Acidogenic biotreatment of wastewater containing 2 nitroaniline and copper

ACIDOGENIC BIOTREATMENT OF WASTEWATER CONTAINING 2-NITROANILINE AND COPPER KRISTHOMBU B. S. N. JINADASA NATIONAL UNIVERSITY OF SINGAPORE 2003 ACIDOGENIC BIOTREATMENT OF WASTEWATER CONTAINING 2-NITROANILINE AND COPPER KRISTHOMBU B. S. N. JINADASA (B.Sc.Eng. (Hons.) University of Peradeniya, Sri Lanka) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING DEPARTMENT OF CIVIL ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2003 ACKNOWLEDGEMENTS I am deeply grateful to all the support and encouragement given to me during the period of my research work at National University of Singapore (NUS) by my supervisors, Prof. W.J. Ng and Assoc. Prof. M.A. Aziz. Their guidance was crucial to the success of my research work at NUS. I wish to thank all the staff of the Environmental Engineering Laboratory, Department of Civil Engineering. Their friendly and helpful attitudes made my research experience at NUS a very happy and memorable one. I would also like to express my appreciation to the National University of Singapore for awarding a research scholarship which enabled me to pursue a higher degree. TABLE OF CONTENTS Page TABLE OF CONTENTS ii SUMMARY vi LIST OF FIGURES viii LIST OF TABLES x LIST OF PLATES xii LIST OF ABBREVIATIONS xiii CHAPTER ONE INTRODUCTION 1.1 Background 1.2 Objectives of the Study 1.3 Scope of the Study 1.4 Experimental Protocol CHAPTER TWO LITERATURE REVIEW 2.1 Fundamentals of Anaerobic Wastewater Treatment 2.1.1 Phases of anaerobic biotreatment process 2.1.2 Substrate flow in anaerobic system 2.2 Inhibitory Substances 10 2.3 Persistent Organic Compounds 12 2.4 Nitroaromatic Compounds 12 2.4.1 Inhibitory characteristics of nitroaromatic compounds 13 2.5 2-Nitroaniline 14 2.6 Biotreatment of Nitroaromatic Compounds under Anaerobic Conditions 16 2.6.1 Feasibility of acidogenic pretreatment of wastewater 19 ii 2.6.2 Significance of acidogenic conditions in the reduction of nitroaromatic compounds 20 2.6.3 Effect of other substitutes in aromatic ring in reduction of nitroaromatic compounds 22 2.7 Heavy Metals 23 2.7.1 Sources 23 2.7.2 Copper 24 2.8 Effects of Heavy Metals on Microorganisms 26 2.9 Effect of Heavy Metals on Acidogenic Biotreatment 28 2.9.1 Activity factor 28 2.10 Need for Research 2.10.1 Biotransformation studies CHAPTER THREE 29 30 MATERIALS AND METHODS 3.1 Reactor Description 31 3.1.1 Reactor operation 32 3.1.2 Acidogenic reactor feed 33 3.2 Anaerobic Toxicity Assay (ATA) Test 35 3.2.1 Experimental setup 35 3.2.2 Operating protocol 36 3.2.3 Experimental procedure 37 3.3 Preparation and Analysis of Samples 38 3.3.1 Identification of metabolites 40 3.3.2 Quantification of metabolites 40 3.4 Track Runs with Acidogenic Sequencing Batch Reactor 41 3.5 ISO Inhibition Test (Aerobic) 42 3.5.1 Experimental setup 42 iii 3.5.2 Test procedure 43 3.5.3 Computation of inhibition 45 3.5.4 Determination of EC50 value 45 3.5.5 Influent and effluent inhibitions 45 3.6 Microbiological Observations CHAPTER FOUR 46 RESULTS AND DISCUSSIONS 4.1 Acidogenic Biotreatment of Wastewater Containing 2-Nitroaniline 48 4.1.1 Biodegradation of 2-Nitroaniline 48 4.1.2 49 Biodegrdation pathway of 2-Nitroaniline 4.1.3 Effect of 2-Nitroaniline on biogas production 58 4.1.4 Effect of 2-Nitroaniline on Volatile Fatty Acids (VFAs) production 62 4.1.5 Comparison of influent and effluent inhibitions 65 4.1.6 Anaerobic Sequencing Batch Reactor (anSBR) Track Run with 2-Nitroaniline 66 4.2 Acidogenic Biotreatment of Wastewater Containing 2-Nitroaniline and copper 69 4.2.1 Effect of copper on Volatile Fatty Acids (VFAs) production 69 4.2.2 Effect of copper on biogas production 72 4.2.3 Effect of copper on biodegradation of 2-Nitroaniline 75 4.2.4 Influent and effluent inhibitions 76 4.2.5 anSBR Track Run with 2-Nitroaniline and copper 77 4.3 Determination of EC50 Values of 2-Nitroaniline and copper in Aerobic System 80 4.4 SEM Observations of Microorganisms 81 iv CHAPTER FIVE SUMMARY, CONCLUSIONS AND RECOMMENDATIONS 5.1 Summary 84 5.2 Conclusions 85 5.3 Recommendations 86 REFERENCES 87 v SUMMARY Laboratory investigations were carried out to evaluate the feasibility of acidogenic pretreatment of wastewater containing 2-nitroaniline and copper. The experiments were conducted using the Anaerobic Toxicity Assay (ATA) Test, Anaerobic Sequencing Batch Reactor (anSBR), and Aerobic Inhibition Test. From the experimental results, it was found that acidogenic biotreatment process could remove 2-nitroaniline effectively. Removal efficiency was around 95% when the concentration of 2-nitroaniline was 12.5 mg/L. This suggested that the acidogenic phase could remove 2-nitroaniline effectively at least in the short term. At 12.5 mg/L 2-nitroaniline, biogas production was found to be higher than that of the control. This indicated the possibility of stimulatory effects by 2-nitroaniline on the acidogenic process at low concentrations. The inhibition potentials of the influent and effluent at varying feed concentrations of 2-nitraoniline were determined by the oxygen consumption rate measured in accordance with International Standards Organisation method ISO 8192-1986-E. There was a significant reduction in inhibition of the effluent compared to that of the influent. This reduction in inhibition is presumed due to the conversion of the potentially inhibitory organics in the wastewater into less inhibitory or non-inhibitory byproducts. Metabolites were identified as 2-methylbenzimidazole and o-phenylenediamine using gas chromatography mass spectrometry and quantified by high performance liquid vi chromatography. Around 80% of the 2-nitroaniline was recovered as metabolites and other end products. 2-nitroaniline was transformed to its corresponding amine and then further transformed to 2-methylbenzimidazole under acidogenic conditions. The identification of o-phenylenediamine and 2-methylbenzimidazole was confirmed by comparing their mass spectral fragmentation patterns against standards. The effect of copper on the acidogenic process was studied using the Anaerobic Toxicity Assay (ATA) Test and an Anaerobic Sequencing Batch Reactor (anSBR). Copper inhibition on bacterial activity was estimated by considering volatile fatty acids (VFAs) production. Propanoic acid was most severely affected followed by pentanoic acid, butanoic acid and ethanoic acid with increasing concentrations of copper. The removal of 2-nitroaniline was observed to be around 90 % and 78 % when the copper concentrations were mg/L and 25 mg/L respectively with 2-nitroaniline acclimated biomass in the anSBR track runs. The acidogenic process could, therefore, be an attractive alternative for upgrading present aerobic treatment processes which are not effective at removing nitroaromatic compounds such as nitrobenzenes, nitroanilines and nitrophenols from industrial wastewaters. These compounds could be in a wastewater containing heavy metals such as copper. vii LIST OF FIGURES Title Page Figure 1.1 Schematic diagram of experimental protocol Figure 2.1 Three phases of the anaerobic biotreatment process Figure 2.2 Flow chart illustrating the mechanism of anaerobic biotreatment system Figure 2.3 Typical effect of an inhibitory compound on biotreatment 11 processes Figure 2.4 Reduction pathway of nitrobenzene to aniline under anaerobic conditions 16 Figure 2.5 Significant pathways in acidogenic conditions relate to the reduction of nitroaromatic compounds 21 Figure 3.1 Schematic diagram showing the experimental set-up of laboratory anSBR system 31 Figure 3.2 Procedure for preparation and analysis of samples 38 Figure 3.3 Schematic diagram of ISO inhibition test apparatus assembly 43 Figure 4.1 Removal efficiency of 2-nitroaniline at concentrations of 12.5~100 mg/L 49 Figure 4.2 Mass spectra identification of o-phenylenediamine (Reactor sample) 50 Figure 4.3 Mass spectra identification of 2-methylbenzimidazole (Reactor sample) 51 Figure 4.4 Mass spectra identification of o-phenylenediamine (Using standard chemicals) 52 Figure 4.5 Mass spectra identification of 2-methylbenzimidazole (Using standard chemicals) 53 Figure 4.6 Degradation of 2-nitroaniline and possible metabolic products in acidogenic biotreatment 54 Figure 4.7 Possible biotransformation pathway for 2-nitroaniline to o-phenylenediamine 54 viii Chapter Table 4.7 Reduction in dissolved COD in the Acidogenic reactor (2-nitroaniline = 100 mg/L, Cu 25 = mg/L) Dissolved COD at start (mg/L) Effluent dissolved COD (mg/L) Dissolved COD reduction (%) 8026 7435 7.36 % Table 4.8 Inhibition test results for Track Run (2-nitroaniline = 100 mg/L, Cu = 25 mg/L) Blank Influent Effluent Respiration rate (mg/L·min) 0.57 0.37 0.42 Inhibition (%) 35.09 26.32 4.3 Determination of EC50 values of 2-nitroaniline and copper in aerobic system The inhibitory effects of 2-nitroaniline and copper on aerobic system were examined because these compounds would not be removed totally by acidogenic pretreatment process. The inhibition was calculated according to the procedure specified by ISO 8192-1986(E) Method B, and plotted in Figure 4.26. The EC50 values for 2-nitroaniline and cooper were found to be around 60 mg/L and 30 mg/L respectively. It was found that at lower concentration (Cu[...]... containing 2- nitroaniline ISO Inhibition Test (aerobic) for 2- nitroaniline Anaerobic Toxicity Assay Test (2- nitroaniline) Inhibition on acidogenisis by 2- nitroaniline Biodegradation pathway for acidogenic pretreatment of 2- nitroaniline Biotreatment of wastewater containing 2- nitroaniline and copper Anaerobic Toxicity Assay Test (2- nitroaniline and copper) Inhibitory effect of copper on acidogenic biotreatment. .. 2- nitroaniline and copper The following are the sub-objectives of this study: 1 To study the feasibility of treating wastewater containing 2- nitroaniline using the acidogenic process 2 To examine the biodegradation pathway of 2- nitroaniline in the acidogenic process identifying and quantifying the metabolites 3 To study the inhibitory effect of copper on acidogenic biotreatment of a wastewater containing 2- nitroaniline. .. effect of persistent organic compounds and heavy metals The second part of this study was, therefore, carried out to investigate the possible inhibitory effects of copper on acidogenic biotreatment of a wastewater containing 2- nitroaniline 2 Chapter 1 1 .2 Objectives of the study The main objective of this study is to investigate the feasibility of acidogenic pre-treatment of wastewaters containing 2- nitroaniline. .. Nitrobenzene 123 0.081 2- Nitroaniline 138 0.014 3 -Nitroaniline 138 0.03 2- Phenylenediamine 108 18. 92 3-Phenylenediamine 108 65.7 Phenol 94 13.83 2- Aminophenol 109 3 .21 3-Aminophenol 109 18.81 4-Aminophenol 109 14 .22 2, 4- Diaminophenol 124 0 .28 3 2- Nitrophenol 139 0.089 3-Nitrophenol 139 0.115 4-Nitrophenol 139 0.061 20 .5 2. 5 2- Nitroaniline 2- nitroaniline may be released to the environment through the wastewater. .. al, 1997) The physical and chemical properties of 2- nitroaniline are shown in Table 2. 3 Table 2. 3 Physical and chemical properties of 2- nitroaniline (NIST 20 01) Chemical abstract number (CAS #) Synonyms Analytical method Molecular formula 88744 2- Nitroaniline Benzenamine, 2- Nitro o -Nitroaniline Reverse Phase HPLC and EPA METHOD 827 0C by GCMS C6H6N2O2 NH2 Chemical structure NO2 Chemical intermediate... activity and copper concentration 71 Figure 4 .20 Effects of copper on cumulative total gas production 73 Figure 4 .21 Relative gasification activities with time 74 Figure 4 .22 Effect of increasing concentration of copper on carbon dioxide production 75 Figure 4 .23 Biodegradation of 2- nitroaniline with varying copper concentrations Biodegradation of 2- nitroaniline during REACT in anSBR cycle 76 Figure 4 .25 ... Variation of MLVSS and MLSS during REACT in anSBR cycle 79 Figure 4 .26 Inhibition at different concentrations of 2- nitroaniline and copper 81 Figure 4.17 Figure 4 .24 67 78 ix LIST OF TABLES Title Page Table 2. 1 Components in a carbohydrate-fed anaerobic biotreatment system 10 Table 2. 2 Molecular weight and IC50 values of some aromatics to methanogenic activity 14 Table 2. 3 Physical and chemical properties of. .. performance profile of the anSBR during a cycle 3 Determination of EC50 values of 2- nitroaniline and copper in aerobic system to assess inhibitory effects 4 Microscopic observations of microbial populations under varying concentrations of 2- nitroaniline and copper to determine morphology 4 Chapter 1 1.4 Experimental Protocol A schematic diagram of study sequences is shown in Figure 1.1 Biotreatment of wastewater. .. pathway for 2- nitroaniline to 2- methylbenzimidazole in presence of aliphatic carboxylic acids 55 Figure 4.9 Quantification of metabolic products 57 Figure 4.10 Variation of cumulative total gas production with varying concentrations of 2- nitroaniline 59 Figure 4.11 Effect of increasing concentration of 2- nitroaniline on carbon dioxide production 60 Figure 4. 12 Effect of increasing concentration of 2- nitroaniline. .. inhibitions at different 2- nitroaniline concentrations 66 Table 4 .2 Reduction in dissolved COD in the acidogenic reactor 68 Table 4.3 Inhibition test results (2- nitroaniline = 100 mg/L) 68 Table 4.4 Variation of Activity Factor with increasing 2- nitroaniline concentration 72 Table 4.5 Variation of Activity Factor with increasing Copper concentration 72 x Table 4.6 Comparison of influent and effluent inhibitions . 23 2. 7.1 Sources 23 2. 7 .2 Copper 24 2. 8 Effects of Heavy Metals on Microorganisms 26 2. 9 Effect of Heavy Metals on Acidogenic Biotreatment 28 2. 9.1 Activity factor 2. 10. with 2- Nitroaniline 66 4 .2 Acidogenic Biotreatment of Wastewater Containing 2- Nitroaniline and copper 69 4 .2. 1 Effect of copper on Volatile Fatty Acids (VFAs) production 4 .2. 2 Effect. FOUR RESULTS AND DISCUSSIONS 4.1 Acidogenic Biotreatment of Wastewater Containing 2- Nitroaniline 48 4.1.1 Biodegradation of 2- Nitroaniline 4.1 .2 Biodegrdation pathway of 2- Nitroaniline