screening and study on microorganisms degrading biopolymers in vietnam

Table of contents INTRODUCTION 1 CHAPTER 1: LITERATURE REVIEW 4 1.1. PLASTIC WASTE POLLUTION 4 1.1.1. Plastic waste pollution in the world 4 1.1.2. Plastic waste pollution in Vietnam 5 1.1.3. Treatment of plastic waste 6 1.1.3.1. Landfill 6 1.1.3.2. Recycling 6 1.1.3.3. Incineration 7 1.2. BIODEGRADABLE PLASTICS 8 1.2.1. Biodegradable plastics 8 1.2.1.1. Poly(Lactic Acid) (PLA) 11 1.2.1.2. Poly(3-Hydroxybutyrate) (PHB) 12 1.2.1.3. Poly(ε-Caprolactone) (PCL) 13 1.2.2. Applications 13 1.2.2.1. Medicine and pharmacy 14 1.2.2.2. Packaging 15 1.2.2.3. Agriculture 15 1.2.2.4. Others fields 16 1.3. THE DEGRADATION OF BIOPOLYMERS 17 1.4. MICROORGANISMS DEGRADING BIODEGRADABLE POLYMERS . 19 1.4.1. Microorganisms degrading PLA 19 1.4.2. Microorganisms degrading PHB 22 1.4.3. Microorganisms degrading PCL 23 CHAPTER 2: MATERIALS AND METHODS 25 2.1. MATERIALS 25 2.2. CHEMICALS 25 2.3. EQUIPMENTS 26 2.4. METHODS 26 2.4.1. Isolation of biopolymer-degrading microorganisms 26 2.4.2. Screening biopolymer-degrading microorganisms 27 2.4.3. Identification of biopolymers-degrading strains 27 2.4.3.1. Gram staining method 27 2.4.3.2. Observation under scanning electron microscopy (SEM) 28 2.4.3.3. Extraction of genomic DNA from bacteria 29 2.4.3.4. Amplification of the 16S rDNA PCR reaction 29 2.4.3.5. Agarose gel electrophoresis 30 2.4.3.6. Sequencing 30 2.4.3.7. Effect of culture conditions 31 2.4.3.8. Utilization of of sugars 31 2.4.3.9. Activity of some extracellular enzymes 31 2.4.4. Study degradation of biodegradable polymers by isolated strains 32 2.4.4.1. Growth experiment in the PLA, PHB or PCL containing media 32 2.4.4.2. Measurement of the PLA, PHB or PCL residual weight 32 2.4.4.3. Determination of TOC in culture broth 32 2.4.4.4. Degradation experiment with biopolymer film 34 2.4.4.5. Statistical analysis 34 CHAPTER 3: RESULTS AND DISCUSSION 35 3.1. ISOLATION AND SCREENING PLA, PHB, PCL-DEGRADING ORGANISMS 35 3.2. PLA-DEGRADING MICROORGANISMS 37 3.2.1. Identification of strains G5 and Cz1 37 3.2.1.1 Morphology of strain G5 and Cz1 38 3.2.1.2. 16S rDNA sequencing of strain G5 39 3.2.1.3. Biochemical and physiological characteristics of strains G5 and Cz1 41 3.2.2. PLA degradation by S. thermoflavus G5 and P. citrinium Cz1 44 3.3. PHB-DEGRADING MICROORGANISM 47 3.3.1. Identification of strain B2 47 3.3.1.1. Morphology of strain B2 47 3.3.1.2. Sequencing 16S rDNA gene of strain B2 48 3.3.1.3. Biochemical and physiological characteristics of strain B2 49 3.3.2. PHB degradation by B. gelatini B2 52 3.4. PCL-DEGRADING MICROORGANISM 54 3.4.1. Identification of strain B1 55 3.4.1.1. Morphology of strain B1 55 3.4.1.2. Sequencing 16S rDNA gene of strain B1 56 3.4.1.3. Biochemical and physiological characteristics of strain B1 57 3.4.2. PCL degradation by Br. agri B1 60 3.5. DEGRADATION OF POLYMERS BY ISOLATED STRAINS 61 CONCLUSIONS 64 FURTHER STUDY 65 REFERENCES 66 WEB REFERENCES Master thesis 2010 Trinh Thi Van Anh 1 INTRODUCTION 1. The urgency of the subject: In recent years, environmental pollution is a major concern of many countries around the world. Pollution caused by plastic waste seems to increase. Use of polymer products that derived from biological sources replaced synthetic plastic products is a new direction in reducing environmental pollution. Bio-plastics aggregated from many different types of polyesters as poly(lactic acid) (PLA), poly(3-hydroxyrate (PHB), poly(ε-caprolactone) currently have attracted much attention because of their potential application in many fields such as: in packaging, agriculture, medicine, biodegradable plastics and other areas. Biodegradable polymers that were capableof degradation by both microorganisms and enzymes are currently considerable as the sustainable recycling method for polymers. Microorganism is one of the factors affecting the degradation of bio-polymers. Polymer-degrading microorganisms could be found in different environments such as soil, sea, water, compost, activated sludge… [55]. In this connection, scientists are now focusing on isolation and selection of microorganisms that are able to increase polymer degradation. Recently, various investigations of microbial degradation of polymers have been published [23], [56], [63]. In Vietnam, environmental pollution caused by plastic waste is an alarming issue. The increasing use of bio-polymer products in day life promotes both research and application of polymer-degrading microbes to reduce environmental pollution. Thus, isolation and selection of strains that can degrade bio-polymers were initially carried out. However, information about microorganisms degrading biopolymers and application is still very limited [32], [33], [34]. The topic: "Screening and study on microorganisms degrading biopolymers in Vietnam" was therfore inplemented in order to contribute to solving the existing plastic pollution problem in Vietnam. Master thesis 2010 Trinh Thi Van Anh 2 2. The aim of the study - Screen microorganisms that are capable of degrading PLA, PHB and PCL. - Classify the isolated strains and study optimal conditions for the growth of these strains. - Biopolymer-degradation activity of the isolated strains. 3. Content of the study Screening microorganisms capable of degrading PLA, PHB and PCL by clear-zone methods and by measuring the growth of microorganisms in the medium supplemented with polymers. Identification of isolated strains based on the phenotypic, biochemical and physiological characteristics together with 16S rDNA sequencing. Study the polymer degradation of the isolated strains by measuring the total organic carbon (TOC) and measuring the loss of polymer residual in the culture broth. 4. Practical applicability In the future, we intend to apply these isolated strains in degradation of biopolymers in the compost. 5. Contribution of the study It is the first time strains capable of degrading PLA, PHB and PCL were screened in Vietnam. Based on the phenotypic, biochemical and physiological characteristics together with 16S rDNA sequencing, strains G5 and Cz1 that were capable of degrading PLA were indentified as Streptomyces thermoflavus and Penicillium citrinum, respectively. Thus, this is the first publication about strains belonging to genera Streptomyces and Penicillium that were capable of degrading PLA. It is also the first time thermophilic members of genus Bacillus that were capable of degrading PHB and PCL were published. Based on the phenotypic, biochemical and physiological characteristics together with 16S rDNA sequencing, these strains were identified as Bacillus gelatini and Brevibacillus agri. Master thesis 2010 Trinh Thi Van Anh 3 6. Distribution of thesis Thesis contained 85 pages, 5 tables, 35 figures, and 74 references. Beside introduction, conclusions, futher study, reference and appendix, thesis concluded 3 chapters: Chapter 1: Literature review Chapter 2: Materials and methods Chapter 3: Results and discussion Master thesis 2010 Trinh Thi Van Anh 4 Chapter 1: LITERATURE REVIEW 1.1. PLASTIC WASTE POLLUTION 1.1.1. Plastic waste pollution in the world Environmental pollution is one of the biggest subjects in nowadays. There are many kinds of pollution, such as air, water, noise, soil pollution…, among them, solid waste pollution is the most pressing issues. The quantity of solid waste is greatly increasing due to the increase of population, development activities, and changes in life style. Plastic waste of all kinds presents a significant and costly form of pollution. In the United Kingdom, plastics made up around 7% of the average household dustbin. The amount of plastic waste generated annually in the United Kingdom was estimated to be nearly 3 million tons in 2002. It was estimated that 56% of all plastics waste is used packaging, three-quarters of which is from households, however only 7% of total plastic waste has been recycled [67]. This situation was similar in the United State, India and many other countries [68], [71]. In the world, annually 100 million tons of plastics has been used, thus causing a significant problem for the environment [49]. The plastic pollution causes a range of environmental impacts. Plastic production requires significant amount of resources, primarily fossil fuels, both as a raw material and energy source for the manufacturing process. According to World Watch Institute (United State), it takes 430,000 gallons of oil to produce 100 million plastic bags. In the world, 4% of the annual oil production is used as a feedstock for plastic production and additionally 3-4% as energy source for manufacturing process [67], [69]. Plastic production also involves the use of potentially harmful chemicals, which are added as stabilizers or colorants. Many of these have not undergone environmental risk assessment and their impact on human health and the environment is currently uncertain. In developing countries, plastic bags are used to carry food by citizens - as they are cheap, convenient and air tight. Liquid, spicy or Master thesis 2010 Trinh Thi Van Anh 5 fatty food items are packed in colored plastic bags, and carcinogens are likely to be generated during chemical reactions taken place in the bags, due to temperature variations. Colored nylon bags used to contain food, can infect contents with metals like lead and cadmium, which harm human’s brains and lungs [38], [67]. The disposal of plastic products also contributes significantly to their environmental impact. The presence of plastic in our environment is killing many animal species [69]. According to Greenpeace, more than 1 million birds and 100,000 marine mammals are estimated to perish each year by either eating or becoming trapped in plastic waste. Sea turtles, whales and dolphins are among sea animals being directly affected by plastic waste products, often mistaking plastic bags for food, causing slow and painful deaths to these animals over a prolonged period of time [69]. 1.1.2. Plastic waste pollution in Vietnam Pollution caused by solid waste is an alarming issue in Vietnam. Untreated waste now affects the environment, land, water as well as people's health. According to a report, the quantity of solid waste in all cities increased significantly, from 1478 tons in 2000 at Lamson landfill (Hanoi) to 2540 tons in 2004. At this time, the total amount of solid waste released in Vietnam is around 28 million tons per year. In particular, in 2004, the amount of solid waste was 15.5 million tons. It is predicted that the amount in 2015, 2020, and 2025 would be approximately 43.6, 67.6, and 91.6 million tons, respectively [70]. Plastic waste was a significant portion of the total solid waste. It is estimated that plastic waste occupied approximately 10% of municipal solid waste [70]. A large portion of plastic waste pollution came from plastic bags. Because buyers found them comfortable to use while sellers used them as an effective advertising tool and companies enjoy diversifying the design of their plastic bags to attract more customers, thus the bags themselves were used more and more popular. According to Ministry of Natural Resources and Environment of Vietnam, each household uses five plastic bags per day, and that amounts to 90 million bags in the entire country. Among them, only 3-4% of plastic in Vietnam is recycled, while the Master thesis 2010 Trinh Thi Van Anh 6 rest is dumped or buried into soil. This situation leads to severe pollution and affect to the citizen life. 1.1.3. Treatment of plastic waste There are several ways to treat the pollution of the plastic waste in the world and in Vietnam. 1.1.3.1. Landfill A landfill is a site for the disposal of waste materials by burial and is the oldest form of waste treatment. Historically, landfills have been the most common methods of organized waste disposal and remain so in many places around the world. Landfills may include internal waste disposal sites (where a producer of waste carries out their own waste disposal at the place of production) as well as sites used by many producers. Many landfills are also used for other waste management purposes, such as the temporary storage, consolidation and transfer, or processing of waste material (sorting, treatment, or recycling). In the past, plastic waste was not separated from waste. It was dumped at the landfill and remained a long time after, so a large number of adverse impacts may occur. Because most plastics are non-degradable, they take a long time to break down, possibly up to hundreds of years [73]. Over time they go through a process of light degradation and break down into smaller pieces that cannot be converted by any known organism and as such remain as plastic in landfills, rivers and oceans. With more and more plastic products, particularly plastics packaging, the landfill space required for plastics waste is a growing concern. Thus, plastic bags can choke the earth, they are making soil unfertile, contaminate ground and water through leaching of toxic substances. Recently, modern landfills in industrialized countries are operated with controls to attempt manage the problems, the plastic waste is not dumped to the landfill, instead of, it is isolated from rubbish and treated in special way. 1.1.3.2. Recycling Recycling used to prevent waste of potentially useful materials. Recyclable materials include many kinds of glass, paper, metal, plastic, textiles, and electronics. Master thesis 2010 Trinh Thi Van Anh 7 It reduces the consumption of fresh raw materials, reduces energy usage, reduces air pollution (from incineration) and water pollution (from landfilling) by reducing the need for "conventional" waste disposal, and lower greenhouse gas emissions as compared to virgin production. The United States Environmental Protection Agency (EPA) has concluded that recycling reduced the country's carbon emissions by a net of 49 million metric tons in 2005 [72]. In the United Kingdom, the Waste and Resources Action Programme stated that Great Britain's recycling reduced CO 2 emissions by 10-15 million tons a year [72]. However, this work was often difficult or too expensive (compared with producing the same product from raw materials or other sources), therfore only a small quantity of plastic waste has been recycled. 1.1.3.3. Incineration Fig 1. Incinerator in Hinwil, Switzerland [73]. Fig 2. Incinerator in Nam Dinh province, Vietnam [74]. Incineration is a waste treatment technology that involves the combustion of organic materials and substances. In developed countries, incineration of waste materials converts the waste into incinerator bottom ash, flue gases, particulates, and heat, which can in turn be used to generate electric power. The flue gases are cleaned before they are dispersed in the atmosphere. In developing countries, this solution is only used as burn materials. Incinerators reduce the mass of the original waste by 80–85 % and the volume (already compressed somewhat in garbage trucks) by 95-96 %, depending upon composition and degree of recovery of materials such as metals from the ash for recycling [73]. Thus, it reduces the necessary volume for disposal significantly. [...]... count and the clear zone methods using emulsified polyester agar plates are very efficient methods in the evaluation of the population of polymer -degrading microorganisms in the environment [35] By applying the clear zone method, it was confirmed that the population of aliphatic polyester -degrading microorganisms at 30 and 50°C decreased in the order of PHB = PCL > PBS > PLA [41] 1.4.1 Microorganisms degrading. .. ensilage and trays with seeds are applications mentioned for biopolymers Containers such as biodegradable plant pots and disposable composting containers and bags are other agricultural applications The pots are seeded directly in the soil, and break down as the plant begins to grow In marine agriculture, biopolymers are used to make ropes and fishing nets They are also used as support for the marine cultures... degraded into low molecular weight oligomers, dimers and monomers and finally mineralized to CO2 and H2O [55] The ecological and taxonomic studies on the abundance and diversity of polymer -degrading microorganisms in the different environment are necessary because they are responsible for the degradation of plastic materials Polymers are degraded in the soil by the action of a wide variety of microorganisms. .. health and the environment by emitting toxic chemicals such as sulfur dioxide and even dioxin [38] It is the reason why, incinerators have been used with the limitation in many countries 1.2 BIODEGRADABLE PLASTICS With the advances in technology and the increase in the global population, plastic materials have found wide applications in every aspect of the life and industries However, most conventional... minutes at 4oC - Rinse again by CaCodylate buffer Trinh Thi Van Anh 28 Master thesis 2010 - Remove water by ethanol with different concentrations: 30o, 50o, 70o, 90o, 100o Each concentration in 10 minutes - Create a well on the sole and drop the sample above into the well - Sample was golden-plated on JFC-1200 machine in 30 seconds at 30 mA - Scanning on the electron microscopy JSM-5421LV (Japan) in. .. minutes It was cooled to 60oC and poured into plates To overlay polymers film onto agar plates, polymers were added to chloroform solution (0.01 g/ml) and 250 µl solution was spread on the agar plates [22] Agar MSM medium containing emulsified polymers were used for screening polymers -degrading microorganisms Polymers emulsion was prepared by dissolving 2g PLA, PHB or PCL in 20 ml chloroform by sonication... car doors and dashboards (Toyota Internet site) Starch-based polymers are used as additive in the manufacturing of tires (Fig 5) It reduces the resistance to the movement and the consumption of fuel and in fine greenhouse gas emissions [66] Electronics: PLA and kenaf are used as composite in electronics applications Compact disks based on PLA are also launched on the market by the Pioneer and Sanyo... well in the medium containing 1% PLLA and the molecular weight of PLLA decreased by 35.6% after 3 days incubation with shaking at 60°C A newly isolated PLLA -degrading thermophile Geobacillus sp strain 41 was reported [61] The time course of PLLA degradation was monitored at 60°C for 20 days and degradation was confirmed by the change in molecular weight and viscosity of the residual polymer The PLLA -degrading. .. sludge, fresh and marine water (including deep sea), estuarine sediment, and air [20], [21] It reported for the first time the PHB -degrading microorganisms from Bacillus, Pseudomonas and Streptomyces species [8] From then on, several aerobic and anaerobic PHB -degrading microorganisms have been isolated from soil (Pseudomonas lemoigne, Comamonas sp Acidovorax faecalis, Aspergillus fumigates and Variovorax... fermentation of biomass on feedstock including sucrose and corn and tapioca starches It is expected that PLA produced by fermentative processes will replace many conventional plastics produced from petrochemicals Industrial lactic acid-producing microorganisms mainly produce L-lactic acid at high concentration of over 100 g/l, with low production of D-lactic acid At high concentration and purity, only a . application is still very limited [32], [33], [34]. The topic: " ;Screening and study on microorganisms degrading biopolymers in Vietnam& quot; was therfore inplemented in order to contribute. environmental pollution. Thus, isolation and selection of strains that can degrade bio-polymers were initially carried out. However, information about microorganisms degrading biopolymers and. strains and study optimal conditions for the growth of these strains. - Biopolymer-degradation activity of the isolated strains. 3. Content of the study Screening microorganisms capable of degrading