past 30 years [8]. Lastly, recycling and recovery has become a market-driven practice, in turn leading to their selectivity [7]. Economical and manufacturing problems arise because the current trend in packaging materials involves using neither non-recyclable nor non-reusable polymers [6]. Therefore, the researchers in this study propose another disposal method for plastic wastes. A plausible option is through the use of biological systems. To back this proposal, considerable work concerning the suggested method has been conducted by different researchers. One is by Gu, J.D. et al. [9] where they have worked on some strains of bacteria and fungi to degrade both natural and synthetic plastics. Another study is by Chandra and Rustgi [10] which showed significant biodegradation results of linear low-density polyethylene starch blends in a soil environment by a mixed fungal inoculum. Several other researches [11, 12, 13] demonstrated that partial biodegradation of PE was also possible. A problem with their work, however, is that although PE biodegradation has been extensively studied, their results were based on PE blended with starch [14]. Only few reports that deal with microbial degradation of pure polyethylene glycol (PEG) and low density polyethylene (LDPE) exist [15, 16]. Phil J Health Res Dev March 2015 Vol.19 No.1, 50-59

Therefore, the aim of this study is to isolate microorganisms capable of degrading plastics, particularly low density polyethylene (LDPE) and polyethylene glycol (PEG). This is the first study in the Philippines which intends to evaluate the PEG and LDPE-degrading capabilities of bacteria isolated from a dumpsite. This is also the first study to use Fyrite® Gas Analyzer equipment in measuring the evolved CO2 levels of the plastic polymer-degrading isolates. A research work focusing on the isolation of bacteria capable of plastic degradation will provide a baseline project for bioremediation studies in the Philippines. The microorganisms isolated in this study can be subjected to further research and be used in decreasing the amounts of the PEG and LDPE plastics in the Payatas Dumpsite waste stream. The general objective of the study is to determine if there are plastic degrading-bacteria existing in Payatas Dumpsite, Quezon City. Specific objectives include (1) isolation of the bacteria capable of degrading plastic specifically polyethylene glycol (PEG) and low density polyethylene (LDPE); (2) identification of the plasticdegrading bacteria until species level; and lastly, (3) evaluation of the bacteria's capability to degrade LDPE films by measuring the CO2 evolution through FYRITE® Gas Analyzer and by surface visualization through scanning electron microscope (SEM) microphotographs.

Methodology Sample Collection At Post 5 Old Mound Payatas (N 14 ° 40' 33.749'' E 121 ° 2'37.321'), grab soil samples were taken from a pit with an approximate depth of 10-20 cm while grab leachate samples were taken from a creek. Samples were placed in sterile containers. Sample temperature and pH were measured in situ. The samples were kept at a temperature of 4°C and were brought to the laboratory for microbial analysis. Isolation of PEG-degrading bacteria Sample enrichment was done by adding fifty grams of the soil sample to 25mL de-ionized sterile water with 0.1 g of PEG powder, and 1.001 mL antifungal Nystatin™. The solution was incubated for one week at room temperature. The enriched sample was diluted several times and 0.1 mL 51


from each dilution was spread plated on Mineral Salt Medium (MSM) with PEG. This medium was specially formulated to provide an environment where the suspended polymer, PEG, was the sole carbon source of the microorganism. The plates were incubated for a week at 30°C. Resulting colonies were purified by streaking onto fresh MSM+PEG medium. The inoculated plates were then incubated at 30ºC for 72 hours. A clearing zone produced by the bacterial colonies signifies vigorous PEG degradation activity. Isolates which produced prominent clearing zones were then taken to be identified. Identification of the isolates The isolates were described using cultural, morphological, and biochemical characterizations. The isolates which showed prominent clearing zones were identified by biochemical tests, and subjected to VITEK 2TM identification system. The biochemical tests include the Catalase Test where an inoculum of each isolate was swabbed on a clean glass slide and a drop of 3% hydrogen peroxide (H2O2) was added to each of the bacterial inoculum. Presence of bubbles indicated a positive result for the test. In addition, the Oxidase Test was done for the second isolate to confirm its identity. On a sterile filter paper, a drop of oxidase reagent from BioMérieux was stained with the bacterial inoculum. Blue to purple coloration indicated a positive result for the test.

Surface Visualization using Scanning Electron Microscope (SEM) Randomly selected LDPE films from the control and treatment groups were viewed using SEM to visualize the surface of each film after exposing it to the plasticdegrading bacteria.

Results Samples The soil samples from the study site had an average pH of 7.8 and an average temperature of 33.3°C during the sample collection. The average leachate sample temperature was 30.83°C. Isolation Four bacterial isolates showed prominent clearing zones around their colonies (Figure 1).

Partial Evaluation through CO2 Evolution Test LDPE films (thickness: 50 microns) weighing 0.2 g each were sterilized in a beaker with a solution containing 70 mL Tween 80, 10 mL bleach, and 983 mL distilled water. The solution was stirred for 30 to 60 minutes and then transferred to another beaker containing distilled water and was shook for an hour. The plastic strips were aseptically transferred to an ethanol solution 70% v/v for 30 minutes. In each 250mL Erlenmeyer flask, a mixture of 100 mL sterile liquid MSM, an inoculum of each isolate, and an LDPE film were placed. The mouth of each flask was sealed using six layers of sterile aluminum foil. For one week, the flasks were placed on a rotary shaker at 150 rpm. A negative control set-up was also included, which only contained a sterile LDPE strip and the liquid MSM only. The set-ups were done in triplicates. All the treatments were incubated at room temperature for one week. The amount of headspace CO 2 evolved in each flask was measured by the equipment, Fyrite ® Gas Analyzer. 52

Figure 1. Isolates (a) EC-3 (Bacteria 1); (b) EC-7 (Bacteria 2); (c) EC8 (Bacteria 3); and (d) EC-5 (Bacteria 4) that showed clearing zones as represented by the yellow arrow in MSM+PEG plates after 24 hours of incubation at 30ºC. Phil J Health Res Dev March 2015 Vol.19 No.1, 50-59


Table 1 summarizes the characterization of the four selected isolates and Figure 2 shows the photomicrographs of the Gram staining of the four isolates. Table 1. Morphological and cultural profile of the four isolates which showed prominent clearing zones Morphological Cultural Code Characteristics Characteristics EC-3 Gram (+) cocci cream colored (Bacteria 1) opaque colonies, punctiform, convex elevation, entire margin EC-7 Gram (+) bacilli yellow white (Bacteria 2) colored opaque colonies, punctiform, flat elevation, entire margin EC-8 Gram (-) bacilli white colored (Bacteria 3) translucent colonies, circular, convex elevation, entire margin EC-5 Gram (-) bacilli orange colored (Bacteria 4) opaque colonies, punctiform, raised elevation, entire margin

Identification Biochemical characterization All isolates yielded a positive result, as formation of bubbles upon addition of H2O2 was observed (Figure 3). This indicates that all the isolates are capable of using oxygen as part of their metabolism.

a

b

c

d

Figure 3. Presence of bubbles as positive result for the catalase test of the four isolates: (a) Kocuria kristinae; (b) Dermacoccus nishinomiyaensis or Kytococcus sedentaris; (c) Pseudomonas stutzeri; (d) Acinetobacter haemolyticus.

Using VITEK 2 for Identification of the Isolates Using microbial growth-based technology, the equipment VITEK 2TM identified the four isolates. Table 2 presents the summarized identification results. The Identification Message Level indicates the % probability of the identification levels. The first isolate (EC-3) had a 94% probability as Kocuria kristinae, while the third (EC-8) and fourth (EC-5) isolates were identified as Pseudomonas stutzeri and Acinetobacter haemolyticus, respectively. In some cases, 2-3 taxa are presented as the probable identities of the unknown microorganism. This occurs when

a

b

c

d

Figure 2. Photomicrographs of the four isolates upon Gram staining. (a) EC-3 (Bacteria 1), a Gram positive coccus which appears in clusters; (b) EC-7 (Bacteria 2), a Gram positive rod; (c) EC-8 (Bacteria 3), a Gram negative rod which occurs singly; and (d) EC-5 (Bacteria 4), a Gram negative rod. Phil J Health Res Dev March 2015 Vol.19 No.1, 50-59

53


the biopattern of a microorganism is a representative of a collective taxon or closely-related organisms. This is called Mixed Taxa Identification and an example of which is the second isolate (EC-7) which had 97% probability to be either Dermacoccus nishinomiyaensis or Kytococcus sedentaris. To identify which between them is the identity of the isolate, ® the oxidase test using oxidase reagent from BioMérieux was employed. The positive test confirmed that the organism was D. nishinomiyaensis (Figure 4). Table 2. VITEK 2 identification and probability of the four isolates from the enrichment culture

Code

Bacteria

Probability

EC – 3 Kocuria kristinae

94%

EC – 7 Dermacoccus nishinomiyaensis

97%

EC – 8 Pseudomonas stutzeri EC – 5 Acinetobacter haemolyticus

91% 91%

Only 3 samples were submitted to the laboratory: one for the control group and two for the treatment groups which were chosen randomly. The treated groups were plastic films exposed to Kocuria kristinae and Dermacoccus nishinomiyaensis. It was observed that surface alterations in the form of dents and etching were present in the inoculated LDPE films. However, none of these were found in the control treatment. Table 3. Determination of the levels of carbon dioxide (Co2) evolution after seven (7) days of incubation with LDPE film samples

Isolate Acinetobacter haemolyticus Dermacoccus nishinomiyaensis Kocuria kristinae Pseudomonas stutzeri Control

Fyrite Reading (in percent)

Equivalent CO2 in Parts per Million (ppm)

0.75

7500

0.25

2500

0.25

2500