Effects of CeO 2 and ZnO Nanoparticles on Anaerobic Digestion and Toxicity of Digested Sludge

Transcription

1 Effects of CeO 2 and ZnO Nanoparticles on Anaerobic Digestion and Toxicity of Digested Sludge by Nguyen Minh Duc A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Environmental Engineering and Management Title Page Examination Committee: Prof. Chettiyappan Visvanathan(Chairperson) Dr. Oleg Shipin Dr. Gabor Hornyak Nationality: Previous Degree: Scholarship Donor: Vietnamese Bachelor of Science in Biotechnology University of Dalat, Vietnam Deutscher Akademischer Austausch Dienst (DAAD), Germany AIT Fellowship Asian Institute of Technology School of Environment, Resources and Development Thailand May 2013 i

2 Acknowledgements I would like to express my deepestand profound gratitude to my advisor, Prof.C. Visvanathan for his valuable guidance and supports and also his constant encouragement that help me to complete this thesis. Working with him not just only develops my scientific working method, but also greatly boosts my confidence and passion to continue this profession. I also would like to express my gratefulness to the examination committee members,dr. Oleg Shipin and Prof. Louis Hornyak,for their valuable advices and suggestions. I like to express my special thank to Dr. Jega Jegatheesan of Deakin University, Australia for providing nanoparticles and experimental supports for this research work. I am grateful for the DAAD Scholarship that financially supports me to complete my master degree here. My special thanks give to all Professor Visu s Research Team members, Mr. Le Minh Truong, Mr. Nguyen Hong Phuc, and Mr. Paul Jacob for their constant assistances, encouragement and above all, for being my friends. I am thankful to all the administrative and laboratorial staffs of the Environmental Engineering and Management Program for their support. Many thanks are also extended to staffs and researchers in the Center of Excellent in Nanotechnology and Biotechnology Laboratory for their guidance. Finally, I would like to send my deepest gratitude to my grandmother, my parents, my brother and everyone in my family for their love and care that give me inspiration and encouragement to complete my master program in AIT. ii

3 Abstract With the rapid growth of nanotechnology in past decade, nanoparticles, such as CeO 2 and ZnO, are now widely commercialized in many products. The industrialization and commercialization of NPs have made the release of these compounds to the environment and wastewater treatment plants become inevitable. However, many researchers have found that conventional WWTPs can effectively remove NPs from the wastewater.it means that the higher the removal efficiency of NPs, the higher NPs exist in the waste sludge. Therefore, NPs can accumulate to a very high concentration in the waste sludge. However, information about impact and toxicity of NPs on sludge treatment stream is still very restricted. As a result, this research aimed to study abouteffects of CeO 2 and ZnO NPs on sludge anaerobic digestion process, sludge dewatering process, and toxicity of sludge to bacteria and plants. The result showed that CeO 2 and ZnO NPs could cause inhibition to the biogas production of anaerobic digestion system. The exposure concentration of ZnO at 1,000 mg/l caused greatest inhibition to the biogas volume (65.3%) and the methane composition (40.7%), as compared with controlled sample. In addition, at tolerable exposure concentration of ZnO, the system could overcome the inhibition effect after 14 days of incubation. In the other hand, CeO 2 at low concentration of 10 mg/l could increase the generated biogas volume by 11%. The positive effect of CeO 2 at low concentration was also observed on bacterial toxicity test. The ZnO NPs was more toxic to bacteria than CeO 2 NPs at same exposure concentration. However, the bacterial toxicity of both nanoparticles were reduced when they were applied on the sludge. Moreover, after went through anaerobic digestion process, the bacterial toxicity was again lessen. Additionally, required time to dewater the digested sludge was increased proportionally with the exposure concentration of nanoparticles. Finally, the accumulation of CeO 2 and ZnO NPs on sludge made the digested sludge become unsuitable to be used as biosolid, since the contaminated digested sludge caused great inhibition on root growth and seed germination of plants. In conclusion, CeO 2 and ZnO nanoparticles greatly impactedthe anaerobic digestion system by inhibiting the biogas production process. Moreover, they made digested sludge become difficult to dewater. Besides, the toxicity of nanoparticles still remained even afteranaerobic digestion process that could inhibit the bacterial viability and seed germination and root growth of plants. iii

4 Table of Contents Chapter Title Page Title Page Acknowledgements Abstract Table of Contents List of Tables List of Figures List of Abbreviations i ii iii iv vi vii viii 1 Introduction Background Objectives of Study Scope of Study 2 2 Literature Review 3 2.1Nanoparticles Definition Classification of nanoparticles Application of nanoparticles Manufacture of nanoparticles Cerium oxide nanoparticles Zinc oxide nanoparticles Nanoparticles in Environment Nanoparticles in Wastewater Treatment Plants Release of nanoparticles in wastewater treatment plants Removal of nanoparticles in wastewater treatment plants Toxicity of Nanoparticles Toxicity of nanoparticles on various organisms Toxicity mechanism of nanoparticles Anaerobic Digestion Introduction Anaerobic digestion process Factors influence anaerobic digestion process Parameters indicate performance of anaerobic digestion Sludge Dewatering Effect of Nanoparticles on Anaerobic Digestion Future Outlook of Nanotoxicity 19 3 Materials and Methods Introduction Preparation and Characterization of Nanoparticles Preparation and Characterization of Sludge Preparation of Substrate Biochemical Methane Potential Test Experimental procedure Analytical method and calculation Capillary Suction Time 27 iv

5 3.7 Microbial Toxicity Test Culture of bacteria Toxicity test procedure Calculations of microbial toxicity test Phytotoxicity Test 29 4 Results and Discussions Nanoparticles Characteristics Effects of Nanoparticles on Anaerobic Digestion Optimization of BMP test Effects of nanoparticles on biogas production Effects of nanoparticles on methane production Effects of nanoparticles on sludge characteristics Effects of Nanoparticles on Sludge Dewatering Effects of Nanoparticles on Bacterial Viability Bacterial toxicity of nanoparticles Bacterial toxicity of sludge containing nanoparticles Effects of Nanoparticles on Plants 48 5 Conclusions and Recommendations Conclusions Recommendations for Future Study 52 References 53 Appendix A 57 Appendix B 59 Appendix C 65 Appendix D 67 Appendix E 70 Appendix F 73 Appendix G 75 v

6 List of Tables Table Title Page 2.1 Nanomaterials in Nanotechnology Nanoparticles Classification Groups Applications of Nanoparticles in Various Fields Sources of Nanoparticles in Environment Fates of NPs in Various Unit Operations in WWTPs Toxic Effects of Nanomaterials on Different Organisms Advantages and Disadvantages of Anaerobic Digestion Dewatering Performances of Various Techniques Effects of CeO 2 and ZnO NPs on Anaerobic Digestion Future Outlooks on Nanotoxicity Analytical Parameters and Testing Methods Particles Size of CeO 2 and ZnO Nanoparticles Characteristics of Sludge Mixture Before and After AD Characteristics of Digested Sludge at Various NPs Exposure Inhibitions of NPs to Seed Germination and Root Growth 48 vi

7 List of Figures Figure Title Page 2.1 Nanoscale materials Nanoparticles manufacturing methods Wastewater and sludge treatment processes in WWTPs Sedimentation of nanoparticles into the sludge of WWTPs Toxicity mechanisms of nanoparticles Steps of anaerobic digestion process Overall research methodology Modified bottle for BMP test CST apparatus Overall microbial toxicity test procedure Size distribution of CeO 2 nanoparticles Size distribution of ZnOnanoparticles Biogas production of BMP test at various I/S ratios Methane gas compositions of various I/S ratio samples Effects of CeO 2 and ZnO nanoparticles on biogas production Total volume of biogas production Possible mechanisms to overcome nanoparticles toxicity Methane composition of various samples Total volume of methane production of various samples Effects of NPs on sludge dewatering Protective mechanism of bacteria to overcome NPs toxicity Bacterial toxicity of CeO 2 nanoparticles dispersion Bacterial toxicity of ZnO nanoparticles dispersion Cytoprotective mechanism of CeO 2 NPs Bacterial toxicity of CeO 2 NPs on sludge supernatant Bacterial toxicity of ZnONPs on sludge supernatant Comparison of CeO 2 NPs toxicity of different samples Comparison of ZnO NPs toxicity of different samples Germination index of different samples Toxicity of digested sludge on bacteria and plant 50 vii

8 List of Abbreviations AD BMP CFU CNTs CODs CST DI DLS DNA ENMs ENPs EPS GC GI I/S LB NMs NOM NPs QDs ROS RPM STP TS UASB UV VFA VS WWTPs Anaerobic Digestion Biochemical Methane Potential Colony Forming Unit Carbon Nanotubes Chemical Oxygen Demand soluble Capillary Suction Time Deionized Dynamic Light Scattering Deoxyribonucleic Acid Engineered Nanomaterials Engineered Nanoparticles Extracellular Polymeric Substance Gas Chromatography Germination Index Inoculum/Sludge Luria-Bertani or Lysogeny Broth Nanomaterials Natural Organic Matter Nanoparticles Quantum Dots Reactive Oxygen Species Rounds Per Minutes Standard Temperature and Pressure Total Solid Upflow Anaerobic Sludge Blanket Ultraviolet Volatile Fatty Acid Volatile Solid Wastewater Treatment Plants viii

9 Chapter 1 Introduction 1.1 Background With the rapid growth of nanotechnology in past decade, nanoparticles (NPs) are now widely commercialized in many products. NPs have been used in paint, coatings, catalysts, biomedicine, cosmetics, skin creams, toothpastes and many other applications. Unique physicochemical characteristics (e.g. magnetic, optical and electrical features) make the use of NPs ideal in manufacture industries. Cerium oxide (CeO 2 ) and Zinc oxide (ZnO) nanoparticles have been popular in recent years and they have been applied widely in many fields. CeO 2 NPs have been used as fuel catalyst to reduce harmful emission from engine combustion. Moreover, these have been used as fuel cell electrolyte, antioxidant, semiconductor, UV absorber, coating and polishing chemical. ZnO NPs have been widely commercialized in consumer products, such as antibactericide coating, sunscreen and other industrial, medical and military applications. The industrialization and commercialization of NPs have made the release of these compounds to the environment becomes inevitable. NPs could be released either from the production process or consumption of products to industrial and municipal wastewater treatment plants (WWTPs). Recently, fate and transport of NPs in WWTPs and the toxicity of NPs with biological activities of various treatment processes have been investigated. These researches showed that NPs cause inhibition effects on biodegradation, nitrification and anaerobic digestion process (Liu et al., 2011; García et al., 2012). However, many researchers have found that conventional WWTPs can effectively remove NPs from wastewater. Agglomeration and adsorption of nanoparticles play a major role in the sedimentation of NPs in the sewage sludge, which resulted in low concentration of NPs in effluent (Brar et al., 2010). It means that the higher the removal efficiency of NPs in biological treatment, the higher NPs exist in the sludge. Therefore, NPs can accumulate to a very high concentration in the waste sludge. However, impact and toxicity of NPs on sludge treatment stream is still an abandoned area of research. Settled sludge from sedimentation tank is collected and treated in various steps before being sent to landfills or reused in other applications. Anaerobic digestion of waste sludge from biological process is a popular technique because of its economic and environmental benefits. The production of biogas from anaerobic digestion process is the main advantages of this technique. However, it has been reported that nanoparticles in sludge can cause adverse effects to the biogas generation of anaerobic digestion (García et al., 2012). Therefore, the inhibition effect of nanoparticles on performance of anaerobic digestion needs to be investigated. Moreover, the digested sludge after anaerobic digestion is usually dewatered and then applied as soil conditioner, compost and other applications. However, nanoparticles accumulated in sludge can make the sludge become toxic and inappropriate to apply as biosolid. Therefore, information about phytotoxicity and bacterial toxicity of digested sludge contaminated with nanoparticles is essential to have insights about the reusability of waste sludge. In addition, the effect of nanoparticles to the dewaterability of digested sludge is still unknown. So whether or not nanoparticles in sludge can hinder the sludge dewatering process, toxicity of nanoparticles in sludge is eliminated during anaerobic digestion or it causes inhibition effect on bacteria and plants, these are still questions that need to be answered. 1

10 The biochemical methane potential (BMP) test is a standard method to determine the methane yield of anaerobic digestion process and to monitoring anaerobic toxicity. This is a cheap, simple and reliable technique to estimate the rate and amount of methane production in the process (Chynoweth et al., 1993). By using the conventional capillary suction time (CST) test, dewaterability of digested sludge can be investigated. In addition, microbial toxicity test is the common method to assess and monitor the environmental pollution and toxic chemicals. Escherichia coli (E. coli) has been widely used as model organism to test the toxicity of chemical and sludge. Phytotoxicity of nanoparticles is commonly tested by studying the inhibition on seed germination and root growth. Tomato (Solanum lycopersicum) and mung bean (Vigna radiata) have been popularly used as test species and were recommended by US EPA(EPA, 1996). In this study, effects of CeO 2 and ZnO NPs on the methane gas production of sludge in anaerobic digestion process were investigated. Impacts of these nanoparticles on sludge dewatering process were also studied. The toxicity of sludge before and after anaerobic digestion were estimated, in order to know whether the toxicity of NPs could be eliminated by anaerobic digestion or not. Phytotoxicity of digested sludge contaminated with nanoparticles was studied to have conclusion on the reusability of digested sludge. Therefore, results obtained from this study helped us to understand fate and effects of CeO 2 and ZnO NPs on the whole sludge treatment process. 1.2 Objectives of Study The main objectives of this study are: 1. To evaluate the effect of CeO 2 and ZnO nanoparticles on biogas production from anaerobic digestion of sludge. 2. To evaluate the effect of CeO 2 and ZnO nanoparticles on dewaterability of digested sludge. 3. To evaluate the bacterial toxicity and phytotoxicity of digested sludge containing CeO 2 and ZnO nanoparticles. 1.3 Scope of Study To achieve above objectives, the scope of this study is set as follow: 1. Biochemical Methane Potential (BMP) test was conducted in laboratory scale at 30 o C to imitate the mesophilic anaerobic digestion process. The sludge wastaken from UASB reactor of Singha Beer Factory. 2. E. coli was used as indicator bacteria to assess the bacterial toxicity of the sludge. 3. Seeds of tomato (Solanum lycopersicum) and mung bean (Vigna radiata) were used in phytotoxicity test. 4. CeO 2 and ZnO NPs concentration of 10; 100; 500 and 1,000 mg/l were used in this study. 2

11 Chapter 2 Literature Review 2.1 Nanoparticles Definition Nanotechnology is the new areaa of development that works on matters at nanoscale (i.e. size range from 1 to 100 nm). It manipulates on nanomaterials (NMs), which is defined as material having at lease one dimension in nanoscale.table 2.1 shows examples of nanoscale materials in comparison with other common materials in term of size. Figure 2.1 Nanoscale materials According to the British Standards Institution (BSI, 2007), nanomaterials can be divided into three groups depending on number of dimensions at nanoscale. The Table 2.1 shows examples of some NMs of each group. Nanoparticles (NPs) can be simply defined as particles that have diameter smaller than 100 nm. From the definition of British Standards Institution,NPs are particles thatt have three dimensions sized from 1 to 100 nm. Due to there small size, NPs have large surface area to volume ratio, which results in higher reactivity and unique surface properties. This distinctive feature of NPs makes it become popular in manufacture of various consumer products. Table 2.1 Nanomaterials in Nanotechnology NMs Dimension(s) at nanoscale Examples Nanolayers Nano-objects Nanoparticles 1 Nanolayer 2 Nanowire, nanotube, nanorod Quantum dots, fullerenes, 3 metal and metal oxides NPs 3

12 2.1.2 Classification of nanoparticles There are various ways to classify nanoparticles based on different bases. However, the classification of NPs based on chemical basis is suitable for environmental studies. The characteristics of NPs depend greatly on their chemical origin, which affects their fate and behavior in environment (Stone et al., 2010; Farré et al., 2011). The four classification groups of NPs are showed on Table 2.2. Table 2.2 Nanoparticles Classification Groups Carbon Inorganic Organic Composites Carbon black Carbon Nanotubes Fullerenes Source: (Stone et al., 2010) Carbon NPs Metals Metals oxides 4 Polymers Dendrimers Surfactant coatings Quantum dots Fullerenes are molecules composed entirely of carbon, in the form of a hollow sphere (buckyball) or tube (carbon nanotube). Fullerene C60 (buckminsterfullerene) is the first fullerene to be discovered in 1985(Kroto et al., 1985). After that, the development in carbon nanoparticles mainly focused on the synthesis and application of carbon nanotubes (CNTs). Carbon NPs have outstanding thermal and electrical conductivities that allow it to be applied in varied fields. It has been used in fuel cell electrodes, capacitors, coatings, membrane filter, and electronic devices(farré et al., 2011). Inorganic NPs This group contains zero-valent metal NPs and metal oxide NPs. Zero-valent iron NPs can be used for the remediation of contaminated ground water, sediments and soils. However, silver nanoparticles is mostly applied in consumer products because of their bactericidal properties(klaine et al., 2008). Metal oxides NPs areone of the most studied and applied nanoparticles because of their unique features. TiO 2 and ZnO NPs can be found widely in sunscreens, cosmetics, paint, solar cell and bottle coatings due to their photolytic properties that can bloc UV light and transparency in nano formed. Cerium oxide (CeO 2 ) is mainlyused as a combustion catalyst in diesel fuels to improve emission quality. It also can be found in solar cells, gas sensors, and glass polishing. Organic NPs Organic NPs is usually made from polymer or branched polymer (i.e. dendrimers). Because of their biodegradability characteristic, organic NPs have been applied in many medical applications. These applications include drug delivery systems, polymer film and chemical sensors. Table 2.4 shows some very common application of organic NPs in medical field.

13 Composite NPs Composite nanoparticles are made from more than two elements, which could be either organic or inorganic basis. Quantum dots (QDs) are the most widely applied composite NPs, which is made from multi-inorganic components. The reactive core of quantum dots gives it magnificent optical characteristic. QDs are used in imaging application or applied in solar cell and telecommunications Application of nanoparticles Nanoparticles have special optical, physical and chemical characteristics. Properties of particles at nanoscale change in unpredictable ways that makes them different with same substance at bigger size. Special characteristics with high reactivity of nanoparticles make them become ideal for variety of fields, such as energy, electronic, medical and consumer products. Nanoparticles can contribute to produce stronger, lighter, cleaner, smarter and more efficient materials and products. Table 2.3 shows some applications of nanoparticles in various fields. According to The Project on Emerging Nanotechnology (2011), the number of consumer product from nanoparticles has been increasing dramatically within just 5 years from 2006 to By October 2011, the inventory incudes 1,317 products, which is five time more than that of March From the inventory of products that utilize nanomaterials, silver particles are the most commonly used NPs followed by carbon, titanium, silica, zinc and gold nanoparticles. According to the report of Organisation for economic co-operation and development (OECD), zinc oxide and cerium oxide nanoparticles are among 13 representativemanufactured nanomaterial(oecd, 2010). The list consists of: fullerenes (C 60 ), single-walled carbon nanotubes (SWCNTs), multi-walled carbon nanotubes (MWCNTs), silver nanoparticles (nano-ag), iron nanoparticles (nano-fe), titanium dioxide (TiO 2 ), aluminium oxide (Al 2 O 3 ), silicon dioxide (SiO 2 ), dendrimers, nanoclays, gold nanoparticles (nano-au), cerium oxide (CeO 2 ), and zinc oxide (ZnO). These are manufactured nanomaterials that commercialized, have high production volume and have hazardous reports. Therefore, to study moreabout toxicity and impact of CeO 2 and ZnO nanoparticles on other systems is crucial. More information about cerium oxide and zinc oxide nanoparticles is available in part and Manufacture of nanoparticles Nanoparticles can be manufactured from various techniques. However, it can be categorized into two groups, namely top-down and bottom-up strategies, which is showed in Figure 2.2(Ju-Nam and Lead, 2008). The top-down technique is the physical method, in which macro or micro scaled particles are size-reduced into nanoparticles. There are different techniques to reduce the size of bulk particles, such as grinding in ball mill or laser beam technique. In the other hand, the bottom-up technique is the chemical method to create nanoparticles from chemical reactions. After reaction time, the nanopowder is harvested by centrifuge the solution and then dry it into powder.for example, the sol-gel method (or wet chemical technique) is the mostly used technique to manufacture nanoparticles, especially metal oxide like zinc oxide and cerium oxide. This is a cheap, simple technique that gives a highly controlled nanoparticles. 5

14 Table 2.3 Applications of Nanoparticles in Various Fields Field Consumer Product Electronics Environment Energy Food Applications TiO 2, ZnO NPs is used in sunscreens to block UV from sunlight without leaving white residue on the skin Silver nanoparticles is applied in toothpaste, textiles ZnONPs added in paint to increase scratch resistance, surface hardness, UV resistance, corrosion protection and antiseptic effect Using Quantum Dots to replace the fluorescent dots used in current displays Transistors made from CNTs to minimize transistor size while increase efficiency Zero-valent Iron NPs clean up organic solvents in groundwater Reduce power loss in electric transmission by using wires containing CNTs, which have lower electric resistance Transparent solar cells made from Silver nanowires and Titanium dioxide NPs that can be used as window glass Zinc oxide NPs can be incorporated into plastic packaging to block UV rays and provide anti bacterial protection, while improving the strength and stability of the plastic film. Silica NPs is used to provide a barrier to gasses or moisture in plastic film used for packaging. This could reduce the possibly of food spoiling or drying out. Drug Delivery: Nanodiscs deliver chemotherapy drugs directly to cancer cells Medicine Therapy Techniques: Use Buckyballs to trap chemicals generated during allergic reaction Diagnostic Techniques: CNTs and Gold NPs can attach to proteins, allowing detection of diseases at early stage Anti-Microbial Techniques: Nanocrystalline Silver is used as antimicrobial agent for the treatment of wounds Space Using carbon nanotubes to make the cable needed for the space elevator or manufacture space ship. Source: (Hawk's Perch Technical Writing, 2007) 6

15 Figure 2.2 Nanoparticles manufacturing methods Cerium oxidenanoparticles Cerium oxide (Ceria) is an oxide of the rare earth metal cerium. It is a pale yellow-white powder with the chemical formula CeO 2. Cerium also forms Cerium (III) oxide, Ce 2 O 3, but CeO 2 (i.e. Cerium (IV) oxide) is the most stable phase at room temperaturee and under atmospheric conditions (Robinson et al., 2002). Cerium oxide nanoparticles have cubic (fluorite) structure that can be synthesize from various method, namely precipitation, ball milling, hydrothermal, pyrolysis and sol-gel method (Ju-Nam and Lead, 2008). Cerium oxide nanoparticles have been popular in recent years and they have been applied in many fields. The most popular application of CeO 2 NPs is as diesel fuel catalyst to reduce harmful emission from engine combustion. Cerium oxide nanoparticles have high oxygen-storing capacity that allow them to take and release free oxygen without decomposed. During that process, CeO 2 is reduced to Ce 2 O 3 that is re-oxidized when the combustion have enough oxygen again. Therefore, it can effectively reduce harmful NOx and CO 2 emissions as well as reduce fuel consumption and emission of soot particles of diesel engines from incomplete combustion (Selvan et al., 2009). Moreover, CeO 2 NPs are used as fuel cell electrolyte, antioxidant, and UV absorbents. CeO 2 has been a widely used industrial material for cleaning and polishing of silicon wafers that used in semiconductor manufacture. Because of its UV filter features without photocatalytic activity, ceria has been developed to replace ZnO and TiO 2 used in sunscreen. In addition, there are studies showed that ceria nanoparticles can act as antioxidant to protect cells against oxidative stress or inflammation (Xia et al., 2008). Therefore, it can be possibly applied in medical applications. There are many contrasting view about the effect of cerium oxide nanoparticles with biological system. Some researches found that cerium oxide have the protective effect for mammalian cell, since it can act like free radical scavenger to reduce ROS (Xia et al., 2008; Amin et al., 2011). In contrast, engineered cerium oxide nanoparticles have been reported to be toxic for bacteria (Thill et al., 2006; Pelletier et al., 2010) and inhibit activated sludge and anaerobic digester (García et al., 2012; Gomez-Rivera et al., 2012). Toxic mechanism of cerium oxide nanoparticles is suggested due to the oxidative stress when the nanoparticles attach to the membrane of bacteria (Thill et al., 2006). 7

16 2.1.6 Zinc oxide nanoparticles Zinc oxide has been widely applied in industries for centuries ago. It has interesting physical and chemical characteristics, like high electrical and thermal conductivity, high thermal stability, UV absorption, antimicrobial and stable at normal ph (Moezzi et al., 2012). The most application of zinc oxide is in rubber industry, where it is used to improve the durable and thermal resistance of tires. Recently, with the development of nanotechnology, zinc oxide nanoparticles, which have a smaller particle size as compare with industrial zinc oxide, have been developed and applied in various field to exploit its unique features. Basically zinc oxide nanoparticles posses chemical and physical characteristics same with its bulk size material. However, nanosize particles have higher reactivity and their own unique features, especially optical features that make them become interesting in emerging technologies. There are several techniques to manufacture zinc oxide nanoparticles, namely pyrolysis, sol-gel, chemical vapor deposition. Zinc oxide can be deposit on thin film, grow to nanowire, nanorod or made into nanoparticles (Ju-Nam and Lead, 2008). There are three crystal structures of zinc oxide, namely hexagonal wurtzite, cubic zincblende and cubic rock salt. However, in normal condition, the hexagonal wurtzite structure is commonly observed(özgür et al., 2005). Zinc oxides nanoparticles have excellent physical features (e.g. high thermal, electrical conductivity, UV light absorption), chemical features (e.g. stable at neutral ph, bactericidal activity) (Moezzi et al., 2012). Moreover, it can be manufactured at reasonable cost that makes ZnO NPs commercially available in many applications.zno nanoparticles have a wide and direct band gap that make it becomes ideal to apply in semiconductor, light emitting diodes (LEDs), chemical sensor and solar cells. The UV absorption characteristic made zinc oxide nanoparticles widely utilized in sunscreen, cosmetic and other health care product. At nanoscale, zinc oxide becomes transparent in visible light range while it blocks all radiation of UV-A and UV-B from the sun. This fact will protect the skin and material surface from damage from UV radiation from the sun. The antibacterial activity of ZnO is utilized in medical applications and personal care products as well as in paint and coating industry. Since zinc oxide nanoparticle can strengthen material, protect from UV damage, anti-bacteria, it can be found in coating products, textile and plastic film(moezzi et al., 2012). Zinc oxide nanoparticles have been reported in many researches to be toxic for bacterial, algaland mammalian cell(adams et al., 2006; Xia et al., 2008; Jiang et al., 2009). These findings put a question on the safety of zinc oxide nanoparticles on consumer products, especially in sunscreen when it is applied directly on human skin. However, there are studies concluded that this nanoparticles just stay at the skin surface and do not penetrate deep underneath the skin that can cause damage to the skin cells(burnett and Wang, 2011). Nevertheless, the fact that sunscreen and other cosmetics applied on human will be washed out and eventually end up in sewer and finally to wastewater treatment plant and released to environment. The same fate will happen with other zinc oxide nanoparticle related products. In recent year, many researches have been focused on fate, behavior and effect of engineered nanoparticles on wastewater and wastewater sludge. Zinc oxide nanoparticles have been reported to be toxic and inhibit the methane gas generation of anaerobic digestion (Luna-delRisco et al., 2011; Mu and Chen, 2011). However, it is still lack of data 8

17 to conclude the mechanism of the toxicity effect and the toxic of the contaminated digested sludge to environment. 2.2 Nanoparticles in Environment Nanoparticles could come from both natural and anthropogenic sources. Table 2.4 shows main sources of nanoparticles in the environment. However, due to the rapid expansion of nanotechnology, engineered nanoparticles (ENPs) have been manufactured and applied widely in many industries. This fact leads to the constant discharge of ENPs to the environment that their possible impacts to human health and environment remain a controversial topic. Since the generation of natural nanoparticles is uncontrollable and they exist in a relative small amount as compared with ENPs, majority of studies on characteristics and impacts of nanoparticles has been focused on engineered nanoparticles. Therefore, in most of study, the term engineered nanoparticles (ENPs) is referred shortly as nanoparticles (NPs). Table 2.4 Sources of Nanoparticles in Environment Environment Natural Sources Anthropogenic Sources Air Water Soil Volcanic eruption Wearing of rock and dust volatilization Hydrothermal vent Nucleation processes Metal-sulfide NPs Hydrous iron Manganese oxide Nanominerals Organic material aggregates Biogenic activities Combustion process Industrial emission Emission from production processes and consumption Deposition from atmosphere Effluent from production processes and consumption Deposition from atmosphere Sorption and transport from aquatic system Spillage from production processes and consumption Source:(Farré et al., 2011) Nanoparticlesfrom human activities will then be eventually released to the environment, intentionally or accidentally.intentionally release of nanoparticles is a controlled action with characterized particles, like the use of iron nanoparticles for remediation of groundwater for instance. However, the unintentionally disposal of nanoparticles is the section that have drawn attention of scientists. Since the fate, behavior and amount of nanoparticles released accidently remain unknown. Nanoparticles can be disposed to the environment from the manufacture process in factory or from the utilization of nanoparticle-based consumer products. 9

18 Released nanoparticles will then contaminate air, water and soil. They can remain in the environment for a long time, poison the habitat andcan be up taken by organisms. Nanoparticleswill be bioaccumulated, enter the food chain system and cause hazardous for plants and living organisms. The impact of nanoparticles in water is more focused than in air and soil. However, to quantify the concentration of nanoparticles in natural water is not a simple work. Since natural colloid can affect the behavior and speciation states of particles (e.g. from dissolved to colloidal and to particulate states). The data about concentration of separated nanoparticles in the environment is not available yet. However, there is report stated that approximately concentration of nanoparticles in natural water is in the range of 1 to 100 µg/l (Klaine et al., 2008). The fate and behavior of nanoparticles in the environment are affected by various environmental factors (e.g. light, ph, ionic strength, natural organic matter, etc.)(klaine et al., 2008). Various influences can affect the physical, chemical or bioavailablepropertiesof released nanoparticles in the nature. In order to assess the risks of nanoparticles and nanomaterial, we must scrutinize the possible mobility, transformation, and interaction with other materials of nanoparticles (Farré et al., 2011). 2.3 Nanoparticles in Wastewater Treatment Plants Release of nanoparticles in wastewater treatment plants Besides of the release of nanoparticles directly to the environment, which is discussed above, large amount of NPs can be found in wastewater treatment plants (WWTPs). From the production process in industries, nanoparticles aretransferred to industrial WWTPs.Moreover, NPs have been commercialized in many commonly used consumer products, like cosmetics, personal care, sunscreens, toothpaste, food additives, clothes, paint, cleaner chemical and etc. (The Project On Emerging Nanotechnologies, 2011). Therefore, nanoparticles from the consumption of these products can be unintentionally washed down to the sewage and end up in wastewater treatment plants. As a result, in recent years, fate and transport of nanoparticles in WWTPs has raised interest of researchers, and the need to understand the impact of these emerging materials to other systems is crucial Removal of nanoparticles in wastewater treatment plants Once nanoparticles is transported to WWTPs, they will go through various unit operations from physical, chemical to biological treatment processes. Each unit operation has different impact on NPs and different removal efficiency of NPs from the wastewater. Table 2.5 shows fate of NPs in various unit operations in WWTPs (Brar et al., 2010).Figure 2.3illustrates wastewater and sludge treatment processes of a typical WWTPs. Screening seems to be inadequate to remove nanoparticles, even when fine screen is used. Although some small amount of nanoparticles can be trapped in cloths, plastic bags, debristhat stopped in bar screen, this amount is too small and can be negligible. In the first sedimentation tank, nanoparticles can be trapped in colloids and suspended particles and settle down with them. By using coagulant and flocculants, the removal efficiency can be increased considerably. Moreover, the agglomeration of nanoparticles that change nano- 10

19 sized particles into colloids and then particulates is an important mechanism that affects the fate and behavior of nanoparticles in wastewater treatment plants. Table 2.5 Fates of NPs in Various Unit Operations in WWTPs Unit Operations Bar screens and grit removal Primary sedimentation tank Activated sludge process Secondary sedimentation tank Source: (Brar et al., 2010) Treatment Technique Remove debris and large particles by screening and settling Sedimentation of particles by gravitational force. Settling velocity is an exponential function of particle size. Biomass will be provided with air and throughout mixing to degrade organic compound Sludge from biological process will be settled down and supernatant will be transferred to next step Removal Efficiency Very low Low-Medium None Highest Removal Mechanism Small amount of NPs can be removed by adsorb onto large particles Agglomeration of NPs in to bulk size and settle down. Coagulant and flocculants will promote NPs sedimentation NPs aggregate with each other and adsorb on biomass. NPs will be settled down with sludge. Entrapment of NPs in settling sludge. Figure 2.3Wastewater and sludge treatment processes in WWTPs 11

20 In the biological treatment, nanoparticles are well mixed with biomass in the reaction tank. During this process, nanoparticles can be adsorbed to or trapped in biomass, which is basically microorganism and solid particles. Many factors affect the adsorption of nanoparticles to biomass. Same in natural water, characteristics and contents of the media in aeration tank (e.g. ph, ionic strength, temperature, organic matter, extracellular polymeric substances )can affect the behavior of nanoparticles. Basically, it affects the surface charge of nanoparticles (i.e. Zeta potential), therefore, affects the tendency to adsorb to certain material. In the end, nanoparticles agglomerate to become bigger and also adsorb to biomass during this biological treatment step. Eventually, when moved to the sedimentation tank, nanoparticles arethen settled down with the biomass. Other remaining free nanoparticles can be meshed with the settling sludge to settle down. Figure 2.4 shows various mechanisms that make nanoparticles settle down into the sludge of WWTPs. As a result, nanoparticles in the influent wastewater will be effectively removed from wastewater by conventional wastewater treatment plant (Brar et al., 2010; Gomez-Rivera et al., 2012). The higher removal efficiency, the higher nanoparticles accumulate in the settled sludge. Therefore, the concentration of nanoparticles accumulate in the sludge from time to time will be increased and cause severe adverse effects on sludge treatment steps (e.g. anaerobic digestion, sludge dewatering, sludge applications ). Figure 2.4Sedimentation of nanoparticles into the sludge of WWTPs 2.4 Toxicity of Nanoparticles Toxicity of nanoparticles on various organisms With the rapid development of nanotechnology in the last decade, the safety of manufactured nanomaterials was studied more serious by scientists. Because of itsclarge surface area per unit volume, nanoparticles are much more active than that particle at bulk or particulate size. As a result, nanoparticles possess new properties, which cause toxic to living organism and human. Because of its nano-scaled, nanoparticles are easily to be exposed to human and organism bodies through inhalation, ingestion and dermal contact. 12

21 A number of authors have published literature on characterization, behavior, and toxicological information of nanomaterials (Brar et al., 2010). Most of researches are focused on commercialized nanomaterials that were manufactured and applied widely, such as carbon nanotubes (CNTs), fullerene and metal oxides. The Table 2.6 shows toxic effects of different nanomaterials on various organisms ranged from bacteria to aquatic and soil species. Table 2.6 Toxic Effects of Nanomaterials on Different Organisms Target Organisms Nanomaterials Toxic Effects References n-c 60 Antibacterial to various types of bacteria (Lyon et al., 2006) Single-walled CNTs Antibacterial to E. coli (Kang et al., 2007) Quantum dots Damage membrane of E.coli and Bacillus subtilis due to oxidative activities (Hardman, 2006) Silver (Ag) Antibacterial to Gram negative bacteria (Morones et al., 2005) Bacteria TiO 2 Antibacterial to E. coli by photocatalytic activities and create ROS. (Rincon and Pulgarin, 2004) CeO 2 Antibacterial to E. coli, Bacillus subtilis (100% dead at 230 mg/l CeO 2 ) (Thill et al., 2006; Pelletier et al., 2010) ZnO Antibacterial to E. coli, Bacillus subtilis, and Pseudomonas fluorescens (100% dead at 20 mg/l ZnO) (Jiang et al., 2009) Aquatic organisms Soil organisms n-c 60 TiO 2 Multiwalled CNTs Zn and ZnO LC50 for D. magna is 7.9 mg/l. NOEC and LOEC is 0.2 and 0.5 mg/l, respectively. LC50 for D. magna is 5.5 mg/l. NOEC and LOEC is 1.0 and 2.0 mg/l, respectively. 100% death at 10mg/L Reduce root elongation of ryegrass. No effect on seed germination Reduce seed germination and root elongation of corn, cucumber, lettuce, radish and ryegrass. IC50 = 50mg/L for radish and 20mg/L for ryegrass (Lovern and Klaper, 2006) (Lovern and Klaper, 2006) (Lin and Xing, 2007) (Lin and Xing, 2007) 13

22 2.4.2 Toxicity mechanism of nanoparticles Although toxicity mechanism of each nanoparticles is not confirmed, there are various mechanisms that may cause damage to exposed organism. Possible causes of toxicity include damage cell membrane, protein, DNA; interrupt electron transportation; release toxic components; or release reactive oxygen species (ROS) (Klaine et al., 2008). Figure 2.5Toxicity mechanisms of nanoparticles: (a) release ROS; (b) release ion; (c) directly damage cell structure Possible toxicity mechanisms of nanoparticles are illustrated in Figure 2.5 above. The primary mechanism of nanoparticle toxicity involves reactive oxygen species (ROS) production. ROS consist of free radicals, such as superoxide anion ( O - 2 ) and hydroxyl radical ( OH). These are strong oxidants that can oxidize DNA, protein, cell membrane, and other cell components. These ROS are also normally formed in metabolism process that cause cell aging.however, when exist at high concentration, they willcause oxidative stress, inflammation, and cell death. High reactivity of nanoparticleswillrelease large amount of ROS. Some nanoparticles,like TiO 2 and ZnO for examples, even generate higher amount of ROS when exposed to sunlight, in the reaction called photocatalysis(rincon and Pulgarin, 2004; Brunner et al., 2006). Release of toxic components is also a main mechanism of nanotoxicity. For examples, release of heavy metal CdSe from core of quantum dots or release of Ag + ion from Ag NPs are reported to cause cytotoxicity(klaine et al., 2008). In the case of zinc oxide, toxicity of this nanoparticles is result from dissolution of particles that create soluble Zn (i.e Zn 2+ ion) and from production of ROS, especially when it is exposed with light near UV range. In the study on effect of ZnO on activated sludge activities, at same concentration, Zn 2+ is the most toxic, followed by nano-sized ZnO and then bulk-sized ZnO. Moreover, the toxicity of ZnO nanoparticles and ZnO bulk particles are suggested to be came from the soluble Zn 2+ ion when ZnO was dissolved (Liu et al., 2011). The nanotoxicity mechanism is still a controversial topic. However, normally nanoparticles cause toxicity by multiple mechanisms at the same time. For example, Ag NPs interrupt the permeability of cell membrane, cease the membrane respiration, release toxic Ag + ions, and penetrate inside the cell to cause damage to DNA(Sondi and Salopek-Sondi, 2004). 14

23 2.5 Anaerobic Digestion Introduction Anaerobic digestion (AD) is a biodegradable process in the condition of less or no oxygen. This is one of the oldest techniques in wastewater treatment process. Waste sludge from primary and secondary sedimentation tank is collected and thickened before sent to anaerobic digestion. In this process, mass of thickened sludge will be reduced significantly due to biodegradation process of anaerobic bacteria. The main advantage of this sludge treatment technique is the generation of methane gas, which can be used as energy source. Other advantages and disadvantages of anaerobic digestion system are shown in Table 2.6. Table 2.7Advantages and Disadvantages of Anaerobic Digestion Advantages Methane gas generated is energy source Significant reduce sludge mass (30-65%) Digested sludge is free of odors Digested sludge use as biosolid Highly remove pathogen Source: (Turovskiy and Mathai, 2006) Anaerobic digestion process Disadvantages High capital cost Long detention time Sensitive anaerobic bacteria Supernatant need to be treated again The anaerobic digestion consists of fourmain degradation steps. Each step involve in activities of different types of microorganism with their specific environmental condition. However, each of four steps exists in synergetic interactions to form anaerobic reaction chain. The Figure 2.6 shows schematic basic steps of anaerobic digestion processes involving four continuous steps: hydrolysis, acidogenesis, acetogenesis, and methanogenesis. In the first phase of anaerobic digestion, complex matters are broken down into soluble organic matters in a step called hydrolysis. Carbohydrates, proteins, and fats are converted into sugars, amino acids, and fatty acids, respectively. This is an important step, since it converts organic matter into a smaller and soluble compound, which can penetrate through cell membrane of bacteria. Bacteria complex in the system will then use these soluble compounds to produce intermediates for next steps of the process. Therefore, this is the rate-controlled step of the whole system. If this step not function well, the serial impact will affect the system (Parkin and Owen, 1986). After hydrolysis reactions, soluble organic matters are converted into volatile fatty acids, hydrogen and carbon dioxide. Volatile fatty acids are mainly acetic, propionic and lactic acids, with a small portion of propionic, butyric and valeric acids. Reactions are processed by acid-forming bacteria, which is quite tolerant to the change of ph and temperature. Moreover, these acidogenic bacteria are facultative bacteria that can use dissolved oxygen in the system. This fact protects the strict anaerobic methanogenic bacteria that involve in the next step (Turovskiy and Mathai, 2006). 15

24 Figure 2.6 Steps of anaerobic digestion process The third step is acetogenesis, where volatile fatty acid and other intermediates from acidogenic reaction are converted to carbon dioxide, hydrogen and acetic acid. The acetogens, which are hydrogen-producing bacteria, involve in this phase. Hydrogens produced are then consumed by methanogenic bacteria in the reaction to convert CO 2 to CH 4. This will help create the balance in the system, since high amount of hydrogen will inhibit the acetogenic reactions(parkin and Owen, 1986). Methanogenesis is the last phaseand also the most important phase of anaerobic digestion. This is the phase to produce methane from acetate and also H 2 and CO 2. About 70-75% of methane production is from acid acetic. Methane gas is insoluble in water and it will leave the system after formed. Carbon dioxide is also formed, some amount released as gas, the rest is converted to bicarbonate alkalinity. As a result, a gas mixture of approximately 60% methane and 40% carbon dioxide is generated from the destruction of organic matter(turovskiy and Mathai, 2006) Factors influence anaerobic digestion process The rate of anaerobic digestion reactions depends on environmental factors. Some main factors that affect the anaerobic process are retention time, temperature, ph, alkalinity, toxic materials(turovskiy and Mathai, 2006). Moreover, waste composition, volatile solid content, carbon to nitrogen ratio, organic loading rate, and mixing also affect the performance of anaerobic digestion. The retention time is a crucial factor that affects the anaerobic system. Bycontrol the sludge retention time (SRT) and hydraulic retention time (HRT), bacteria in the system have enough sufficient time to metabolize and decompose organic matter effectively. In 16

25 the anaerobic system have recycle, the feeding rate and removal rate is controlled to balance the amount of bacteria population in the system.the retention time ranges from 10 to 40 days, depend on operation temperature (mesophilic or thermophilic), different technologies, and waste sludge composition. Temperature is a key parameter that controls the rate of anaerobic process and the proper operation of system. Anaerobic digestion is divided into two categories based on operation temperature (i.e. mesophilic and thermophilic anaerobic digestion). Optimum temperature of mesophilic digestion is 35 o C, favorably in the range of 30 to 38 o C. Whereas, thermophilic digestion work at higher temperature of 50 to 57 o C (Turovskiy and Mathai, 2006). Thermophilic system can work at higher organic loading rate, shorten the reaction time and eliminate pathogen more effective. Nevertheless, high energy consumption and sensitiveness of thermophilic bacteria are reasons that make this system unpopular, as compared with mesophilic digestion.however, in both systems, fluctuation in temperature during the process can easily cause failure to the system. Temperature should not change more than 1 o C/day. Another important factor affects the performance of anaerobic system is ph. Optimum ph of the anaerobic system is in the range of 6.8 to 7.4(Khanal, 2008). Methanogens are more sensitive to change of ph than the rest bacteria species. During the start up period of the anaerobic digestion, fast growing acidogens prosper and cause ph drop down to as low as 5.0 due to the formation of fatty acids. Therefore, addition of sodium bicarbonate to adjust the ph and increase buffer is needed when conducting anaerobic digestion system. The activities of methanogenic bacteria can raise ph of the system as high as 8.0 by the generation of ammonia. However, these two phases occur simultaneously and stable the ph of environment in the neutral level. Moreover, activities of methanogens also produce carbon dioxide, ammonia and bicarbonate, which are alkalinity that prevents the fluctuation of ph. Although there are many substances caused toxic for anaerobic digestion system, the main concerns are heavy metals, metal cation, and ammonia. For examples, ammonia nitrogen over 3,000 mg/l, Zn 2+ ion at 1 mg/l can strongly cause inhibition in anaerobic digestion(wef, 1998) Parameters indicate performance of anaerobic digestion Major parameters indicating performance of anaerobic digestion system are gas production, destruction of organic matter, ph and alkalinity. Based on these parameters, we can estimate the performance of the digestion system and identify the inhibited effect of toxic materials caused to the system. Among all parameters, gas production is the most obvious indicator. Biogas is the final product of anaerobic digestion. Therefore, the volume of biogas generation and its composition are indicators for the performance of the process. Since biogas is generated from a reaction chain started from break down organic matter. Thus, high amount of biogas generate indicate high amount of organic matter destruction. Normally, it is expressed as ml of biogas per gram of volatile solid (ml biogas/gvs). Moreover, since biogas is used as energy source, the higher amount of methane composition in biogas, the higher efficiency of the system. As a result, percentage of methane in biogas and rate of methane production are important aspects to monitor. The methane content is from 55 to 75% of biogas, typically 60%. The rest composition is mainly CO 2, with trace amount of other 17

26 gases (e.g. H 2, N 2, NH 3 ). Hence, the increase of CO 2 leads to reduction of methane content and indicate malfunction of the anaerobic digestion. If the main objective of anaerobic process is waste disposal, the destruction of organic matter is the key parameter. One of advantages of anaerobic digestion is mass reduction due to destruction of organic matter from activities of microorganism. The destruction of organic matter in waste sludge is measured by percentage of volatile solid reduction during the digestion process. However, different substrates and operation systems resultin different destruction ability of microorganism. Monitor ph level of the system is the basic but most important parameter to identify the performance of system. The ph in the digester should remain in the range of 6.8 to 7.4 to optimize the system performance. Alkalinity should be maintained above 1,000 mg/l as CaCO 3 to give the buffering ability for the system and keep ph above 6.8. The VFA/Alkalinity ratio is also important and it should be kept within the range of 0.1 to 0.25.At this ratio, the amount of volatile fatty acids not exceeds the buffering capacity of the system If this ratio exceeds 0.4, the system starts to become sour(khanal, 2008). 2.6 Sludge Dewatering After anaerobic digestion, handling digested sludge is the next process. Normally digested sludge will be dewatered to reduce the moisture content of the sludge in order to reduce the storing and transportation cost. In this process, sludge with solid concentration of around 5% will be dried up to a sludge cake with 20% of dry solid. The dry level of dewatered sludge depends on different technique applied. The dewatering processes that are commonly used include mechanical processes such as centrifuges, belt filter presses, pressure filter and sand drying bed. Different technique will give different solid concentration, flow rate, chemical demand and side stream (Turovskiy and Mathai, 2006). The table 2.8 gives the performance of various dewatering techniques. Table 2.8 Dewatering Performances of Various Techniques Dewatering Techniques Inlet Percentage of dry solid (%) Outlet Drying bed Lagoons Belt Press Filter Press Centrifuge Incereration Source: (Bratby, 2006) 18

27 The sludge dewaterability depends on various factors. Extracellular polymeric substance (EPS), which is secreted by microorganisms are the major components of sludge flocs, isimportant factor that influences the dewaterability of sludge. High amount of EPS will increase the viscosity of the waste sludge and therefore makeit difficult to dewater. Various methods have been investigated as potential pretreatment technologies to enhance sludge dewaterability, such as the addition of calcined aluminum salts, alkaline pretreatment, ultrasonication, electrolysis, and microwave irradiation. Although these methods may have high dewatering potentials, their application has been limited by factors including sludge volume increase and high energy requirement for operation, as well as complexity of implementation. 2.7 Effect of Nanoparticles on Anaerobic Digestion In recent years, inhibition effects of nanoparticles on different unit operations of wastewater treatment plan have been focused, especially on biological process. Due to antibacterial characteristic of wide range of nanoparticles, the effect of them on biological process has drawn interest of researchers. Nanoparticles were found to inhibit the activities of aerobic and anaerobic bacteria. Cerium oxide NPs was found to be most toxic for the heterotrophic, ammonia oxidizing and anaerobic bacteria. At concentration of 630 mg/lof CeO 2, it caused 100% inhibition to the biogas production of anaerobic digestion (García et al., 2012). The effects of zinc oxide on different phases of anaerobic digestion process were also examined. The result showed that strong inhibition occurred for hydrolysis and methanogenesis steps (Mu and Chen, 2011). As compared with other tested nanoparticles (e.g. TiO 2, Al 2 O 3, SiO 2, Ag, Au NPs), cerium oxide and zinc oxide nanoparticles showed greatest inhibition of anaerobic digestion(mu et al., 2011; García et al., 2012). However, the number of research on effect of these two NPs on anaerobic digestion of waste sludge is still restricted with variation in tested method. Table 2.9 compares result of recent researches on CeO 2 and ZnO NPs on anaerobic digestion. 2.8 Future Outlook of Nanotoxicity Study on toxicity and effect of nanoparticles on environment and other systems are crucial when applications from nanotechnology widely commercialize. However, there are still some aspects of nanotoxicity that is not scrutinized yet. Studying these aspects will give us a broader and clearer idea of how nanoparticles behave and affect on other systems. Table 2.10 shows future outlooks recommendation from other papers. 19

28 Table 2.9 Effects of CeO 2 and ZnO NPs on Anaerobic Digestion NPs Size (nm) CeO 2 12 Preparation Method Oxidize Ce(NO 3 ) 3 and stabilize in HMT Test Method Effects References Batch test in 50 days with mesophilic and thermophilic anaerobic digestion and measure gas volume by pressure transmitter CeO 2 at 640 mg/l cause 90% inhibition to biogas volume of mesophilic and thermophilic anaerobic digestion. Mesophilic: EC 0 =160mg/L, EC 50 =260mg/L Thermophilic: EC 50 = 320mg/L (García et al., 2012) ZnO 140 ± 20 Disperse nanopowder with surfactant SDBS Continuous test with SRT=20days in total 100 days. Gas measure by glass syringe. Compare ZnO and Zn 2+ effect ZnO = 1,30, and 150 mg/gtss cause 0, 18.3, and 75.1% inhibition on biogas volume Zn 2+ = 1.2, 11.6, and 17.6 mg/l (amount released from ZnO at concentrations above) cause 0, 9.4, and 63.8% inhibition on biogas volume (Mu and Chen, 2011) ZnO Nano: Bulk: 1,000 Disperse nanopowder in DI water Batch test in 14 days and measure gas volume by pressure transmitter Inhibition on methane gas production Nano ZnO = 120 and 240 mg/l cause 43 and 74% inhibition on biogas volume Bulk ZnO = 120 and 240 mg/l cause 18 and 72% inhibition on biogas volume Nano ZnO: EC10, 20, 50 = 19.5, 28.2, and 57.3 mg Zn/L Bulk ZnO: EC10, 20, 50 = 39.8, 53.6, and 101 mg Zn/L (LunadelRisco et al., 2011) 20

29 Table 2.10 Future Outlooks on Nanotoxicity Data Gap and Future Outlook Nanoparticles on disposed dried sludge can disperse in air and cause air hazard NPs can bind to reactor, pipe and basin in WWTPs NPs can be destroyed or not in sludge stabilization, incineration. Leachate of NPs on sludge in landfill Bacteria is protected by EPS in sludge from NPs toxicity Effect of environmental characteristics (ph, ionic strength, humic acid, NOM) on NPs toxicity Extend experiment time to examine the adaptation ability of bacteria in anaerobic digester to NPs toxicity Study toxicity mechanism by research on effect of NPs on production of intermediates (H 2 and VFA) Need more study on effect of NPs on surface water, marine, sediment and soil ecosystem and food web. Lack data on concentration and form of NPs have been released to environment Need testing guideline to standardize method of studying nanotoxicity, so data from different studies can be comparable Study on degradeability, mobility and bioavailability of NPs in environment References (Brar et al., 2010) (Klaine et al., 2008; Jiang et al., 2009; Brar et al., 2010) (Luna-delRisco et al., 2011) (Klaine et al., 2008) (Farré et al., 2011) (Stone et al., 2010) 21

30 Chapter 3 Materials and Methods 3.1 Introduction This study investigated effects of CeO 2 and ZnO NPs on biogas production, dewaterability of sludge from anaerobic digestion process, along with microbial toxicity and phytotoxicity of the digested sludge. The Biochemical Methane Potential testwas used to assess the extent and composition of biogas production at different exposure concentrations of CeO 2 and ZnO NPs. By using this test, the inhibition effect of NPs toward biogas production of anaerobic digestion process was assessed. Characteristics of sludge mixture before and after digestion were analyzed to scrutinize impacts of NPs on the process.dewaterability of digested sludge from each sample was estimated and compared using Capillary Suction Time test. The toxicity of sludge was estimate by microbial toxicity test used E. coli as indicator bacteria. The microbial toxicity was compared between sludge before and after digestion to study the ability of anaerobic digestion to restrain toxicity of NPs. Moreover, phytotoxicity of digested sludge was estimated by using the seed germination test. The Figure 3.1 shows the overall research methodology of this study. Figure 3.1 Overall researchmethodology 22

31 3.2 Preparation and Characterization of Nanoparticles CeO 2 nanopowder was purchased from Antaria Ltd. Company (Product name: Ceria dry). ZnO nanopowder is a product of Nanosun (Product name: Nanosun Zinc Oxide P99/30). Stock solution of CeO 2 and ZnO NPs at concentration of 5g/L was prepared by dispersing nanopowder into MiliQ water (Conductivity of 18.2 MΩ/cm at 25 o C). The stock dispersion was then sonicated (53KHz at 35 o C)for 30 minutes to break aggregates and totally disperse the solution. The nanoparticle dispersion was kept in dark condition by wrapping the bottle in aluminum foil, in order to prevent the photocatalytic reaction of nanoparticles. The stock dispersion of CeO 2 and ZnO NPswere stored at 4 o C and used within 24 hours. The 10mg/L nanoparticles dispersion was prepared from stock solution to be used for particle size analysis. The average particle size and size distribution of CeO 2 and ZnO NPs were determined bydynamic light scattering technique usingzetasizer machine (Nanoseries Model S4700, Malvern Instrument, UK). Each sample was measured three times and data was shown as mean ± standard deviation. 3.3 Preparation and Characterization of Sludge The anaerobic sludge was taken from UASB reactor of Singha Beer Company (Phathumthani Brewery Co., Ltd). This source of sludge was chosen as inoculum since it has high organic content with low residues and toxic chemicals that can affect results of the study. The collected sludge was allowed to settle at 4 o C for a duration of 24 hours and the supernatant water part was removed, leaving the thickened sludge at the bottom. Later, the concentrated sludge was brought to room temperature and ph, TS, and VS were analyzed. The analytical methodsarepresented on Table Preparation of Substrate Substrate was added to the sludge as nutrient to optimize the digestion process and increase the biogas production volume in order to facilitate the comparison. The substrate solution was prepared with the COD:N:P ratio of 250:5:1, which is optimum for anaerobic process (Metcalf and Eddy, 2004). Glucose was used as a sole carbon source, NH 4 HCO 3 and KH 2 PO 4 were added at suitable amount to maintain the COD:N:P ratio. Stock solutions of glucose, NH 4 HCO 3,and KH 2 PO 4 were prepared separately and stored at 4 o C in dark bottles up to one month.the amount of added substrate was determined based on the suitable Inoculum:Substrate ratio (I/S ratio) in term of g VS/ g COD. 3.5 Biochemical Methane Potential Test Experimental procedure The biochemical methane potential (BMP) test was conducted following method of Owen et al. (1979) with modification. The 500 ml Duran glass bottleswere used in this test as batch reactor. The working volume of the bottle was 300 ml filled with anaerobic sludge, substrate, nanoparticlesdispersion and in the end DI waterwas added to have an equal 300 ml working volume of all bottles. The volume of sludge (i.e. inoculum) used for the BMP bottle waschosen to be200g (approximately 200mL). The amount of added substrate was calculated from the suitable I/S ratio in term of g VS/g COD.I/S ratio of 1,2 and 3 were 23

32 tested to confirm the most appropriate condition for the later BMP test with nanoparticle exposure. Before nanoparticles dispersion was added, all bottles were covered by aluminum foil to create dark condition, which prevented the photocatalytic reactions of nanoparticles. The new prepared nanoparticle dispersion wastaken by micropipette from the stock solution, which was continuously stirring by magnetic bar, and added drop by drop to the BMP bottle that was also stirring. This method ensured the best dispersion and mixture of nanoparticles into the sludge. A hole in the cap of each bottlewas made to install rubber stopper.silicon gel was used to air-tightly seal both side of the stopperto the cap.the thick rubber stopper allowed needle to be injected many times to measure biogas volume. Holes made by injecting needleswere automatically healed and air leakage was minimal. After filled all components, the bottle was capped tightly and the oxygen inside was completely flushed out by N 2 gas in 5 minutes.nitrogen gas was flushed through the bottle bytwo needlesinjected to the rubber stopper. After that, the gas pressure inside the bottle wasreleased to equilibrium with ambient pressure. Figure 3.2 shows the modified bottle for BMP test with components inside. Figure 3.2 Modified bottle for BMP test All bottles were then incubated in an insulated cabinet placed in ambient condition. The temperature inside the cabinet was 30±2 o C. Bottles were manually shaken 3 times per day to ensure contact of constituents and homogeneousness of the sample. Everyday at constant time, the volume of generated biogas was measured by injecting a 20mL syringe into the rubber stopper. The higher gas pressure inside the bottle pushed the piston up, until equilibrium with ambient pressure. Only one type of syringe (20 ml) was used in this study to ensure the uniformity of measurement, since different sizes of syringe showed different results to a same pressure. New syringes and needles were changed frequently to prevent blockage of needles and higher resistance of used syringes. The final volume was confirmed by moving the piston out of its final position and it should come back to that level. 24

33 The generated biogas volumewas recorded with temperature and pressure at the measured time. The experimentwas conducted during 40 days, which was enough retention time to digest almost all organic matter. Every 7 days, the biogas composition was measured by Gas Chromatography. A 2mL glass syringe was used to take 0.3mL of biogas from each sample and immediately injected to the Gas Chromatography. The biogas composition was measured in term of percentages of CH 4, CO 2, N 2, CO, and O 2. Four different exposure concentrationsof 10, 100, 500, and 1,000mg/L of CeO 2 and ZnO nanoparticles were tested. Blank sample, in which nanoparticlesdispersion was replaced with DI water, was used to compare the result and show the impact of nanoparticles. All samples weretriplicate to ensure data reproducibility Analytical method and calculation The mixture inside BMP bottleswerecharacterized before and after incubation time. The parameters ph, TS, VS, alkalinity, VFA, and soluble CODwereanalyzed by methods described in Table 3.1. The volume of generated biogas wasmeasured and converted to the volume at standard temperature and pressure (STP), which is at 0 o C and 1 atm, by Equation 3.1. Where: V s = Gas volume at STP (NmL) V m = Gas volume measured (ml) T s = Standard temperature (T s = 273 K) V s =V m T s. P m T m. P s Eq. 3.1 T m = Measured ambient temperature at sampling time ( K) P s = Standard pressure (P s = 1,013 mbar) P m = Measured ambient pressure at sampling time (mbar) The composition of biogas (% CH 4, CO 2,and traced gases) was determined by Gas Chromatography (GC) equipped with thermal conductivity detector. By multiplying the percentage of CH 4 determined by the GC with biogas volume at STP, the volume of methane generated at STP was calculated. The cumulative biogas and methane production were calculated by adding the volume of generated biogas or methane day by day. 25

34 Table 3.1 Analytical Parameters and Testing Methods Parameter Unit Analytical method Equipment/Technique Possible Interferences Prevent interferences/ Precaution ph - ph meter ph meter Temperature Calibrate meter before use and measure sample at room temperature COD soluble mg/l Closed reflux Filter sample through GF/C paper + Titration Chloride, Nitrite Add HgSO 4 to eliminate chloride and sulfamic acid to eliminate nitrite Alkalinity mg/l as CaCO 3 Total Alkalinity Titration Soap, oily matter and suspended solid Allow additional time between titration VFA mg/l Total VFA Distilation + Titration TS mg/l Dry at 105ºC Oven VS mg/l Ignite at 550ºC Furnace Eluting organic acids and some synthetic detergents Loss of volatile organic matter during drying Loss of volatile inorganic salts like (NH 4 ) 2 CO 3 Titrate right after the sample cools down enough to prevent loss of volatile acids Dry for 1h until constant weight Ignite for 1h until constant weight Source: (APHA et al., 2005) 26

35 3.6 Capillary Suction Time The dewaterability of digested sludge from each sample were estimated by Capillary Suction Time (CST) instrument as described in Standard Method (APHA et al., 2005). The test was triplicated for each sample to have a reproductive data. Figure 3.33 shows the design of CST apparatus. Figure 3.3 CST apparatus The CST test determined the rate of water released from sludge.a volume of 10mL of digested sludge was fed into astainless steel cylinder that restedon a sheet of chromatography paper (Whatman Chromatography Paper Grade 17, size 7x9cm). The paper extracted liquid from the sludge by capillary action. The required time for liquid to travel a specific distance was recorded automatically by monitoring the conductivity change occurring at two sensorss placed in contact with the chromatography paper. From the Figure 3.3, the two sensors are noted as 1A, 1B and 2. When the water reached sensors 1A or 1B, the clock started to run and it stopped when the water came to sensor 2. The time was indicated in the monitor box in term of seconds. The elapsed time indicated the dewater rate of that sludge. The smaller the time needed, the faster the sludge could be dewatered. Each sample was measured 3 times and the data was shown as mean ± standard deviation. 3.7 Microbial Toxicity Test Culture of bacteria The model bacteria used in this E.coliwas prepared, distributed 1month. test wasescherichia coli (E. coli). The glycerol stock of into 1.5mL eppendorf tubes and stored in freezer up to The bacteria were pre-cultured in 10 ml Luria-Bertani medium (LB broth, Miller) for 12 hours at 37 o C. After that, 0.5 ml (5% v/v) of culture broth was inoculated to new LB broth 27

36 and the cell culture washarvested at approximately mid-exponential growth phrase (around 4 hours) to optimize the viability of bacteria(k. L. Lin and Chen, 2007) After that, 1 ml of fresh cell culture was diluted with 9 ml of 0.85% NaCl solution(8.5 g/l NaCl) and then continued likewise to make a serial dilution (i.e. 10-1, 10-2, 10-3, of initial cell culture). The saline solution 0.85% NaCl was used instead of DI water to prevent the bacteria die-off due to the hypotonic effect. After that, 0.1mL of different dilutionswerespread on LB agar plate and incubate at 37 o C in 16 hours to identify the cell concentration. Diluent with cell concentration of cell/ml was then used for the toxicity test Toxicity test procedure The overall toxicity test procedure shows on Figure 3.4. Figure 3.4Overall microbial toxicity test procedure The microbial toxicity test was designed to study the toxicity of sludge before and after digestion on bacteria. Moreover, to understand about the influence of solid in the sludge on the toxicity, the toxicity of the sludge supernatant (liquid part of the sludge after removing solid) was also tested. The sludge supernatant was obtained by taking the liquid part after centrifuged the sludge at 5,000rpm in 10 minutes. In addition, the toxicity of nanoparticles dispersion was also studied to have a reference. Therefore, this test gave useful data about the toxicity of nanoparticles and how it changed when nanoparticles was mixed in the sludge and when the sludge went through anaerobic digestion process. 28

37 The sludge and sludge supernatant at different exposure concentrations of nanoparticles were first sterilized in autoclave (121 o C at 15psi for 15 minutes) to eliminate all bacteria inside. After that, 9 ml of sludge or sludge supernatantwas mixed with 1 ml of E. coli culture solution in a sterile test tube. This E. coli solution was prepared before to have the cell concentration of cell/ml. The test tubes were then vigorously mixed by Vortex machine and incubated at 37 o C in 1 houras contact time. A controlled test with 1mL of E. coli culture and 9 ml of 0.85% NaCl solution was also prepared.the test on toxicity of nanoparticles dispersion was conducted in the same way, by mixing 9ml of nanoparticles dispersion at different concentrationswith 1mL of E. coli culture. After contact time, 0.1 ml of the mixture in each tube was spread on LB agar petri plate. Each samplewas triplicated to have reproducible data. After that, all petri plateswereinvertedincubated at 37 o C for 16 hours. The number of colonies formed in each plate was counted and the inhibition effect was calculated. The data was presented as mean ± standard deviation (n=3) Calculations of microbial toxicity test The population of viable bacteria cell was calculated from the Equation 3.2. C x = Total CFU Number of plates Dilution factor Eq. 3.2 Where: C x : cell concentration of different samples(cfu/ml) The inhibition effects of sludge, sludge supernatant and nanoparticles dispersion at different nanoparticles concentrations on viability of E. coliwere calculated from the Equation 3.3. Where: Inhibition â% = C x C o 100 Eq. 3.3 C x : cell concentration after exposed to nanoparticles (CFU/mL) C o : cell concentration in control test (CFU/mL) 3.8 Phytotoxicity Test The phytotoxicity test gaveinsights about the toxicity of digested sludge containing nanoparticles to the germination of seed and elongation of root.this test followed the method of Lin and Xing (2007). Seeds of two plants were used in this test: tomato (Solanum lycopersicum) and mung bean (Vigna radiata). The seeds were purchased from the East-West Seed Company, Thailand. Digested sludge samples from BMP test after digestion periodwere centrifuged at 5,000 rpm in 10 minutes to remove the solid part. The supernatant was collected and used for this phytotoxicity test. Ten seeds were placed in the filter paper that placed in a petri plate. Each seedwasplaced 1cm away from each other. After that, 5mL of sludge supernatant was evenly transferred 29

38 into the petri plate.the control sample was prepared by applying 5mL of DI water on the filter paper with 10 seeds rested on top. All plates were then incubated in dark condition at 25 o C in 5 days. After 5 days, the number of germinated seeds was counted and the total length of all sprouts was measured. The data was presented as mean ± standard deviation (n=3). The Germination Index (GI) was calculated from the data of seed germination and root growth to have the overall comparison. Low percentage of GI indicated greater inhibition effect of contaminated digested sludge. The Germination Index of each sample was calculated from the Equation 3.4. Seed germination of sample % x Root length of sample (mm) GIâ% = Seed germination of control % x Root length of control (mm) x 100 Eq. 3.4 Since the germination index represents the proportion of seed germination and root length as compared with the controlled sample, the inhibition effect of NPs on plant growth was calculated follow the Equation 3.5. Inhibition (%) = 100% - GI (%) Eq

39 Chapter 4 Results and Discussions This chapter isdivided into five parts,namely:nanoparticles characteristics, BMP test, CST test, microbial toxicity test, and phytotoxicity test. In the first part, characteristics of tested nanoparticles are presented.the BMP test comprises of BMP verification, effectsof NPs on biogas and methane, and sludge characteristics analysis part.the microbial toxicity test comprises of test on nanoparticles and test on sludge containing nanoparticles. All results and discussions are presented in each part with correlation with each other. Therefore, results from various tests gave an interdisciplinary approach to understand impacts of CeO 2 and ZnO nanoparticles to sludge treatment processes, namely anaerobic digestion, sludge dewatering, and digested sludge applications. 4.1 Nanoparticles Characteristics The CeO 2 and ZnO nanoparticles dispersions were characterized by measuring particle size and size distribution. The particles size data (hydrodynamic diameter) of CeO 2 and ZnO measured by DLSis shown in Table 4.1 as mean ± standard deviation (n=3). The Figure 4.1 and 4.2 show particle size distribution of CeO 2 and ZnO nanoparticles, respectively. Table 4.1 Particles Size of CeO 2 and ZnO Nanoparticles Samples ph Particle Size(nm) CeO ±2 ZnO ± 7 Figure 4.1 Size distribution of CeO 2 nanoparticles 31

40 Figure 4.2 Size distribution of ZnOnanoparticles The data showed that CeO 2 have the smaller size than ZnO. The average diameter of CeO 2 nanoparticles measured by DLS technique was around 192 nm. In the other hand, ZnO nanoparticles had particle diameter of 850 nm approximately. Moreover, the particle size of ZnO was widely distributed with some particles that had size bigger than 1,000 nm (Figure 4.2). Since both CeO 2 and ZnO nanoparticles were prepared by dispersing nanopowder into DI water without using any surfactant, the size of nanoparticles were quite large. High particle size of nanoparticles was also resulted in other researches (Xia et al., 2008). In the research of Luna-delRisco et al. (2011), bulk-sized ZnO (1,000 nm) was less toxic for the biogas production than the nano-sized ZnO (50-70 nm).the difference on toxicity was explained by the fact that nanoparticles ZnO had a higher solubility than the same substance at bulk size. However, using stabilizer or surfactants to achieve a smaller size nanoparticles had it own disadvantages. Surfactants was indicated to be toxic for the bacteria and could inhibit bacterial activities(garcía et al., 2012). Therefore, in this research, no surfactant was used in the preparation of nanoparticles. However, the size of nanoparticles used for this research was still in the acceptable range as compared with nanoparticles characteristics of other studies. 4.2 Effects of Nanoparticles on Anaerobic Digestion In order to estimate the impact of CeO 2 and ZnO nanoparticles on performance of anaerobic digestion of sludge, the BMP test was conducted. Before working on BMP test with exposure of NPs, optimum condition for BMP test was conducted. After that, the best condition for BMP test was used to examine impacts of CeO 2 and ZnO NPs on anaerobic digestionsystem. Effects of CeO 2 and ZnO NPs on the system were determined based on impacts on biogas volume production and methane composition of biogas. The three parts, namely optimization of BMP test, effects of NPs on biogas production, and effects of NPs on methane production, are presented in sequence in following parts Optimization of BMP test The BMP test was designed to estimate the biogas production of anaerobic digestion. Therefore, in order to use BMP test to monitor toxicity of nanoparticles, the optimum condition for BMP test was tested in advance. The optimum condition was decided based on best performance of differentinoculums to sludge ratios (I/S ratios). Sludge (i.e. 32

41 inoculum) was taken from UASB reactor of Singha Beer Company. Substrate was a mixture of glucose, NH 4 HCO 3, and KH 2 PO 4 at COD:N:P ratio of 250:5:1. Various I/S ratios of 1, 2, and 3 were tested to estimate the best condition for BMP test. In addition, the blank sample, in which substrate was replaced by DI water, was also conducted. The calculation of amount of constituents need to be added in each sample was shown in Appendix A. The BMP test was conducted in 40 days. The biogas volume was measured everyday and the biogas composition was measured after 15 days of incubation. The result of cumulative biogas volume production at STP is shown in Figure 4.3 From the graph, it shows that the lower the I/S ratio, the higher the gas production is achieved. After 40 days of incubation, the total biogas production of sample I/S 1, 2, and 3 were 2,100 ml, 1,993 ml, and 1,637 ml, respectively. The supplied substrate gave the boost to the biogas production in first 5 days of incubation period. However, biogas production of sample I/S 1 was inhibited during the startup period. After about 10 days, the biogas production began to recover and reached over 2,000 ml of biogas at the end of incubation time. To scrutinize the problem, characteristics of sludge mixture of each sample were analyzed. The Table 4.2 presents the detailed characteristics of sludgesubstrate mixture of different samples before and after BMP test. Biogas Production of Various I/S Ratios Cumulative Biogas Volume at STP (Nml) Blank I/S 1 I/S 2 I/S Day(s) Figure 4.3 Biogas production of BMP test at various I/S ratios Except sample I/S 1, all samples had ph of sludge mixture before and after anaerobic digestion in the range of 7 to 7.5, which is suitable for anaerobic digestion system(khanal, 2008). As mentioned in section above, the optimum range of ph for anaerobic digestion is 6.8 to 7.4. The sample with I/S ratio of 1 was acidified after incubation time. The ph of this sample dropped from 7.86 at the beginning down to 5.49 after 40 days. This could be due to excess amount of substrate was given to the system. Acidogenic bacteria can rapidly convert substrate to volatile fatty acids that cause ph drop in the system. Methanogenic bacteria, in contrast, are slow growing bacteria that cannot convert enough volatile fatty acids into methane and carbon dioxide. This resulted in high amount of accumulated fatty acids in the system that caused system failure due to acidification. The total VFA amount of this sample rose up to 1,500 mg/l, which was threefold of other 33

42 samples. Therefore, the VFA:Alkalinity ratio of sample I/S 1 was up to 5, which was far above the recommended level of below 0.4 that was noted in section above. After 15 days of incubation, the biogas compositions of all samples were measured and the data of methane composition is shown in Figure 4.4. From the figure, we can see that just sample I/S 2 and I/S 3 have high methane composition, which was 64.5% and 60.5%, respectively. Blank sample did not have supplied substrate that resulted in low biogas volume production and low methane composition also. Sample I/S 1 was acidified, which resulted in low methane composition and high CO 2 production of 69.4% (Appendix C). This high amount of CO 2 could be soluble in the sludge mixture and caused ph drop in the system also. Table 4.2 Characteristics of Sludge Mixture Before and After AD Parameters Blank I/S 1 I/S 2 I/S 3 Before After Before After Before After Before After ph TS (%) VS (%) CODs (mg/l) Total Alkalinity (mg/l as CaCO 3 ) Total VFA (mg/l) VFA/ Alkalinity CODs removal (%) ,400 12,500 17, , , , ,500 1,500 2,500 2, , In conclusion, substrate at I/S ratio of 2 showed best performance, which was illustrated through high amount of biogas generated and methane composition. As a result, substrate at I/S ratio of 2 was used for further BMP test with nanoparticle exposure. 34

43 Methane Composition (%) Methanee Composition of Various I/S Ratios Blank I/S 1 I/S 2 I/S 3 Sample Figure 4.4 Methanee gas compositions of various I/S ratio samples Effects of nanoparticles on biogas production CeO 2 and ZnO nanoparticle dispersions at concentration of 10, 100, 500, and 1,000 mg/l were applied on BMP test to analyze inhibition effects on anaerobic digestion. The volume of generated biogas was measured daily for 40 days. The results of cumulative biogas production though incubation time of different samples are illustrated in Figure 4.5. CeO 10 2 CeO CeO CeO (NPs concentrations were in term of mg/l) Figure 4.5Effects of CeO 2 and ZnOnanoparticles on biogas production 35

44 From the Figure 4.5, it shows that exposed different concentrations of nanoparticles resulted in different amount of biogas production. Every samplehad biogas production less than the blank sample, except the sample with CeO 2 exposure at 10 mg/l. The blank sample, as decided from the previous experiment, was the sample that consists of sludge and substrate at I/S ratio of 2. When CeO 2 nanoparticle dispersion was added at low concentration of 10 mg/l, it stimulated the biogas production performanceabout 11% as compared with the blank sample. However, increase exposure concentration of CeO 2 NPs above 100 mg/l resulted in decrease in biogas production potential. This finding relatively matched with the data from study of García et al.(2012). In that study, García et al. found that CeO 2 NPs do not have inhibition effect on biogas production of anaerobic digestion at concentration less than 160 mg/l. Therefore, the lowest observed effect concentration of CeO 2 NPs to biogas production in this study was lower than data recorded in previous study of García et al. (2012). It is worth to mention that up to the time this research finished, the study of García et al. (2012) is the only available reference about effect of CeO 2 NPs toward biogas production. The research of García et al. (2012)also mentioned that biogas volume was reduced 50% and 90% at exposure concentrations of CeO 2 NPs of 260 mg/l and 640 mg/l, respectively. However, in this study,at exposure concentrations of CeO 2 NPs at 100, 500, and 1,000 mg/l, the results of biogas production through 40 days of incubation were almost similar. The inhibition on total biogas production volume of CeO 2 NPs at 100, 500, and 1,000 mg/l were 32.2%, 32.3%, and 35.1%, respectively. The results of total generated biogas volumeof different samples are shown on Figure 4.6. Biogas Volume at STP (NmL) Total Volume of Biogas Production Blank CeO2 2 CeO2 2 CeO2 2 CeO CeO2 2 ZnO ZnO Samples (NPs concentrations were in term of mg/l) Figure 4.6Total volume of biogas production In contrast with CeO 2 NPs, exposure of different ZnO NPs concentrations resulted in greatly differences in inhibition effects on biogas production. From the Figure 4.6, we can see that at every exposure concentration, ZnO NPs caused inhibition to biogas generation of the BMP test. At lowest exposure concentration of 10 mg/l ZnO NPs, the reduction of biogas volume was 8.4% as compared with blank sample.at high concentration of 1,000 mg/l ZnO NPs, the generated biogas volume was reduced up to 65.3%. The inhibition ZnO 100 ZnO ZnO

45 effect of ZnO NPs from this experiment was less than previous study when ZnO NPs at 240 mg/l could inhibit the biogas volume by 74%(Luna-delRisco et al., 2011). However, in the research of Luna-delRisco et al. (2011), the incubation period was just 14 days, which was too short as compared with 40 days period in this study. Sample ZnO NPs 100 mg/l and 500 mg/l showed comparatively same result on biogas production through 40 days of incubation. In the first 2 weeks, the biogas productions of these two samples were hindered that resulted low biogas production. However, from day 15 th, the inhibition effect was overcame and the biogas production increased to reach the same level as sample ZnO 10 mg/l at the end of incubation period. Therefore, the total volume of biogas production after 40 days of sample ZnO NPs 10, 100, and 500 mg/l were similar (Figure 4.6). This fact was also mentioned in research ofluna-delrisco et al. (2011) about the adaptation of anaerobic bacteria to toxicity of nanoparticles. It was mentioned in that research that at exposure ZnO NPs concentration under 120 mg/l, the biogas production started to recoverafter 11 days of incubation. The recovery could come from eitherinfluence of environmental factors or adaptation of microorganism. Adsorption of inhibitors to organic substances, precipitation of inhibitors, or ionic interactions are examples of environmental influences that reduce the toxicity effect to the system. In the other hand, microorganism had various detoxification mechanisms, such as release enzymes or adjust microbial metabolism(gadd and Griffiths, 1977). As a result, bacteria can adapt themselves to a tolerable toxic environment. The illustration of possible mechanisms to overcomenanoparticles toxicity is shown in Figure 4.7. Figure 4.7 Possible mechanisms to overcome nanoparticles toxicity Effects of nanoparticles on methane production The biogas compositions of samples were measured by the Gas Chromatography at day 7 and day 40 of the incubation period. The result of methane compositionof each sample is shown in Figure

46 Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO 1000 (NPs concentrations were in term of mg/l) Figure 4.8 Methane composition of various samples Methane compositions of every sample were relatively stable between day 7 and day 40 of the incubation period. Except sample ZnO NPs 1,000 mg/l, the rest samples had methane composition of between 55% to 66%, which is in the range of good performance anaerobic digestion system(parkin and Owen, 1986). Sample with high exposure concentration of ZnO NPs at 1,000 mg/l showed great inhibition effect on methane gas composition. At the end of the incubation period, the methane composition of this sample was 35.4% and carbon dioxide composition of 56.6% (Appendix C). This fact indicated unbalance in the metabolism of anaerobic digestion system, which was reflected in the characteristics of digested sludge mixture that isdiscussedin section The data of biogas composition shown that CeO 2 and ZnO NPs did not affect the biogas composition unless very high concentration of NPs was applied, which was the case of ZnO NPs at 1,000 mg/l. The total methane production of each sample was calculated by multiplying the total biogas volume with the methane composition at day 40. The Figure 4.9 shows total volume of generated methane from different samples. From the Figure 4.9, we can see that the CeO 2 and ZnO NPs had both positive and negative effect on methane production of anaerobic digestion. At low concentration (e.g. CeO 2 and ZnO NPs at 10 mg/l), nanoparticles helped to increase the methane production of anaerobic digestion system. In detail, CeO 2 NPs at 10 mg/l raised total methane volume by 17.4% as compared to blank sample. Sample ZnO NPs 10 mg/l and 100 mg/l also slightly increased methane production by 2.5% and 2.0%, respectively. The positive effect of nanoparticles at low concentration was also mentioned in previous studies. In the study of Xia et al. (2008), CeO 2 NPs showed protective effect to mammalian cellsagainst ROS. Experiments from that researchconfirmed that CeO 2 NPs could act like antioxidant to neutralize the oxidative effects of ROS. 38

47 Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO 1000 (NPs concentrations were in term of mg/l) Figure 4.9Total volume of methane production of various samples However, increase exposure concentration of CeO 2 NPs above 100 mg/l could significantly reduce methane production of the system. At exposure concentration of CeO 2 NPs of 100, 500, and 1,000 mg/l, the methane production reduced 28, 34, and 35%, respectively. In the other hand, ZnO NPs just caused inhibition to total methane production at concentration of 500 and 1,000 mg/l. At 500 mg/l of ZnO NPs exposure, the methane production slightly reduced by 6%. But when increase the ZnO NPs concentration to 1,000 mg/l, 79% reduction in methane volume was observed. In conclusion, from the result of BMP test, CeO 2 NPs caused inhibition effect to the biogas and methane gas production from exposure concentration of 100 mg/l. In the other hand, ZnO NPs showed greatest inhibition effect on both biogas and methane volume at concentration of 1,000 mg/l. At lower exposure concentration, the system either not be affected or could adapt to the toxicity after a certain time Effects of nanoparticles on sludge characteristics The characteristics of sludge mixture before and after BMP test were analyzed to have a deeper insight about effects of NPs to the anaerobic digestion system. The detailed sludge characteristics of each sample are presented in Appendix D. As mentioned in section 2.5.4, in order to monitor the performance of anaerobic digestion system, ph, VFA/Alkalinity ratio, and COD removal efficiency are indicators. The results of these parameters of sludge mixture after BMP test are presented in Table 4.3 below. 39

48 Table 4.3 Characteristics of Digested Sludge at Various NPs Exposure Samples ph VFA/Alkalinity CODs Removal (%) Blank CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO100 mg/l ZnO 500 mg/l ZnO 1,000 mg/l According to Table 4.3, sample ZnO 1,000 mg/l was the only sample that showed bad performance through sludge characteristics and CODs removal efficiency. The CODs removal efficiency of this sample was just 38.5%. This removal efficiency was correlated with the low biogas production volume of this sample. Moreover, the balance between VFA and alkalinity amount was also broken. The VFA:Alkalinity ratio of sample ZnO 1,000 mg/l was 1.4, which was greatly above the recommended level of less then 0.3. The high value of this ratio indicated acidic condition in the system, which was clearly confirmed by ph 5.9 of the digested sludge mixture. This fact could be because ZnO NPs at high concentration could kill large number of methanogens, which were more sensitive and vulnerable than acidogens (Turovskiy and Mathai, 2006). As a result, generated VFA could not be converted to methane by methanogens and eventually accumulated in the system that caused ph drop.in addition, when ph drop under 6.8, all methanogens, which were very sensitive to ph change, were eliminated from the system. From the Figure 2.2 of literature review part, if methanogens not exist in the system, volatile fatty acids, acetic acid, H 2 and CO 2 will be accumulated because cannot be converted to methane and carbon dioxide. This resulted in low ph condition in the system and also low biogas and methane production that were discussed in section and In conclusion, high exposure of ZnO NPs at 1,000 mg/l caused severe inhibition in biogas production potential by affecting the balance between acidogenesis and methanogenesis. Except sample ZnO NPs 1,000 mg/l, inhibition effects of the rest samples could not be identified through characteristics of digested sludge. The percentage of CODs removal slightly reduced when increased the exposure concentration of NPs. However, ph and VFA:Alkalinity of these samples remained in the preferable range. As a result, the best indicator for effects of nanoparticles on anaerobic digestion is generated biogas volume. Moreover, methane composition can be considered to have a deeper insight. 40

49 4.3 Effects of Nanoparticles on Sludge Dewatering The digested sludge after BMP test was then used to study the effects of nanoparticles on sludge dewaterability. The data of capillary suction time of different samples are shown in Figure CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO 1000 (NPs concentrations were in term of mg/l) Figure 4.10 Effects of NPs on sludge dewatering From the Figure 4.10 above, the negative effect of nanoparticles on sludge dewatering was clearly revealed. Increase the exposure concentration of nanoparticles caused difficulties to the sludge dewatering process. The CST test measured required time to dewater a certain volume of sludge. Samples with higher exposure concentration of nanoparticles took longer time to be dewatered. Applied CeO 2 NPs at 10, 100, 500, and 1,000 mg/l increased the time to dewater by 22, 20, 69, and 213 % as compared with the blank sample. In addition, inhibition effects of ZnO NPs to the sludge dewatering were more severe than that of CeO 2 NPs. The sludge samples with exposure of 500 and 1,000 mg/l of ZnO NPs had the required time to dewater 11 and 13 times longer than the blank sample, respectively.therefore, ZnO NPs could dramatically impact the sludge dewatering process when it was accumulated in the sludge at high concentration. The dewaterability of digested sludge was mentioned to be independent with ph, volatile solid, or ammonia and phosphorus. In the other hand, it depended on concentration of extracellular polysaccharides and extracellular protein(zhou, 2003). The extracellular proteins and polysaccharides, or sometimes are referred as EPS, are substances generated by bacteria. Hypothetically, bacteria in the system could produce high amount of EPS to protect themselves from the toxicity of nanoparticles. It was mentioned in the section and Figure 4.7 that bacteria could adapt themselves to the toxicity of nanoparticles by creating enzymes that could inhibit the nanoparticles. Therefore, producing extracellular polymeric substances, which created a slimy matrix thatcover bacteria cells, could be a useful protection mechanism of bacteria when they were exposed to nanoparticles. The 41

50 Figure4.11 illustrates possible mechanism of bacteria to adapt to nanoparticles toxicity by producing EPS. Figure 4.11 Protective mechanism of bacteria to overcome NPs toxicity Although EPS proteins and polysaccharides help create sludge flocs, but EPS are highly hydrated and could hold large amount of water inside (Neyens et al., 2004). As a result, high EPS could cause high viscosity to the sludge and hold capillary water inside them, which eventually resulted in high CST value. 4.4 Effects of Nanoparticles on Bacterial Viability The bacterial toxicity test has been used widely to study the toxicity of nanoparticles. In this experiment, toxicity of CeO 2 and ZnO nanoparticles dissolution were first tested to have the toxicity data of these nanoparticles. In the second part of this bacterial toxicity test, the toxicity of the sludge that contained nanoparticles at various concentrations were analyzed in order to know effects of anaerobic sludge toward nanoparticles. Moreover, sludge samples before and after BMP test were tested to compare and study the effect of anaerobic digestion system to the toxicity of sludge containing nanoparticles Bacterial toxicity of nanoparticles CeO 2 and ZnO nanoparticles dissolution were prepared follow method described in section 3.2. Nanoparticles dispersionsat different concentrations were then applied on E. coli to test the viability of bacteria after contact with nanoparticles. The resulted data gave us information of toxicity of CeO 2 and ZnO NPs. Figure 4.12and Figure 4.13show bacterial toxicity data of CeO 2 and ZnO nanoparticles dispersion, respectively. 42

51 Bacterial Toxicity of CeO2 NPs Inhibition (%) CeO2 Concentration (mg/l) Figure 4.12Bacterial toxicity of CeO 2 nanoparticles dispersion From the achieved data, ZnO nanoparticles showed higher toxicity to E. coli than CeO 2 NPs. Detailed data on bacterial cell concentration of the test is shown in Appendix E. From the Figure 4.13, ZnO NPs caused 99 % cell death at concentration of 100 mg/l. No bacteria survival was found at exposure concentration of ZnO NPs at 500 and 1,000 mg/l. However, the toxicity of ZnO NPs in this study was less than the result reported by Jiang et al. (2009). In that research, ZnO NPs caused 100 % cell death of E. coli, Bacillus subtilis, and Pseudomonas fluorescens at exposure concentration of just 20 mg/l. Nevertheless, ZnO NPs were also reported to inhibit E. coli viability by 38% and 48% at exposure concentration of 500 mg/l and 1,000 mg/l, respectively (Adams et al., 2006). As a result, toxicity of nanoparticles could be widely varied depends on the testing method of each research. This fact raised the need to have a standard method to test toxicity of nanoparticles, which has been mentioned in other articles and was listed in the Table Bacterial Toxicity of ZnO NPs Inhibition (%) ZnO Concentration (mg/l) Figure 4.13Bacterial toxicity of ZnO nanoparticles dispersion 43

52 From Figure 4.12, CeO 2 NPs at concentration of 50, 100, 500 and 1,000 mg/l caused cell death of about 31, 55, 97, and 100%, respectively. This result was less toxic than the result of Thill et al. (2006), where CeO 2 NPs at 230 mg/l led to 100 % inhibition in bacterial growth. The difference might be the result of different testing procedure and nanoparticles characteristics. Moreover, it is important to note that at low concentration of 10 mg/l, CeO 2 NPshad a positive effect to bacterial viability. At 10 mg/l of CeO 2 NPs, the amount of counted bacteria was 3 % more than that of the blank sample. Even though the difference is marginal, but it was correlated with positive effect of CeO 2 NPs at same concentration to the biogas and methane production volume, which was discussed in section and The positive effect of CeO 2 NPs toward bacterial viability was explained by the ability to act like free radical scavengers of CeO 2. By the same mechanism of superoxide dismutase (SOD), Ce 4+ and Ce 3+ ion can convert ROS (e.g. O 2 - ) to O 2 and H 2 O(Xia et al., 2008). Therefore, oxidizing substances that can cause cell damages were converted to harmless substances. As a result, at suitable concentration, CeO 2 NPs could increase bacteria viability by inactivating ROS. The cytoprotective mechanism of CeO 2 is illustrated in Figure Figure 4.14 Cytoprotective mechanism of CeO 2 NPs Bacterial toxicity of sludge containing nanoparticles The bacterial toxicity of nanoparticles depends on many factors, such as testing method, testing media, environmental conditions, etc. There areseveral researches that have been concluded that environmental factors could greatly affect toxicity of nanoparticles (Adams et al., 2006; Ma et al., 2013). As a result, in order to have a practical view about actual toxicity of nanoparticles on the waste sludge, the bacterial toxicity test with sludge containing nanoparticles was conducted. However, due to the high solid content of sludge mixture, the sludge sample could not be taken accurately by micropipette for the bacterial toxicity test. Solid granules in the sludge blocked the tip of the pipette when the sample was taken. Therefore, the bacterial toxicity 44

53 test with raw sludge mixture that was exposed with nanoparticles could not be done. As a result, the sludge sample was centrifuged to remove the solid part and the supernatantwas used for the toxicity test. Sludge mixture was centrifuged at 5,000 rpm in 10 minutes to settle down solid part, and the supernatant was collected and used to test its toxicity on microorganism. The sludge supernatant of samples at different exposure concentration with CeO 2 and ZnO NPs were tested. The toxicity of samples before and after anaerobic digestion were compared based on percentage of inhibition on bacterial viability. The toxicity data of CeO 2 and ZnO NPs are shown in Figure 4.15 and 4.16, respectively. Overall, sludge before anaerobic digestion was more toxic than sludge after the digestion process. The sludge with exposure of 1,000 mg/l of CeO 2 NPs before anaerobic digestion caused 47.5% of inhibition to bacterial viability. However, the same sample after anaerobic digestion just had 30.4% of inhibition toward bacteria viability. Similarly, sample with 1,000 mg/l of ZnO NPs induced up to 92.3% of inhibition before anaerobic digestion, while after digestion process, this value was just 34.8% CeO 2 NPs Concentration (mg/l) Figure 4.15 Bacterial toxicity of CeO 2 NPs on sludge supernatant 45

54 Bacterial Toxicity of ZnO NPs on Sludge Before AD After AD Inhibition (%) ZnO Concentration (mg/l) Figure 4.16 Bacterial toxicity of ZnONPs on sludge supernatant However, at low exposure concentration of nanoparticles, sample after anaerobic digestion showed higher bacterial toxicity than the same sample before incubation period. From the Figure 4.15, the sample CeO 2 NPs at 10 mg/l before anaerobic digestion had positive effect on bacterial viability. But after going through the anaerobic digestion process, this sample caused toxic to E. coli. The sample ZnO 10 mg/l also showed the increase in toxicity after anaerobic digestion. This observation could be explained by the possibility that other toxic compound could be released during anaerobic digestion process. Therefore, the inhabitants to E. coli growth in this case were toxic substances from the digestion processes instead of nanoparticles. These substances could be products or intermediates from various digestion steps, such asammonia, VFA, H 2 S, or other organic compounds. The Figure 4.15 and 4.16 show that before anaerobic digestion process, the bacterial toxicity of sludge containing nanoparticles was concentration-based. It means that increased the exposure concentration of nanoparticles resulted in higher inhibition effect on bacterial viability. However, this correlation could not be observed in sludge samples after going through anaerobic digestion. As discussed in section about the possibility of microorganism to overcome nanotoxicity, there were many environmental factors and also bacteria reactivities that could affect the toxicity of nanoparticles. Adsorptions of nanoparticles to solid granule, biomass or other organic matters, nanoparticles agglomeration due to change in ionic strength, and etc. could be reasonss that made nanoparticles become bigger in size, less reactive, less bioavailability. As a result, nanoparticles became less toxic for microorganism after a long period staying in the sludge, which went through anaerobic digestion process. This fact could be clearly seen in Figure 4.17 and 4.18 where the toxicity data of CeO 2 and ZnO nanoparticles dispersion, nanoparticles on sludge before and after anaerobic digestion are compared. 46

55 100 Comparision Toxicity of CeO2 NPs Nanodispersion Before AD After AD Inhibition (%) CeO 2 NPs Concentration (mg/l) 1000 Figure 4.17 Comparison of CeO 2 NPs toxicity of different samples Comparision Toxicity of ZnO NPs Nanodispersion Before AD After AD Inhibition (%) ZnO NPs Concentration (mg/l) Figure 4.18 Comparison of ZnO NPs toxicity of different samples From the Figure 4.17, it shows that the cytoprotective effect of CeO 2 NPs at 10 mg/l was not presented in the sludge sample after anaerobic digestion. Moreover, the toxic effect of CeO 2 and ZnO nanoparticleswass highest whene. coli was exposed to nanoparticles in DI 47

56 water. The inhibition effect was reduced when E. coli was exposed to sludge containing nanoparticles, and the toxicity of the sludge sample that went through anaerobic digestion was lowest. This fact was clearly illustrated at high exposure concentration of NPs (i.e. at 500 mg/l and 1,000 mg/l). Since at high exposure concentration, the inhibition effect toward bacterial viability was mainly come from nanoparticles toxicity. In the other hand, at low exposure concentration, the inhibition effect could be the result of other toxic substances as discussed previously. In conclusion, the bacterial toxicity of nanoparticles could be greatly reduced when nanoparticles was applied in the sludge. In addition, the toxicity of nanoparticles on the digested sludge was again diminished. However, the experiments showed that even nanoparticles were applied on sludge and went through anaerobic digestion process, the toxicity of nanoparticles toward bacteria still existed. 4.5 Effects of Nanoparticles on Plants As discussed in the previous section, CeO 2 and ZnO nanoparticles caused inhibition to bacterial viability even when they were applied on the sludge and went through anaerobic digestion process. Therefore, in order to have the insight about toxicity and environmental impact of digested sludge containing nanoparticles, the phytotoxicity test was conducted. From this test, effects of digested sludge containing different concentration of CeO 2 and ZnO nanoparticles toward seed germination and root elongation were revealed. Seeds of two plants, namely Vigna radiata (mung bean) and Solanum lycopersicum (tomato), were used as tested species. The result of inhibition effects of different digested sludge samples on plants is shown on Table 4.4. Table 4.4 Inhibitions of NPs to Seed Germination and Root Growth Mung Bean Tomato Samples Inhibition on Seed Germination (%) Inhibition on Root Growth (%) Inhibition on Seed Germination (%) Inhibition on Root Growth (%) CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO 100 mg/l ZnO 500 mg/l ZnO 1,000 mg/l The data from Table 4.4 shows that nanoparticles had greater impact on root elongation than seed germination. Every sample had inhibition effect on root growth more than 50 % 48

57 as compared with control sample.basically, the inhibition effect increased with the increase of nanoparticles concentration on the sludge. Therefore, nanoparticles that existed in the sludge made the sludge become toxic for plant growth. In addition, the impact of ZnO NPs to seed germination and root growth was greater than that of CeO 2 NPs. Besides, the correlation between exposure concentration of ZnO nanoparticles and inhibition effects to plants were more transparent. Digested sludge contained 1,000 mg/l ZnO NPs inhibited 94.2% and 99.9% on root growth of mung bean and tomato, respectively. While this value of sample CeO 2 at 1,000 mg/l was just 82.5% and 62.1%, respectively. In the research of Lin and Xing (2007), ZnO NPs was also appeared to be the most toxic for seed germination and root growth of various plants. In their research, the inhibition on seed germinationwas just observed at sample of 2,000 mg/l of ZnO. However, the impact on root elongation was significant with 100% inhibition at 200 mg/l of ZnO exposure. Although this study tested on contaminated digested sludge instead of nanoparticles dispersion in research of Lin and Xing (2007),the finding that ZnO NPs were highly toxic for plants and inhibition effect on root growth was more severe than seed germination were verified. In order to have an overall perspective about phytotoxicity of digested sludge containing nanoparticles, the data on seed germination and root growth were combined and presented in germination index.the lowervalue of germination index shows the higher toxicity of that sample toward both seed germination and root growth combined. In the other meaning, the germination index showed the opposite value with inhibition percentage.the germination index of different samples are presented in Figure CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO 1000 (NPs concentrations were in term of mg/l) Figure 4.19 Germination index of different samples 49

58 The germination index data shows that mung bean seeds were more sensitive to nanoparticles toxicity than tomato. The fact that nanoparticles caused inhibitionn to various plant species differently was mentioned in researches before (Lin and Xing, 2007; García et al., 2011). Moreover, the extent of inhibition caused by CeO 2 on tomato was not proportional with the exposure concentration. The inhibition effect of CeO 2 on tomato seeds was highest at sample CeO mg/l. This result showed less toxic when compared with germination index of 0% at exposure of 640 mg/l CeO 2 in research of Garcia et al. (2011). However, in the experiment of Garcia et al. (2011), CeO 2 nanodispersion was used instead of digested sludge. Therefore, it could be indicated that high solid content of the sludge, along with the anaerobic digestion process could reduce the toxicity of nanoparticles. Nevertheless, the existence of nanoparticles in sludge still made the digested sludge become toxic for plant growth. At high concentration of ZnO NPs at 1,0000 mg/l, the digested sludge became highly toxic for plant, with germination index of 3.1% and 0% for mung bean and tomato, respectively. In conclusion, nanoparticles could inhibit the germination of seeds and growth of root even when it existed on the sludgeand went through anaerobic digestion process. The Figure 4.20 compares the inhibition effects of digested sludge containing nanoparticles on bacteria and mung bean. The correlation between inhibition effect of digested sludge and exposure concentration of nanoparticles is clearly shown on the graph. Digested sludge with higher nanoparticles concentration caused greater inhibition on bacteria viability and plant growth. In addition, in both bacterial toxicity test and phytotoxicity test, ZnO NPs caused higher inhibition effect than CeO 2 NPs. In conclusion, nanoparticles that accumulated in sludge could cause toxicity to both bacteria and plants even the sludge was gone through anaerobic digestion process Inhibition (%) Toxicity of Digested Sludge on Bacteria and Plant Mung bean Inhibition Bacteria Inhibition CeO 2 10 CeO CeO CeO 2 1, Samples (NPs concentrations were in term of mg/l) ZnO 10 ZnO 100 ZnO ZnO 1,000 Figure 4.20 Toxicity of digested sludge on bacteria and plant 50

59 Chapter 5 Conclusions and Recommendations In this research, the inhibition effects of CeO 2 and ZnO nanoparticles on the whole sludge treatment process were studied. Overall, CeO 2 and ZnO nanoparticles caused significantinhibition to the anaerobic digestion process, sludge dewatering process, bacterial viability, and plants growth. Specific conclusions that achieved from this research are presented in the following part with recommendations for further studies in this research area. 5.1 Conclusions The BMP test had optimum condition with added substrate at I/S ratio of 2. The optimum performance was shown through high biogas production and highest methane composition. ZnO NPs at 1,000 mg/l caused highest inhibition on generated biogas volume (inhibit 65.3%) and methane composition (inhibit 40.7%) of anaerobic digestion system. At tolerable exposure concentration (ZnO at 100 and 500 mg/l), the inhibition on biogas production could be overcome after 14 days. The recovery could come from environmental factors or bacterial activities in the system. CeO 2 NPs at low concentration (10 mg/l) could increase the biogas production by 11%. Increase the exposure concentration above 100 mg/l caused reduction in biogas volume. The inhibition effect on NPs on methane composition just existed at sample ZnO NPs 1,000 mg/l. The effect was possibly due to acidification of the system that caused ph drop and inhibition on methanogens. Increase the exposure concentration of nanoparticles caused increase in sludge dewatering time. Especially, sample ZnO NPs 500 mg/l and 1,000 mg/l increased the required time to dewater sludge by more than 11 and 13 times, respectively. The reasonable explanation could be due to the generation of EPS, which was triggered by the exposure of nanoparticles. The bacterial toxicity of ZnO NPs was greater than that of CeO 2 NPs. At 100 mg/l of ZnO NPs exposure, 99% of bacterial cells were killed, while this number for sample 100 mg/l of CeO 2 was just 55%. CeO 2 NPs at 10 mg/l had protective effect to bacterial cell (increase 3% of bacterial viability). The cytoprotective effect of CeO 2 at low concentration could come from the antioxidative effect of CeO 2 NPs. When nanoparticles were applied on the sludge, the bacterial toxicity of nanoparticles was reduced due to agglomeration and adsorption of nanoparticles to organic matters and biomass. Moreover, after anaerobic digestion period, the toxicity of nanoparticles was shrunk more also. The accumulation of CeO 2 and ZnO NPs on sludge made the digested sludgebecome unsuitable to apply as biosolid. Since the contaminated digested sludge caused great inhibition on root growth and also seed germination of plants. 51

60 5.2 Recommendations for Future Study Since productions and applications of nanoparticles have been increasing constantly, the need to have a standard method or official guideline about testing nanoparticles toxicityiscrucial. Adetailed standard method about preparation and characterization of nanoparticles, toxicity testing methods, etc. will help researchers to have a comparable result about toxicity of various nanoparticles Concentration of various nanoparticles in fresh water, domestic wastewater and industrial wastewater should be analyzed to have a reference data. From there, actual exposure concentration of nanoparticles and their impacts on different system can be studied. The particle-size effect of metals and metal oxides on anaerobic digestion of sludge should be studied. Since characteristics and reactivities of nanoparticles can be affected when nanoparticles are applied on sludge, so whether or not nanosized particles are more toxic than its bulk-sized counterpart?comparison between inhibition effects ofnanoparticles and its bulk-sized counterpart on anaerobic digestion systemcan be done to answer the question. Adaptation mechanisms of bacteria to nanoparticles toxicity should be investigated by studying relationship between extracellular polysaccharides and proteinsproduction, nanoparticles exposure and bacterial viability. The protective effect of EPS against nanoparticles can be verified by using imaging techniques to observe the adsorption of NPs on EPS. Leachate of nanoparticles from digested sludge to landfill should be studied to have a perspective about life cycle of nanoparticles and possibility to contaminate ground water of nanoparticles. Standard leaching test can be used to study the leachability of nanoparticles. Moreover, the transformation of nanoparticles through soil column should be studied to know the actual fate, mobility and transformation of nanoparticles. 52

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65 Appendix A Calculation of inoculum and substrate amount for BMP test 57

66 Calculation of inoculum and substrate amount for BMP test: Mass of Inoculum = 200 g (%TS = 6.5% and %VS = 78% TS ) => Mass of VS in Inoculum = 200 g x 6.5% x 78% = g VS Mass of COD in Substrate: Table A1Constituents of Substrate in Various I/S Ratios Samples I/S ratio Mass of VS in Inoculum (g) Mass of COD in Substrate (g) Mass of Glucose (g) Mass of NH 4 HCO 3 (g) Mass of KH 2 PO 4 (g) Take example of I/S = 2 to show the calculation steps: I/S = 2 =>Mass of COD needed = g 2 = 5.07 g COD We have 1 g Glucose = g COD (Sawyer et al., 2003) => Mass of Glucose needed = = 4.75 g Glucose We have COD:N = 250:5 => Mass of N needed = 5.07 x = 0.1 g N => Mass of NH 4 HCO 3 needed = 0.1 g x 79 g 14 g = 0.56 gnh 4 HCO 3 We have COD:P = 250:1 =>Mass of P needed = 5.07 x = 0.02 g P => Mass of KH 2 PO 4 needed = 0.02 g x 136g 31 g = 0.09 gkh 2 PO 4 58

67 Appendix B Data of volume biogas production of BMP test 59

68 Table B1 Biogas Volume of BMP Optimization Test Day Average Biogas Volume Cumulative Biogas Volume at STP Temp ( o (ml) (NmL) C) Blank I/S1 I/S2 I/S3 Blank I/S1 I/S2 I/S

69 Table B2 Average Biogas Volume of BMP Test with NPs Exposures Day Temp. ( o C) Average Biogas Volume (ml) Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO

70 Day Temp. Average Biogas Volume (ml) ( o C) Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO (NPs concentrations were in term of mg/l) 62

71 Table B3 Cumulative Biogas Volume of BMP test with NPs Exposures Day Temp. ( o C) Cumulative Biogas Volume at STP (NmL) Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO

72 Day Temp. Cumulative Biogas Volume at STP (NmL) ( o C) Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO (NPs concentrations were in term of mg/l) 64

73 Appendix C Data of biogas composition of BMP test 65

74 Table C1 Biogas Composition of Various I/S Ratio Samples Sample CH 4 (%) CO 2 (%) N 2 (%) O 2 (%) CO (%) Blank I/S I/S I/S Table C2 Biogas Composition of Samples With NPs Exposure Day Sample CH 4 (%) CO 2 (%) N 2 (%) O 2 (%) CO (%) 7 Blank CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO100 mg/l ZnO 500 mg/l ZnO1,000 mg/l Blank CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO100 mg/l ZnO 500 mg/l ZnO 1,000 mg/l

75 Appendix D Sludge characteristics analysis 67

76 Table D1 Characteristics of Sludge Mixture Before and After BMP Optimization Test Parameters Blank I/S 1 I/S 2 I/S 3 Before After Before After Before After Before After ph TS (%) VS (%) CODs (mg/l) Total Alkalinity (mg/l as CaCO 3 ) Total VFA (mg/l) VFA/ Alkalinity VS destruction (%) CODs removal (%) ,400 12,500 17, , , , ,500 1,500 2,500 2, ,

77 Table D2 Characteristics of Sludge Mixture Before and After Anaerobic Digestion With NPs Exposures Parameters Before AD After AD Blank Blank CeO 2 10 CeO CeO CeO ZnO 10 ZnO 100 ZnO 500 ZnO 1000 ph TS (%) VS (%) CODs (mg/l) 11, Total Alkalinity (mg/l as CaCO 3 ) 3,600 4,650 4,750 4,600 4,650 4,550 4,050 4,650 4,950 3,300 Total VFA (mg/l) 840 1,300 1,300 1,100 1,150 1,000 1,250 1,250 1,200 4,600 VFA/Alk VS destruction (%) CODs removal (%) (NPs concentrations were in term of mg/l) 69

78 Appendix E Data of bacterial toxicity test 70

79 Table E1 Data of Toxicity Test with NPs Dispersions Samples Bacteria Concentration (CFU/mL) Inhibition (%) Standard Deviation Blank x CeO 2 10 mg/l x CeO 2 50 mg/l x CeO mg/l 68.7 x CeO mg/l 4.7 x CeO 2 1,000 mg/l 0.3 x ZnO 10 mg/l x ZnO 50 mg/l 30.3 x ZnO 100 mg/l 1.7 x ZnO 500 mg/l ZnO 1,000 mg/l Data shows as mean ± standard deviation (n=3) Table E2 Data of Toxicity Test with Sludge Containing NPs Before AD Samples Bacteria Concentration (CFU/mL) Inhibition (%) Standard Deviation Blank x CeO 2 10 mg/l x CeO mg/l x CeO mg/l 98.0 x CeO 2 1,000 mg/l 93.0 x ZnO 10 mg/l x ZnO 100 mg/l x ZnO 500 mg/l 39.0 x ZnO 1,000 mg/l 13.7 x Data shows as mean ± standard deviation (n=3) 71

80 Table E3 Data of Toxicity Test with Sludge Containing NPs After AD Samples Bacteria Concentration (CFU/mL) Inhibition (%) Standard Deviation Blank 23.0 x CeO 2 10 mg/l 20.0 x CeO mg/l 17.3 x CeO mg/l 16.7 x CeO 2 1,000 mg/l 16.0 x ZnO 10 mg/l 15.0 x ZnO 100 mg/l 18.7 x ZnO 500 mg/l 15.3 x ZnO 1,000 mg/l 15.0 x Data shows as mean ± standard deviation (n=3) 72

81 Appendix F Data of phytotoxicity test 73

82 Table F1 Effect of NPs on Mung Bean Samples Seed Germination (%) Root Length (mm) Germination Index (%) Blank CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO 100 mg/l ZnO 500 mg/l ZnO 1,000 mg/l Table F2 Effect of NPs on Tomato Samples Seed Germination (%) Root Length (mm) Germination Index (%) Blank CeO 2 10 mg/l CeO mg/l CeO mg/l CeO 2 1,000 mg/l ZnO 10 mg/l ZnO 100 mg/l ZnO 500 mg/l ZnO 1,000 mg/l

83 Appendix G Pictures of various experiments 75

84 Figure G1 BMP test with nanoparticles exposure Figure G2 CST machine 76

85 Figure G3 Bacterial toxicity test Figure G4 Phytotoxicity test 77