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BIOLOGICAL TREATMENT OF HIGH WASTEWATER USING YEAST AND BACTERIAL SYSTEMS

by

Nguyen Phuoc Dan

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Engineering

Examination Committee: Prof. C. Visvanathan (Chairman) Prof. Chongrak Polprasert (Co-chairman) Prof. Nguyen Cong Thanh Dr. Josef Trankler Dr. Sudip K. Rakshit

External Examiner: Prof. Ronald E. Simard

Nationality: Vietnamese Previous Degree: Bachelor of Engineering (Civil) Hochiminh City University of Technology (HUT) Hochiminh City, Vietnam Master of Engineering (Environmental Engineering) AIT, Thailand

Scholarship Donor: Swiss Development Cooperation (SDC)

Asian Institute of Technology School of Environment, Resources and Development Bangkok, Thailand December 2001 Acknowledgements

The author wishes to deeply express his gratitude to his advisor, Prof. C. Visvanathan for kindly giving valuable guidance, suggestions and encouragement through his study in AIT. He would like to express his appreciation to his co-advisor, Prof. Chongrak Polprasert for his valuable comments and suggestions provided throughout the research work. The author wishes to express deepest sincere thanks to Prof. Nguyen Cong Thanh, Dr. Josef Trankler, Dr. Sudip K. Rakshit and Dr. A. Sathasivan for their valuable comments, critical ideas and serving as members of examination committee.

A special thank is addressed to Prof. Ronald E. Simard for kindly accepting to serve as External Examiner His constructive and professional comments are highly appreciated.

The author gratefully acknowledges Swiss Development Cooperation (SDC)- EPFL,IGE/GS for his financial support. Grateful acknowledgement is also extended to Nishihara ERSC. for supporting partially experimental equipment. Also acknowledgement is given to the SERD school for financial support on attendance of Conference in Malaysia.

The author is very grateful to Mdm. Visvanathan, Mr. Jonathan Shaw and Mr. Basu for providing comments and editing in English language.

A special thank is extended to Lab Supervisors, Mr. Suwat, Ms. Salaya, Mr. Peter and Mr. Chai, technicians Khun Verin, Khun Tam and others. The author would like to thank his friend, Master student, M.M. Cho, for co-operation of thesis works.

The author is most grateful to his family and CEFINEA’s Director, Prof. L.M. Triet, for mental support during study in AIT.

ii Abstract

This study aimed to compare the performance of aerobic treatment using wild mixed yeast and bacterial culture for high salinity wastewater. The operating conditions of yeast treatment under high salinity such as pH, retention time (SRT) and dissolved (DO) were examined. The comparative evaluation is based on determination of biokinetic coefficients using the respirometric method and treatment efficiency of long-term operation of two laboratory-scale systems.

The biokinetic experiments reveal that yeast culture has a lower observed maximum specific grow rate (Pobs) at low content (20g/L) than that of bacteria. But Pobs of yeasts at higher salt contents (above 30 g/L) did not decline dramatically and had higher value than that of bacteria. The osmotolerant yeast mixture was able to tolerate a wider pH range than bacterial culture. The (COD) removal rate of the yeast mixture was highest at pH values 5.0-5.5.

Two laboratory-scale membrane bioreactor systems were investigated to treat high salinity wastewater containing high organic (5,000 mg/L COD) and salt content (32 g/L NaCl), namely: the Yeast Membrane Bioreactor (YMBR), and Yeast pretreatment followed by Bacterial Membrane Bioreactor (BMBR). In the YMBR system, experimental runs were conducted with a mean biomass concentration of 12 g MLSS/L. Here, the maximum COD removal rate of 0.93 g COD/g MLSS.day was obtained at F/M of 1.5 g COD /g MLSS.d, whereas the BMBR system was operated with a biomass concentration of up to 25 g MLSS/L, resulting in maximum COD removal rate of 0.32 kg COD /kg MLSS.day at F/M ratio of 0.4. In comparison the BMBR, the YMBR could obtain higher COD removal rate at higher organic loading, indicating the potential of the yeast reactor system to treat high salinity wastewater containing high organic concentration.

Transmembrane in the BMBR was progressively increased from 2 to 60 kPa after 12d, 6 d and 2 d at hydraulic retention time (HRT) of 14h, 9 h and 4h, with average biomass concentration of 6.1, 15 and 20 g MLSS/L respectively. By contrast, the transmembrane pressure in YMBR was only increased from 2 to 60 kPa only after 76 days of operation, with an average biomass concentration of 12 MLSS/L and an operating HRT range of 5 - 32 h.

The comparative evaluation of treatment performance of both YMBR and BMBR with the low organic-feed wastewater (1,000 mg/L COD and 32 g/L NaCl) was examined. COD removal of both processes were above 90% at HRT of 5 h. Under the same operating conditions, the YMBR could run under transmembrane pressure 10 times lower than the BMBR with a significantly reduced membrane fouling rate. This may be due to low production of adhesive extracellular polymers (ECP) and the secondary layer formed from large free yeast cells. ECP production of bacterial sludge was increased considerably at high salt contents and high sludge retention time (SRT). For the bacterial sludge, the increase salinity led to increase in ECP value, whereas the ECP content of the yeast sludge was relatively very small.

iii Table of Contents

Chapter Title Page

Title page i Acknowledgements ii Abstract iii Table of Contents iv List of Figures vii List of Tables x Abbreviations xii

1 Introduction 1.1 Background 1 1.1.1 Environmental Concerns 1 1.1.2 Effects of High Salinity on Biological Treatment Processes 2 1.1.3 Salt-Tolerant or Halophilic 3 1.1.4 Membrane Bioreactor Process 3 1.2 Objectives of the Study 4 1.3 Scope of the Study 4

2 Literature Review 6 2.1 Introduction 6 2.1.1 The Seafood Processing Industry 6 2.1.2 Pickled Vegetable Processing 9 2.1.3 Other Saline Wastewaters 13 2.2 Effects of High Salinity on Biological Waste Treatment Process 13 2.2.1 Aerobic Treatment 14 2.2.2 Anaerobic Treatment 17 2.2.3 Nutrient Removal 18 2.3 Application of Halophilic Bacteria for Saline 19 2.4 Yeasts 22 2.4.1 General 22 2.4.2 Applications of Yeasts for Wastewater Treatment 24 2.5 Theoretical Modeling Consideration 33 2.5.1 Growth without Inhibition 33 2.5.2 Growth with Inhibition 35 2.6 Respirometric Method 38 2.6.1 Respirometer 38 2.6.2 Experimental Procedure 38 2.6.3 Determination of Kinetic Constants 40 2.7 Membrane Bioreactor (MBR) 41 2.7.1 Advantage of the MBR Process 42 2.7.2 Main Design Parameters 42 2.7.3 Membrane Fouling 45

3 Methodology 3.1 Biokinetic Study 49 3.1.1 Seed Sludge 51 3.1.2 Acclimation 52 3.1.3 Biokinetic Experiments 52 3.2 Parametric Study 54

iv 3.2.1 pH values 54 3.2.2 Sludge Retention Time (SRT) 55 3.3 Biomembrane Study 56 3.3.1 High COD loading 56 3.3.2 Low COD loading 59 3.4 Sludge Characterization Study 60 3.5 Analytical Methods 60

4 Results and Discussion 4.1 Biokinetic Study 62 4.1.1 Enrichment and Acclimation of Yeast and Mixed Bacterial Sludge 62 4.1.2 Evaluation and Comparison of Biokinetic Coefficients 69 4.2 Parametric Study 73 4.2.1 DO and pH 74 4.2.2 Variation in Mixed Yeast and Bacterial Cultures 78 4.2.3 Effect of SRT on COD and Nitrogen Removal 80 4.3 Biomembrane Study 81 4.3.1 High COD loading 82 4.3.2 Low COD loading 87 4.4 Sludge Characterization Study 93 4.4.1 Culture Study 94 4.4.2 YMBR and BMBR 96 4.4.3 Microscopic Observations of Mixed Yeast Sludge 97 4.4.4 Nutrient Uptake 98

5 Conclusions and Recommendations 5.1 Conclusions 100 5.2 Recommendations 101 Appendix A: Pictures of Experiments A-1 Appendix B: Experimental Data of Acclimation B-1 Appendix C: Experimental Data of Biokinetic Study C-1 Appendix D: Experimental Data of Parametric Study D-1 Appendix E: Experimental Data of Biomembrane Study E-1

v List of Figures

Figure Title Page

2.1 Flow diagram of steamed canned shrimp processing 7 2.2 Flow diagram of Dried and Salted fish processing 9 2.3 Flow diagram of kim chi pickles processing 11 2.4 Variation of COD removal rate with salt contents (Kargi and Uygur, 1996) 15 2.5 Diagram of a nitrogen treatment system 18 2.6 Schematic diagram of percolation reactor (Kargi and Uygur, 1996) 20 2.7 Variation of COD removal rate (R) as function of salt content (Kargi and Dincer, 2000) 20 2.8 Schematic diagram of the biofilter and treatment system (Yang et al., 2000) 21 2.9 Diagram of yeast cell (Salle, 1961) 22 2.10 Budding is a common reproductive process in yeasts 23 2.11 True mycelium (formed by fission) and pseudomycelium (formed by budding) 23 2.12 Specific growth rate of Candida ingens vs DO and VFA concentration (Anciaux et al., 1989) 25 2.13 Traditional carbon and nitrogen removal system can be altered with anaerobic and yeast treatment system (Ortiz et al. 1997) 27 2.14 Schematic diagram of the Yeast Cycle System (YCS) 28 2.15 Comparison between Yeast Cycle System (YCS) and complete mixed (AS) (Nishihara ESRC Ltd., 2001) 29 2.16 SCP from confectionery effluent (Gray, 1989) 31 2.17 The Symba process (Gray, 1989) 31 2.18 Growth curve of microorganisms in a culture 33 2.19 The effects of a limiting substrate on the specific growth rate (Monod model) 34 2.20 Curves of inhibition growth models (n =1: Ghose and Tyagi; n= 0.5: Bazua and Wilke model) 35 2.21 Curves of substrate inhibition growth models 36 2.22 pH and DO models 37 2.23 Schematic diagram of respirometer 38 2.24 Recorder chart with a typical respirogram (Cech et al., 1984) 39 2.25 OUR response in respirometer (Ekama, et al., 1986) 40 2.26 Diagram of membrane bioreactor processes 42 2.27 Diagram of fouling mechanisms (adsorption and deposition) 45 2.28 Schematic illustration of membrane biofouling process (Ridgway and Flemming, 1996). 46 2.29 Schematic diagram of biofloc or biofilm 47

3.1 Flowchart of different phases of experimental study 49 3.2 Flowchart of biokinetic experiments 51 3.3 Schematic diagram of enrichment procedure 51 3.4 Respirometer set-up 53 3.5 Membrane reactor systems in the high COD loading 58 3.6 Schematic diagram of biomembrane reactor 59

4.1 Appearance of yeast cells predominantly grown in glucose-feed wastewater 62 4.2 Acclimation of yeast sludge cultured with glucose at high salt contents 63

vi 4.3 Acclimation of microbial mixed culture with glucose-feed wastewater as function of salt 64 4.4 Typical COD and COD removal profile of mixed yeast batch in glucose-feed wastewater at 32 g salt/L 65 4.5 Variation in COD removal rate versus salt contents in acclimatized yeast and bacterial mixed cultures 66 4.6 Acclimation of yeast and bacterial to fish-protein-feed wastewater containing 32 g/L salt 67 4.7 Predominance of wild yeast strains in the cultures fed with fish-protein wastewater (at 32 g/L salt) 68 4.8 OUR curves of mixed yeast and bacterial sludges feed with 50 mg/L COD and 32 g/L salt (glucose-feed wastewater) 70 4.9 OUR curves of mixed yeast and bacterial sludges feed with 100 mg/L COD and 32 g/L salt (protein-feed wastewater) 70 4.10 Variation in specific growth rate of yeast sludge as function of COD at different salt contents for glucose-feed wastewater 71 4.11 Variation in specific growth rate of bacterial culture as function of COD concentration at different salt contents for glucose-feed wastewater 71 4.12 Inhibition effect of salt contents on mixed yeast and bacterial cultures on glucose- feed wastewater 73 4.13 Inhibition effect of salt contents on mixed yeast and bacterial cultures on protein- feed wastewater 73 4.14 DO and COD changes of yeast batch fed with glucose and protein wastewater at 32 g salt/L 74 4.15 DO and COD changes of mixed bacterial batch fed with glucose and protein wastewater at salt content of 32 g/L 75 4.16 pH changes of yeast culturefed with glucose and protein wastewater at 32 g salt/L 75 4.17 pH changes of mixed bacterial batch fed with glucose and protein wastewaters at 32 g salt/L 76 4.18 Variation in OUR as funtion of initial for mixed yeast fed with protein wastewater at 32 g salt/L 77 4.19 Variation in OUR as funtion of initial pHs for mixed bacterial fed with glucose wastewater at 32 g salt/L 77 4.20 Variation in nitrogen components as funtion of time in the mixed yeast at 32 g salt/L NaCl (Nitrite and nitrate concentration of both feed wastewaters were not dectected) 79 4.21 Variation in nitrogen components vs. time in the mixed bacterial culture at 32 g salt/L NaCl 79 4.22 Variation in MLSS as funtion of SRT 80 4.23 Variation in COD, nitrogen removal and MLSS in funtion of SRT in mixed yeast culture at VLR of 5 kg COD/m3.d (32 g salt/L) 81 4.24 Variation in flux as function of membrane transmembrane pressure (Viscosity of at 26oC = 8.70 x 10-4 kg/m.sec) 82 4.25 Variation in COD, biomass and transmembrane pressure in the YMBR as function of volumetric loading 83 4.26 Variation in COD, biomass and transmembrane pressure in the BMBR as function of volumetric loading 84 4.27 Variation in COD removal in function of volumetric loading rate 86 4.28 Variation in COD removal rate in function of F/M ratio (initial COD = 5,000 mg/L) 86 4.29 Variation in COD, biomass and transmembrane pressure in the YMBR as function of volumetric loading 88

vii 4.30 Variation in COD, biomass and transmembrane pressure in the BMBR as function of volumetric loading 89 4.31 Variation in COD removal as function of HRTs in YMBR and BMBR 89 4.32 Variation in specific growth rate of yeast and bacteria at 32 g salt/L in function of COD 90 4.33 Possible mechanisms for flux enhancement by yeast cells 93 4.34 Variation in ECP and CST in function of salt content 95 4.35 Variation in SVI, SS, ECP and viscosity with salt content in mixed bacterial cultures 96 4.36 ECP contents of mixed yeast and bacterial sludges in YMBR and BMBR 97

viii List of Tables

Table Title Page

1.1 Comparison of pollutant loads from seafood processing and other industries in the Saigon-Dong Nai river catchment area (DOSTE-HCMC and CEFINEA, 1998) 2

2.1 Characteristics of herring waste (Balslev-Olesen et al., 1990) 7 2.2 Characteristics of wastewater from the dried salted fish (Dan, 2000) 8 2.3 Composition of used for canning vegetables (Joslyn and Timmons, 1967) 9 2.4 Raw waste loads and quality of wastewater from some pickling industries 10 2.5 Characteristics of the waste brine from four different kim chi factories located in Suwon city and Kyunggi province, Korea (Park and Choi, 1999) 10 2.6 Wastewater characteristics of from various fishery product and vegetable pickling industries 12 2.7 Characteristics of oil field brine (Dalmacija et al., 1996) 13 2.8 Characteristics of (Pirbazari, 1996) 13 2.9 Adverse effects of high salinity in activated sludge process 16 2.10 Adverse effects of high salinity in anaerobic treatment processes 18 2.11 Summary of adverse effects of high salinity in nutrient removal processes 19 2.12 Effects of using halophilic bateria for high salinity wastewater treatment 22 2.13 Basic composition of Candida utilis yeast biomass (Defrance, 1993) 24 2.14 A comparison between yeast and anaerobic treatment process (Defrance, 1993) 27 2.15 Operating conditions of YCS (Nishihara ESRC Ltd., 2001) 28 2.16 Quality of treated water and efficiency of the YCS for seafood processing wastewater treatment (Nishihara ESRC Ltd., 2001) 29 2.17 Summary of studies on yeast treatment of high salinity wastewater 30 2.18 Kinetic models for inhibition growth (Han and Levenspiel, 1988) 35 2.19 Comparison between biological performances of MBR process and conventional AS process 44

3.1 Composition of glucose-feed wastewater (Defrance, 1993) 50 3.2 Composition of protein-feed wastewater 50 3.3 Operating conditions for high salinity acclimation 52 3.4 Operating conditions for the respirometric experiments 53 3.5 Operating conditions for the pH effect experiments 54 3.6 Operating conditions of the experiments on SRT effect 55 3.7 Difference between the high COD loading and low COD loading 56 3.8 Experimental operating conditions of YMBR and BMBR systems 57 3.9 Composition of the low COD wastewater 59 3.10 Effects of different HRTs and SRTs on yeast and bacterial membrane reactors 60 3.11 Operating conditions for the sludge characterization study 60 3.12 Parameters and their analytical method 61

4.1 Performance of mixed yeast and bacterial batches adapted to glucose-feed wastewater with high salt 64 4.2 Performance of mixed yeast and bacterial sludges adapted to protein-feed wastewater with high salt contents (Initial COD cof 5,000 mg/L). 69 4.3 Biokinetic coefficients of the yeast and bacterial sludges at different salt contents for glucose and protein-feed wastewaters 72 4.4 Variation of parameters during various SRTs (Initial COD of 5000 mg/L) 81

ix 4.5 Operating parameters of the YMBR, BMBR, some yeast treatments, MBR processes treating different wastewaters and conventional AS system 85 4.6 Operating parameters and performance of YMBR and BMBR in high COD loading phase 91 4.7 Values of different parameters during YMBR and BMBR filtration cycle 92 4.8 Yeast and bacterial sludges characterization 94 4.9 Composition of mixed bacterial and mixed yeast sludge 98

x List of Abbreviations

AF Anaerobic Filter AS Activated Sludge BOD Biochemical Oxygen Demand BMBR Bacterial Membrane Bioreactor COD Chemical Oxygen Demand CST Capillary Suction Time DO Dissolved Oxygen DOSTE Department of Science, Technology and Environment ECP Extracellular Polymers EPS Extracellular Polymer Substances ESRC Environmental Sanitation Research Center F/M Food/ ratio HCMC Hochiminh City HRT Hydraulic Retention Time J Permeate flux MBR Membrane Bioreactor MF Microfiltration MLSS Mixed Liquor Suspended Solids MLVSS Mixed Liquor Volatile Suspended Solids N Nitrogen NH3-N Nitrogen NO2-N Nitrite Nitrogen NO3-N Nitrate Nitrogen OUR Oxygen Uptake Rate P Phosphorus SBR SCP Single-Cell-protein Production SRT Sludge Retention Time SS Suspended Solids SSL Spent Sulphite Liquor SVI Sludge Volume Index TDS Total Dissolve Solid TOC Total Organic Carbon TKN Total Kjedahl Nitrogen TS Total Solids TVS Total Volatile Solids U Substrate Utilization Rate UASB Upflow Anaerobic Sludge Blanket UNEP United Nations Environment Programme UF Ultrafiltration VFA Volatile Fatty Acid VLR Volumetric Loading Rate VOC Volatile Organic Carbon VSS Volatile Suspended Solids Y Yield coefficient YCS Yeast Cycle System YR Yeast Reactor YMBR Yeast Membrane Bioreactor 'P Transmembrane Pressure

xi Chapter 1

Introduction

1.1 Background

High salinity wastewater containing high inorganic salt content is mostly generated from industries such as seafood processing, vegetable canning, pickling and cheese processing. Among these, the seafood processing industry is an industrial sector that produces large volumes of saline wastewater with high organic and nutrient concentration. Therefore it causes heavy pollution to receiving . At present, the seafood processing industry plays an important role in South East Asia’s economy. Under stringent environmental regulations, this industry is now facing both high treatment costs and problems in the operation of conventional wastewater treatment plant. These operational problems are linked to high organic loading, high salt content and very large seasonal variation leading to change in waste characteristics.

1.1.1 Environmental Concerns

In seafood processing, the main environmental concern is the use of large amounts of for processing, including washing raw material and products, for cleaning of machines, containers or flushing the working floor, for de-icing, thawing and salt soaking. In general, 90-95% of water consumed is converted into highly polluting wastewater. Frozen seafood processing consumes particularly large volumes of water, ranging from 70 to 120 m3/ton of product, the equivalent of 32-60 m3/ton of raw fish (DOSTE-HCMC and CEFINEA, 1998). The wastewater generated by fish processing factories has high loads of organic and nutrients. This waste is commonly discharged directly into coastal areas. Another + - 2- important aspect of this industrial waste is its high salinity (Na , Cl , SO4 ), caused both by the raw materials and used in various processes. Here, using pre-filtered seawater for processing leads to high salinity in the wastewater, which reduces the biodegradation rate in effluent treatment units (Mendez et al., 1992).

Because factories process a broad range of products with large seasonal variation, pollution characteristics vary significantly both from plant to plant, and even within the same plant. In Ho Chi Minh City (HCMC), the seafood processing sector is one of the major industrial contributors to the heavy pollution to receiving waters. The average BOD5 generally ranges from 1,200 to 1,800 mg/L (COD of 1,600 - 2,300 mg/L) (DOSTE, 1994). In addition, the wastewater contains high levels of suspended solids (150-200 mg/L), and is rich in nutrients with total nitrogen ranging from 70 to 110 mg/L. The pollutant loads from the seafood processing industry and other industries in the Saigon-Dong Nai river catchment area is shown in Table 1.1. These data indicate that seafood processing is a sector that causes considerable pollution to the environment in this part of Viet Nam.

1 Table 1.1 Comparison of pollutant loads from seafood processing and other industries in the Saigon-Dong Nai river catchment area (DOSTE-HCMC and CEFINEA, 1998)

Flow rate Industry m3/day Pollution load, kg/day SS BOD5 TKN Seafood processing 18,900 4,200 28,400 1,700 Pulp and paper 49,200 54,900 104,800 340 Cassava 47,100 30,600 590,000 NA Textiles & dyeing 32,500 5,600 17,300 NA Beverages 15,600 4,400 19,000 630 Latex processing 11,600 2,500 86,600 2,800 Meat processing and slaughterhouse 6,400 4,000 13,300 1,020 Sugar (sugar cane) 5,520 6,900 32,000 72 Vegetable canning 3,700 520 2,700 70 SS – suspended solids NA – None available

1.1.2 Effects of High Salinity on Biological Treatment Processes

Past studies on saline wastewater treatment reveal that salinity decreases BOD5 removal efficiencies, increases effluent due to sludge settling in the secondary sedimentation unit, solid losses, and changes in the mixed liquor floc protozoan population in an activated sludge system (Dalmacija et al., 1996; Woolard and Irvine, 1995; Kargi and Dincer, 1998). Kargi and Uygur (1996) reported many adverse effects of salt on aerobic attach growth such as trickling filter and rotating biological contactors. The efficiency of COD removal decreased significantly with increases in salt contents over 20g/L.

The anaerobic digester were much more sensitive to chlorides than activated sludge processes (Burnett, 1974). Biogas production and COD removal of anaerobic treatment processes such as anaerobic filter, UASB and batch reactor were inhibited significantly at salt content above 30g NaCl/L (Baere et al., 1984; Feijoo et al., 1995). In addition, high salt content also depressed the treatment ability of nitrifying and denitrifying bacteria, even though pre-acclimation had been done (Dahl et al., 1997; Panswad and Anan, 1999).

The adverse effects of high salinity on conventional biological processes can be attributed to high osmotic stress or inhibition of the reaction pathways in the organic degradation process. In addition, high salt content induces cell lysis, which increases effluent solids. The population of protozoa for proper flocculation is also significantly reduced at high salt contents. Here, although salt acclimation can be expected from conventional processes, the extent of adaptation is limited, and thus conventional processes can not be used to treat wastewaters containing more than 3% salt (Woolard and Irvine, 1995).

Currently many saline wastewater treatment are able to overcome the technical problems associated with high salinity by diluting the saline waste stream with fresh water. Nevertheless, this practice is unsustainable, due to continuous pressure on the industries to reduce fresh water consumption.

2 1.1.3 Salt-Tolerant or Halophilic Microorganisms

In order to improve organic and nitrogen removal efficiency, application of salt-tolerant microorganisms in biological treatment of saline wastewater has been investigated experimentally by several researchers (Nishihara ESRC Ltd., 2001; Woolard and Irvine, 1995; Hinteregger and Streichsbier, 1997; Park and Choi, 1999; Kargi and Dincer, 2000). Salt tolerant microorganisms are those which can tolerate high salt content during their growth. This utilization of halophilic microorganisms (e.g. Halobacter halobium) along with activated sludge culture resulted in better treatment performances at salt contents above 2% (Kargi and Dincer, 2000). Woolard and Irvine (1995) studied the treatment of hypersaline wastewater by a moderate halophilic bacterial mixture isolated from soil of a saltern and fed in sequencing batch reactor. They found that over 99% phenol removal was possible from 15% saline wastewater.

In investigating the application of yeast in the food processing wastewater treatment, researchers have investigated this potential in the treatment and reuse of wastes containing solids and high concentrations of salt, fat and antibiotics. Park and Choi (1999) studied the possibility of culturing an osmotolerant yeast, Pichia guilliermondii A9, using waste brine from Kim Chi factory. The growth of Pichia guilliermondii A9 in waste brine was not inhibited by NaCl concentrations of up to 60 g/L. In the Yeast Cycle System, wild yeasts were utilized for treatment of wastewater, and the recovered excess sludge could be reused (Nishihara ESRC Ltd., 2001). This yeast system is used as a pre-treatment to reduce the organic pollutants, followed by a conventional activated sludge process. Primary treatment plays an important role in removal of high organic and nutrient loadings. Moreover, excess yeast sludge that had high protein, vitamins, lipid content could be used as animal feedstuff, mushroom growing or as fertilizer.

1.1.4 Membrane Bioreactor Process

Application of the membrane bioreactor (MBR) concept in high salinity wastewater treatment offers the possibility of overcoming low biodegradation rate and poor sludge settling in the secondary sedimentation tank. MBR process can be operated at high MLSS and thus organic removal can be improved. This results in sludge wastage and plant size reduction (Visvanathan et al., 2000). Moreover, the selection of microorganisms present in the membrane bioreactor is no more dependent on their ability to form biological flocs and settling characteristics.

However, the membrane fouling problems lead to rapid flux reduction in MBR. This secondary effect results in increase in energy consumption, and more frequent chemical cleaning is required. These major problems hinder the widespread application of MBR in effluent treatment processes. The membrane fouling might be the result of (a) the biofilm growth or attachment of bacterial flocs on the upper surface of the membrane, and (b) the deposition of macromolecules at the pore entrances or within the internal pore structure of the membrane. The macromolecules can be protein from wastewater, extracellular polymers (ECP) or long chain organic by-products generated during the biodegradation process.

3 1.2 Objectives of the Study

The overall objectives of this research were: (1) To evaluate variation of biokinetic coefficients of salt-tolerant yeast mixture and bacterial mixture with high salt contents. (2) To find out suitable operating parameters for membrane bioreactor systems using salt- tolerant yeast and bacterial mixture to treat saline seafood processing wastewater. (3) To investigate membrane fouling of both microorganisms in terms of sludge characteristics and ECP production.

1.3 Scope of the Study

To accomplish the above objectives, four studies were carried out:

(1) Biokinetic study. Biokinetic coefficients of mixed yeast and mixed bacterial treatment at high salt contents were evaluated using respirometric experiments. The three salt contents examined were 20, 32 and 45 g/L NaCl. Two feed wastewaters were used, namely glucose-feed wastewater and fish-protein-feed wastewater. In the fish-protein- feed wastewater, commercial tuna fish protein extract was mixed to obtain wastewater composition similar to tuna fish processing wastewater. Whereas, glucose-feed wastewater was composed of glucose as carbon source and inorganic ammonia as the nitrogen source. (2) Parametric study (optimization of operating conditions). The fish-protein wastewater with salt content of 32 g/L was used in this study. a. Optimum pH for mixed yeast and bacterial treatment at a salt content of 32 g/L was evaluated in terms of oxygen uptake rate (OUR) by respirometric experiments. Based on theresponse of maximum OUR at different pH values, the optimum pH range was determined. b. The variation of COD, DO and nitrogen (organic nitrogen, ammonia, nitrite and nitrate) with aeration time in acclimatized mixed yeast and bacterial cultures was monitored. Based on these COD, nitrogen profile data, suitable hydraulic retention time (HRT), organic removal rate and nutrient uptake or nitrogen removal were evaluated. c. Five sludge retention times (5, 7, 10, 20 and 45 days) were investigated for mixed yeast treatment. Every the sludge retention time (SRT) experiment was conducted in two-liter batch reactors with fill-and-draw operation. Based on the COD and nitrogen removal, optimum SRT was suggested. (3) Biomembrane study. This study consisted of two phases: a. High COD loading: Two parallel experimental set-ups were carried out, namely (1) Yeast pretreatment followed by Bacterial Membrane Bioreactor (BMBR), and (2) the Yeast Membrane Bioreactor (YMBR). Fish-protein-extract wastewater with feed COD of 5,000 mg/L and salt of 32 g/L was used. The experimental investigations were carried out by step-wise increase in volumetric loading at SRT of 50 d. b. Low COD loading: The fish-protein wastewater with 1,000 mg/L COD and 32 g/L NaCl was used in this phase. Two experimental set-ups were conducted (1) YMBR and (2) BMBR. Treatment performance of both reactors was investigated at different HRTs and SRTs (5 and 10 days).

4 The process efficiency was evaluated in terms of organic removal and membrane filtration flux for various volumetric loading rates vis-à-vis HRTs. (4) Sludge characterization study: Variation of sludge characteristics with different salt contents (0.5, 15, 32 and 45 g/L) was investigated by using two-liter batch reactors with the fill-and-draw operation. Sludge properties were evaluated in terms of extracellular polymer content, CST, SVI, viscosity and nutrient contents.

5 Chapter 2 2 Literature Review

2.1 Introduction

High salinity wastewaters are usually generated from industries such as seafood processing, vegetable canning, pickling, tanning and chemical manufacturing. Seafood processing factories located in arid zones use treated seawater or reused or recycled water in processing steps such as defrosting, butchering and washing raw materials. Thus, the effluent from these industries contains high salinity, which is approximately the same as that of seawater. In addition, the adoption of waste minimization techniques within these industries has led to reductions in waste volume, while waste concentration has been increased (Woolard and Irvine, 1995). These wastewaters are often difficult to treat with conventional treatment processes such as activated sludge, trickling filter and anaerobic processes. High salinity can cause osmotic stress or inhibit the reaction pathways in the organic degradation process. This results in a significant decrease in biological treatment efficiency or biodegradation kinetics. In addition, high salt content induces cell lysis which causes increased effluent solids. The populations of protozoa and filamentous organisms required for proper flocculation are also significantly reduced at elevated salt content (Burnett, 1974). Therefore, conventional treatment process can hardly meet the effluent standards for high salinity wastewater.

2.1.1 The Seafood Processing Industry

The seafood processing sector contributes serious organic pollution loads and high salinity to receiving waters. This feature leads to difficulty in biological treatment processes (Mendez et al., 1992). The fish processing industry with cooking and brine filling operations normally produces high strength organic matter, high level of oil and grease, and high salt content. Typical water consumption ranges from 18–74 m3/ton of fish processed (Battistoni and Fava, 1994).

Process for canning shrimp is shown in Fig 2.1. In this process, receiving, peeling and washing discharge large quantities of wastewater containing 90% of total COD. High salinity wastewater is generated from precooking or brine treatment. In the precooking operation, shrimp is boiled in brine solution for 3-5 minutes, or it is steamed. These operations curl the meat, extract moisture and develop the pink or red color of the finished product. The salt content of precooking wastewater is in range of 2 - 3% (UNEP, 1999).

Sardine and herring are classified as small, oily fish. These fishes contain a considerable amount of oil or fat located between the skin and the flesh. The hot brine separates the oil from the sardines. The oil rising to the surface of the brine forms a thick oil film on the top which is then skimmed off. After cooking, the cans are taken out the brine and the remaining brine in the cans is drained off. After cooling in a drying chamber, the cans are sealed, washed down and packed.

The sardine and herring processing industry regenerates two kinds of wastewater: (1) sardine and herring brine and (2) wastewater from cleaning and rinsing operations. The flow rate of wastewater from these processes is as large as 6 times that of sardine or herring brine. However, herring brine is a very concentrated wastewater consisting of a mixture of acetic

6 acid, sugar, fish protein and fish oil, as well as a number of spices and salt. The characteristics of the herring brine is shown in Table 2.1.

water water, debris RECIEVING

water ice, water THAWING debris, shells

water, salt water, debris COOKING shells

water water, salt SHELLING meat particles

water water WASHING

water water INSPECTION

water, salt SALTING water, salt

water, salt water, salt CANNING

steam hot water RETORTING

COOLING

to wastewater treatment plant Product

Figure 2.1 Flow diagram of steamed canned shrimp processing

Table 2.1 Characteristics of herring brine waste (Balslev-Olesen et al., 1990)

Parameter Units Average value COD mg/L 90,000 BOD5 mg/L 78,000 Oil/fat mg/L 4,000 Ntotal mg/L 3,000 Ptotal mg/L 1,000 SS mg/L 10,000 VSS mg/L 7,000 Chloride g/L 65 TDS g/L 110 pH 3.8

The canning process for mollusks such as mussel, oysters, clams or scallops also generates large quantities of wastewater with salt content above 2%. The mollusks are shelled and washed with 3 to 6% salt solution. Then they are drained and steamed or cooked for 10 to 15 minutes at 100qC. After inspection and grading, the cooked mollusks are packed in cans

7 with 1 to 2% brine. Mendez et al. (1992) reported that wastewater from processing mollusks contained very high organic, nitrogen and salt content (18.5 g COD/L, 4.0 g N/L and above 2% salt).

The typical processing of dried salted fish is schematized in Figure 2.2. Slime, blood and other contaminating substances of raw fish are washed off using a 3% solution of clean salt in water. This reduces bacterial loads on the fish during subsequent salting. Large fish like mackerel are split open at the ventral side from the head down. All visceral matter and blood are removed. The fish is then cut into large pieces (1.5-2 cm thick, 10 cm wide and 20 cm long). Fishes have an odor of ammonia, the dressed fish, or fish fillets, are soaked in mild brine (10%) and crushed iced for six to 10 hours. This may be followed by salting. After washing in clean brine solution, the eviscerated fish is salted in 21% brine for about 15 hours. Salted fish is placed on bamboo trays and sun dried for two to three days in full sunshine, depending on the size of the fish. Salted fish can also be dried in ovens. Fishes are then packed and stored.

The characteristics of wastewater from the dried salted fish plant are shown in Table 2.2. This wastewater contains very high salt contents, ranging from 17g to 46g NaCl/L. A large volume of wastewater is produced from soaking and washing operations. The volume ranges from 10 to 12 m3/ton of preprocessed fish, and 20-30 m3/ton of iced or fresh fish. The preprocessed fish, namely, is eviscerated or beheaded and cleaning at fishing boats or villages in coastal zone before it is transported to -food processing plant.

Table 2.2 Characteristics of wastewater from the dried salted fish plant (Dan, 2000)

Parameter Unit Concentration Washing + soaking tank Combine Wastewater(*) COD mg/L 5,250 873 SS mg/L 371 119 TDS g NaCl/L 46 17 Cl- g/L 27 10 2- SO4 mg/L 1,240 164 TKN mg N/L 747 128 Total P mg P/L 5 5 (*)Except brine waste from soaking tank

8 wastewater water RECEIVING THAWING ice, debris energy water, ice wastewater, blood EVISCERATION eviscera eviscera, skin water, ice wastewater Energy FILLETING meat particles meat particles

wastewater, salt water, ice WASHING meat particles Offal recovery (animal feed) water wastewater, salt SOAKING 10% salt organics

water wastewater, salt SALTING 21% salt organics

Energy DRYING WWTP for high salinity wastewater

GRADING

Energy PACKING

WWTP(*) Energy COOLING for combined wastewater

Product (*)WWTP – Waste water treatment plant Figure 2.2 Flow diagram of Dried and Salted fish processing

2.1.2 Pickled Vegetable Processing

Salt is used widely in vegetable canning processing to enhance flavor, to preserve, or for conditioning. Therefore this industry in general produce wastewater containing high salt content. The composition of the brines commonly used in canning vegetables is presented in Table 2.3. Table 2.3 Composition of brines used for canning vegetables (Joslyn and Timmons, 1967) Product Brine, g/L Asparagus 21.5 – 24.0 salt Green bean 19.2 – 27.5 salt Cabbage 15.6 – 25.2 salt Beets 24.0 salt, 18 – 24 sugar may be added Peas 21.5 salt and 36 – 48 sugar

Brine waste from fermenting pickles contains high salt content (3 to 20%) and extremely high organic concentrations. This is due to extraction from pickled vegetable tissue during fermentation process. In addition, large amounts of saline wastewater are also

9 generated from washing or rinsing pickles after fermentation and rinsing equipment, soaking or fermenting tanks. Table 2.4 presents waste loads in some pickling industries. EPA (1975) reported that the BOD:N:P ratio of pickles and sauerkraut wastewaters were 100:1:0.2 and 100:4:0.5, respectively. These ratio show that nutrient concentrations in the pickling wastewater are low for microorganism growth (BOD:N:P for bacteria growth is 100:5:1).

Table 2.4 Raw waste loads and quality of wastewater from some pickling industries

Raw waste loads Concentration Category Flow BOD SS BOD SS 5 5 pH m3/ton(*) kg/ton kg/ton mg/L mg/L Pickles: (EPA, 1975) - Fresh packed 7.76 8.61 1.72 - Process packed 8.70 16.7 2.97 - Salting stations 0.96 7.21 0.38 Total 17.4 32.5 5.07 1,500- 135-825 4.3 – 6.3 5,800 (400) (5.3) (3,280) Sauerkraut: (EPA, 1975) - Canning 3.19 3.18 0.55 - Trimming 0.39 1.13 0.17 Total 3.58 4.31 0.72 1,400 – 60 - 630 4.3 – 6.3 6,300 Sauerkraut: - (Woodroof, 1975) 19 6.8 1.36 - (NCA, 1971) 15 6.4 0.45 (*) Ton of raw material

Kim chi pickle which is pickled celery cabbage is a well-known food in Korea, Japan and Vietnam. Figure 2.3 illustrates kim chi processing. Park and Choi (1999) reported that the volume of waste brine produced from a kim chi factory is approximately 0.53 - 0.67 m3/ton of product. Typically, the composition of waste brine contains sugars and other nutrients extracted from the vegetables during fermentation, as well as a high salt content (approximately 10%). Table 2.5 describes characteristics of the waste brine from four different Kim chi factories located in Suwon city and Kyunggi province in Korea.

Table 2.5 Characteristics of the waste brine from four different kim chi factories located in Suwon city and Kyunggi province, Korea (Park and Choi, 1999)

Parameter Factory A Factory B Factory C Factory D pH 5.36 4.91 5.48 5.80 NaCl, g/L 116 95 84 70 BOD5 , mg/L 1,100 1,200 1,060 1,040 COD, mg/L 1,300 1,790 1,550 1,250 TKN, mg/L 25 28 20 25

10 Fresh celery Cabbage

water water, debris RINSING

spoiled leaves/bases CUTTING

Outer leaves Squares and bases

brine brine water, salt FERMENTATION FERMENTATION debris discarded leaves Liquid and bases water water, salt BOILING RINSING

onion, chili water, spices water water, acid CANNING CAN FILLING ginger citric acid

Animal feeds water water, salt water water, salt recovery WASHING WASHING

SEALING SEALING

Kim Chee Juice to WWTP Kim Chee Nappa to WWTP

Figure 2.3 Flow diagram of kim chi pickles processing

The characteristics of high salinity wastewater generated from seafood processing and vegetable pickling industries is shown in Table 2.6. In seafood processing, the main environmental issue concerns the use of large amounts of fresh water for processing, and its emission as wastewater. The volume of wastewater discharged depending on the type of products or raw materials range from 10 to 120 m3/ton of product. In comparison with the vegetable pickling process, a ten-fold increase in organic loading (COD or BOD5) is discharged by the seafood processing industry. However, the salt content of wastewater from vegetable pickling is normally very high, possibly as much as 200 g/L.

11 Table 2.6 Wastewater characteristics of from various fishery product and vegetable pickling industries

Type of product Unit Canned Canned Canned Tuna Fish meal Kim chi Cucumber Sauerkraut(1) sardine shrimp(2) mussel/oyster pickles (3) pickles (1) Water & wastewater m3/ton 9 60 20-120 22 97 0.6 17 4 volume

BOD5 load kg/ton 9 120 60 15 194 0.7 33 4 SS load kg/ton 5-6 54 - 11 - - 5 0.7 Oil & Grease kg/ton 27 42 - 6 - - - - Operation units Off-load, Brine filling, cooking, cooking, Off-load, Fermenting Fermenting, Fermenting, generating saline sauce cooking, washing sauce centrifuging, pickle washing pickle washing wastewater filling/can sealing, can filling/sealin storage washing washing g/ can washing Saline wastewater % of total 39 2 – 2.5 (2) 3 (3) 3 (3) 95 100 56 89 volume volume (seawater) (seawater) TDS of saline g NaCl/L 30-35 20 – 30 (2) 21(3) 23 (3) 100 30 – 200 30 –200 wastewater Sources UNEP, 1999 UNEP, 1999 UNEP, 1999 UNEP, 1999 UNEP, 1999 Choi & Middlebrook Middlebrook Park,1999 1979 1979 (1) ton of raw material (2) Soderquist, 1971 (3) Estimated for waste brine only

12 2.1.3 Other Saline Wastewaters

Wastewater generated from oil field exploitation contains high salt content and is refered to as oil-field brine. Its characteristics are presented in Table 2.7.

Table 2.7 Characteristics of oil field brine (Dalmacija et al., 1996)

Value Parameter Max Min Average COD, mg/L 1,200 200 400 pH 7.6 7.3 7.5 TS, g/L 34.6 29.3 32.3 Phenol, mg/L 0.14 0.01 0.05 Oil, mg/L 315 139 237 Cl-, g/L 17.9 17.4 17.6 - SO4 , mg/L 17.7 9.2 11.9

In coastal areas, when subsurface water rises, infiltration of into sewers can result in high concentrations of chloride and sulfate in wastewater. Therefore, large variation of salinity in domestic wastewater occurs normally in this areas. This can cause salt shocks or adverse effects on conventional biological treatment methods.

Hypersaline wastes are produced in significant quantities in chemical industries such as oil and gas production. These wastes contain organic compounds and high concentrations of salt (>3.5%). High salinity is also found in landfill leachates. Pirbazari (1996) reported that the leachates from domestic waste landfill (Los Angeles) and hazardous waste landfill for chemical and petroleum waste (Niagara) had high strength organic matters and high (TDS). The characteristics of two leachates are described in Table 2.8.

Table 2.8 Characteristics of leachates (Pirbazari, 1996)

Leachate Parameters Unit Domestic waste landfill Hazardous waste landfill COD mg/L 3,050 – 3,450 9,000 – 10,500 BOD5 mg/L 1,505 – 1,710 6,950 – 7,500 TOC mg/L 905 – 965 3,040 – 3,500 SS mg/L 460 – 565 862 – 946 TDS mg/L 5,800 – 6,250 22,600 – 25,900 TKN mg/L 75 - 84 160 – 180 Oil and grease mg/L 60 – 80 pH - 4.3 – 6.0

2.2 Effects of High Salinity on Biological Waste Treatment Process

In wastewater treatment, there are conflicting reports on the influence of salt on the biological processes. Some reports have indicated adverse effects of high salinity, or shocks of NaCl on organic removal efficiency and sludge settleability (Burnett, 1974). Others have reported that constant application of NaCl to biological treatment systems does not upset the organic removal efficiency, and results in good flocculation of the biomass. This shows that acclimation of the biomass and level of salt are important factors that may explain these different observations (Hamoda and Al-Attar, 1995).

13 2.2.1 Aerobic Treatment

Previous studies reported that operation of activated sludge process at salt contents higher than 20 g/L is characterized by poor flocculation, high effluent solids, and a severe decrease in substrate utilization rate (Burnett, 1974).

Microscopic observations of the mixed liquor flocs (Burnett, 1974) showed that alterations in saline wastewater caused alterations in the mixed liquor floc ecology. There was a rapid die-off for rotifers, stalked protozoa and motile ciliate protozoa coincided with a decrease in BOD removal and disruption in performance. After a few days, motile ciliated protozoa were again observed, but rotifers and stalked ciliata were absent.

Tokuz and Eckenfelder (1979) estimated the effects of inorganic (NaCl and Na2SO4) on continuous flow activated sludge with low F/M. The results indicated that the relative high concentration of NaCl (up to 35 g/L) had only a slight effect on the performance of the activated sludge process and the effluent SS did not increase. This was probably due to a decrease in the F/M ratio. A further increase of NaCl over 35 g/L caused sudden increases in effluent SS. The effect of sodium sulfate on the system was not significant. They also observed that the protozoa population decreased gradually and disappeared at salt contents above 35 g/L. The disappearance of the protozoa coincided with the sudden increase in effluent turbidity or SS. Likewise the effects of high concentrations in an activated sludge process was studied by Hamoda and Al-Attar (1995). The results showed that the organic removal efficiency, and the treated effluent quality of activated sludge process did not deteriorate as constant application of NaCl up to 30 g/L. COD removal efficiency ranged from 93 to 99%. However, in order to obtain equivalent substrate removal, three-fold-lower F/M ratios were applied in conventional AS at salt content of 30 g/L compared to those applied in AS at salt-free wastewater (at the same SRT). Thus the substrate removal rate decreases at high salinity. The MLVSS in the activated sludge reactor increased at salt content up to 30 g/L. This result differs from the research conducted by Burnett (1974). An explanation for this may be the long acclimation of microorganisms to the saline wastewater might result in the growth of halophilic microorganisms in the system. Burnett (1974) also noted that, although the substrate utilization rate decreased, the biomass yield obtained was increased at higher NaCl concentrations. This may be due to a change in the efficiency of microbial metabolism and the selection of salt tolerant species in the system. The salt tolerant species may be halophilic micro-organisms such as Zooglea ramugera or Halobacteriaceae which are aerobic heterotrophs.

Dalmacija et al. (1996) reported that the of pollutants and the high salinity (about 29 g/L) of oil-field brine has an unfavorable effect on the activated sludge process. High hydraulic loadings (above 2.5 m3/m3.day) increased the wash-out of the activated sludge from the reactor. The addition of PAC improved the sludge volume index and increased the rate of biodegradation. This is due to the ability of biofilm formation on the activated carbon surface.

Kargi and Uygur (1996) investigated the effects of high salinity on the Rotating Biodisc Contactor (RBC). The results indicated that the rate and efficiency of COD removal decreased significantly with increases in salt content above 10g/L. COD removal efficiency with salt free wastewater was 95%. Due to the adverse effect of salt on microorganisms, the COD removal was down to 60% at 5% salt content. The increase in salt content causes a linear reduction in COD removal rate as shown in Fig. 2.4.

14 5000 h

2. 4500

4000

3500

3000 COD removalrate, mg/m

2500 0.01.02.03.04.05.0

Salt concentration, %

Figure 2.4 Variation of COD removal rate with salt contents (Kargi and Uygur, 1996)

A review of the literature (Table 2.9) confirms the presence of adverse effects of the high salinity on the conventional activated sludge systems. Major problems encountered in the biological treatment of saline wastewater were summarized by Kargi and Dincer (2000). They are: x Limited extent of adaptation: Conventional cultures cannot be effectively used to treat saline wastewaters with salt contents above 3%. x Sensitivity to changes in ionic strength: Shifts in salt content from 0.5 to 2% usually cause disruptions in system performance. Rapid change in salt contents causes more adverse effects than gradual change. Equalization to constant salt content is necessary before biological treatment. x Reduced degradation kinetics: Biological degradation rates decrease with increasing salt content. Therefore, saline wastewaters should be treated at lower F/M ratios. x High effluent SS: Salt content in wastewater reduces the population of protozoa, resulting in low settlability. Salt content in wastewater increases the forces, causing low sedimentation efficiencies.

15 Table 2.9 Adverse effects of high salinity in activated sludge process

Authors Experiment Results Ludzack and Noran (1965) Increasing influent from 100 mg Cl/L Æ 20,000 mg Cl/L (| - Solid losses Æ disrupting clarifier 33 g NaCL/L) over 2 to 3 weeks - 10% loss in BOD5 removal - Inhibiting nitrification

Burnett (1974) Changing TDS up to 35,5 g NaCL/L - Decreasing BOD5 removal from 97% to 25% for 6 days after - Rapid die-off of rotifers & stalked/mobile ciliata protozoa - Turbid in effluent Tokuz & Eckenfelder Operating continuous flow activated sludge with low F/M - Slight effect on BOD removal (1979) ratio at d 35 g NaCl/L - Effluent SS did not increased due to low F/M If salt content > 35g/L - Decreasing the population of protozoa and then disappeared - Increasing effluent SS Hamoda & Al-Attar (1995) Increasing salt content to 10 g/L and 30 g NaCL/L - Decrease in substrate utilization rate - But increasing biomass yield due to selecting salt tolerant species (halophilic bacteria such as Zooglea ramugera, Halobacteriaceace etc.) Dalmacija et al. (1996) Oil-field brine with salt content of 29 g/L - Increasing wash-out of activated sludge as hydraulic loadings > 2.5m3/m3.day Kargi & Uygur (1996) Increasing in influent salt contents over 1% for RBC - Decreasing COD removal rate & efficiency. - COD removal was down to 60% at 5% salt content. - Increasing salt content caused linear reduction in COD removal efficiency.

16 2.2.2 Anaerobic Treatment

Anaerobic process has become one of the most interesting treatment for highly organic polluted wastewaters. However, the presence of salts may cause inhibition and toxicity problems in the methanogenic activity. High salt levels can dehydrate anaerobic bacterial cells because of osmotic pressure.

Anaerobic digester was much more sensitive to chlorides than activated sludge (Burnett, 1974). Baere et al. (1984) examined influence of high NaCl on methanogenic activity on anaerobic filter (AF) process. The AF reactor was filled with ether-based polyurethane foam with a specific surface area of 600 m2/m3. The results showed that initial inhibition occurred at 30g NaCL/L. A shock treatment with 35g/L had a sharp decrease in gas production, which dropped by 65%, and TOC removal efficiency, which decreased from 98% to 70%. The pH dropped significantly after each shock treatment, from about 6.8 in the influent to a pH of 5.4. When Methanosarcina, a halophilic anaerobic bacteria strain, was predominant (>99% of the methanogenic biomass) in the reactor, TOC removal was improved. The methanogenic activity of these bacteria was inhibited at 60 g NaCl/L. TOC removal was less than 20% and the gas production dropped below 15% at 50 g/L.

An anaerobic and aerobic system consisting of an aerobic contactor followed by activated sludge was tested for the biological treatment of high salinity wastewater (Belkin et al. 1993). This wastewater generated from several chemical factories. The mean salt and COD concentration were 32 g/L and 4900 mg COD/L respectively. Low COD removal efficiency (55%) of the whole system was obtained at F/M ratio of 0.56 g COD/gMLSS.d for anaerobic process and 0.28 for aerobic process. The COD efficiency increased to 74% at very low F/M ratios (0.02 and 0.04 for the anaerobic and aerobic process, respectively).

Feijoo et al. (1995) examined the continuous exposure of high salinity in pilot scale UASB and AF reactors. The results shows that the methanogenic activity of both anaerobic processes was reduced by 50% at sodium concentrations above 20g/L. For unadapted sludge, the anaerobic reactors could be shocked in the concentrations ranging from 6 to 13 g/L. In addition, sodium inhibition in process was conducted with batch assays (Feijoo et al., 1995). At low concentrations, sodium is essential for methanogens. The optimum concentrations were reported to be about 0.23 – 0.35 g Na/L. The effect of NaCl on the methanogenic activity depends on the type of sludge. When the sodium concentration was increased by 4 to 10g/L (10 to 25 g NaCl/L), methanogenic activity reduced to 50%. After 40 days of digestion, the relative methanogenic activity of the sludge increased from 0% to 45%. The sludge pregrown in the presence of high salt content showed a higher tolerance to sodium, probably due to the adaptation of methanogenic bacteria to sodium. However, the treatment efficiency after recovery was still low (45%).

17 Table 2.10 Adverse effects of high salinity in anaerobic treatment processes

Authors Experiment Results Baere et al. (1984) AF with surface area of 600m2/m3 - Decrease in gas production (dropped 65%) at 30 g NaCl/L - TOC removal was decreased from 98% to 70% - Decrease in pH from 6.8 to 5.4 at salt content of 60 g/L - Gas production dropped below 15% - TOC removal < 20% Feijoo et al. (1995) UASB and AF - Reducing 50% methanogenic activity at salt content > 33 g NaCl /L - Shocked at concentrations ranging from 10-21 g NaCl/L for unadapted sludge Belkin et al. (1993) Anaerobic and aerobic system at - Low COD removal for whole system (50%) 32 g salt/L - COD removal (70%) could be improved at very low F/M ratio (0.02 for anaerobic and 0.04 for aerobic process Feijoo et al. (1995) Anaerobic batch digestion - Decreasing 50% of methane activity as increasing TDS by 10-25 g NaCl/L.

2.2.3 Nutrient Removal

In the nitrogen removal processes (Fig. 2.5), the oxidation of ammonia to nitrite and then nitrite to nitrate (nitrification process) takes place under aerobic conditions (autotrophic bacteria) and reduction of nitrate to nitrogen gas (denitrification process) occurs under anoxic condition (hetetrophic bacteria). Dahl et al. (1997) found inhibition of the nitrifiers in the case of a rapid increase of chloride concentration. The decrease in nitrification activity resulting from increasing salt content from 16 g NaCl/L to 32 g/L, was approximately 30%.

Figure 2.5 Diagram of a nitrogen treatment system

Panswad and Anan (1999) investigated the effects of various salinity levels on ammonia and nitrate uptake rates of the biological nutrient removal systems (Anaerobic/anoxic/aerobic). In the steady state, the specific ammonia and nitrate uptake rates decreased with increase in chloride concentrations. The total nitrogen removal dropped from 85% to 70% at high salt contents (20 and 30 g NaCl/L). Concurrently, COD removal of the system also was dropped from 90% at 5 g NaCl/L to 71% at 30 g/L. This indicated that the nitrifying and denitrifying bacteria are very sensitive to sudden high salt content even with a high degree of pre-acclimation. Similarly in conventional activated sludge process, acclimation was clearly proven to be an important factor in improving the nitrification and denitrification performance of the system. The phosphorous removal of this system decreased from 38 to 10% with gradually increase in salt content from 0 to 30 g NaCl/L. This indicates that poly-P bacteria have intense sensitivity to high salinity condition.

18 Dincer and Kargi (1999) reported that the salt content reduced the rate and the efficiency of nitrification and denitrification at salt contents above 2% and 1% respectively. Nitrobacter was more adversely affected by high salinity than Nitrosomonas resulting in accumulation of nitrite in the effluent at salt contents above 2%. The denitrification rate seemed to be more sensitive to salt inhibition than nitrification is. A summary of adverse effects of high salinity in nutrient removal is shown in Table 2.11.

Table 2.11 Summary of adverse effects of high salinity in nutrient removal processes

Authors Experiment Results Dahl et al. (1997) Synthetic wastewater, combined - Nitrification and denitrification rates were biological nitrification and reduced with increase in salt content (32 g/L) denitrification lab-scale experiment Dincer and Kargi Activated sludge for nitrification and - Salt concencentrations > 3% resulted in (1999) downflow packed column for significant reductions in performance of both denitrification nitrification & denitrification. Panswad and Lab-scale anaerobic/anoxic/aerobic - Shocked at 70 g NaCl/L. Anan (1999) with synthetic wastewater - Specific nitrate and ammonia decreased as increasing Chloride concentration - Nitrifying and denitrifying bacteria were very sensitive to sudden salt content. - More intense sensitivity of P-bacteria to high salinity

2.3 Application of Halophilic Bacteria for Saline Wastewater Treatment

Removal of salt content from wastewaters by , electrodialysis before biological treatment, is normally expensive. However, because of salt inhibition of bacteria growth, application of conventional treatment processes does not obtain acceptable efficiency. In recent years, several studies have shown that, in utilization of salt-tolerant microorganisms in biological treatment such as halophilic bacteria, yeasts could be a reasonable approach for treatment of high salinity wastewater.

Non-halophilic bacteria grow well in media which contain less than 1% salt content. True halophilic bacteria require salt for survival. These bacteria can be divided into two groups, namely moderate and extreme . The moderate halophiles are microorganisms which grow best in medium containing 3-15% NaCl (0.5-2.5M). While extreme halophiles exhibit optimum growth in media containing 15 – 30% NaCl (Woolard and Irvine, 1995). To tolerate the osmotic forces present in saline environments, halophilic microorganisms accumulate compatible solutes to equalize the ionic strength of the cytoplasm with external environment. Moderate halophiles accumulate a mixture of inorganic cations (K+, Na+) and organic compounds (amino acids, glycerol) for osmosis regulation.

Kargi and Dincer (1996) examined the treatment ability of Zooglea ramigera, a moderate halophilic bacteria strain, at different salt contents using a fed-batch reactor. This is different from sequencing batch reactor (SBR). The fed batch operation involves slow addition of highly concentrated or wastewater into an aeration reactor until the tank is full. With slow feeding, concentrated/toxic wastewater gets diluted inside the reactor, resulting in less inhibition and higher BOD removal rates. COD removal efficiency for salt-free wastewater was about 85%. This was not effected at salt content of 0.5%. However, the efficiency dropped quite significantly with increasing salt contents above 1%, and attained nearly 60% at 5% salt content.

19 In order to estimate the removal efficiency of salt tolerant microorganisms, Kargi and Uygur (1996) used different types of microbial flora, namely Zooglea ramigera and Halobacter halobium. The experiments were conducted using an aerated percolation reactor with 1% salt content (Fig. 2.6). The percolator column was filled with crushed ceramic particles (I = 4 mm) on which microorganisms were immobilized on the medium surface (fixed biofilm).

air pump

Ceramic particles

air diffusor

Feed tank Percolate Effluent reactor tank Figure 2.6 Schematic diagram of percolation reactor (Kargi and Uygur, 1996)

The highest COD removal efficiency obtained (90%) corresponded to the mixed culture of activated sludge and Halobacter halobium. Kargi and Dincer (2000) conducted a further study with Halobacter halobium. This species was cultured along with activated sludge in the fed-batch reactor. The organic removal rate was significantly improved (Fig. 2.7). COD removal of 85% was obtained within 9 hours at high salt contents (3-5%).

400 h 3

300 e, g/e, m t

200 CO D removalra

100 0.0 1.0 2.0 3.0 4.0 5.0 Salt concentration, %

Halobacter halobium+activated sludge

Only activated sludge Figure 2.7 Variation of COD removal rate (R) as function of salt content (Kargi and Dincer, 2000)

Woolard and Irvine (1995) investigated the treatment of phenolic wastewater with extremely high salinity. A moderate halophilic bacteria was seeded in a sequencing batch reactor. Over 99% phenol removal was achieved from 15% saline wastewater. Tellez et al. (1995) evaluated biokinetic coefficients in biodegradation of oil field produced wastewater. It is generated during recovery of natural gas and crude oil from onshore and offshore 20 operations. A commercial bacterium sp. (Petrobac-S) was used in this study. This is a hydrocarbon degrader specially formulated for degrading crude or refined hydrocarbons in moderated saline environments. The result indicated that when TDS was increased from 50 to 100 g/L, the maximum specific growth rate reduced from 0.137 to 0.047 h-1. There was a slight increase in the half-velocity-constant (Ks) at higher salt contents. Ks is substrate concentration at one-half the maximum growth rate. The affinity level for subtrate can be evaluated in terms of KS .

The phenol removal capacity of a new moderate halophilic bacterium, Halomonas sp. was investigated by Hinteregger and Streichsbier (1997). This bacteria consumed phenol as a source of carbon at NaCl concentrations between 10 and 140 g/L. Under optimum conditions, the degradation of 0.1g phenol/L in the aerated reactor was completed after 13 hours at salt contents in the range of 30 and 50 g NaCl/L.

Dincer and Kargi (1999) reported that biological treatment of pickling industry wastewater usually resulted in low COD removal efficiencies because of plasmolysis of cells caused by high salt content (3-5%). Utilization of halophilic microorganisms (e.g. Halobacter halobium) along with the activated sludge culture usually resulted in a better treatment performance. COD removal of 97% was obtained at HRT of 30 hrs and sludge age of 10 days.

An aerobic, submerged biofilter, coupled with a trickling filter was investigated to treat emulsified diesel fuel wastewater with high salinity (2% salt) (Yang et al., 2000). Figure 2.8 illustrates the schematic diagram of this system. The biofilter was randomly packed with plastic media particles. The salt-tolerant-bacteria were isolated from the sediments on an . This system could give high removal efficiency (TOC removal > 90%) at volumetric loading of 1.5 kg TOC/m3.d. The biodegradation of captured VOCs in the trickling filter was effective (68% removal). The adsorption of VOCs was accomplished by countercurrent flow of the gas and liquid phases through media bed. Based on the biodegradability tests at high salt contents of 3.4% and 4.0%, the authors postulated that the bacterial mixture could undergo high salinity up to 4.0%.

DO meter

pH meter

Biofilter Trickling fliter

Feed tank

Air blower

Figure 2.8 Schematic diagram of the biofilter and trickling filter treatment system (Yang et al., 2000) 21 Table 2.12 Effects of using halophilic bateria for high salinity wastewater treatment

Authors Experiment Results Kargi and Dincer Feed batch reactor with Zooglea - Not affected at salt content of 0.5% (| 5 g/L) (1996) ramigena - The efficiency dropped fast at increasing salt contents above 1%. Kargi & Uygur Percolator with Zooglea ramigena, - Zooglea ramigera culture obtained COD efficiency (1996) Halobacter halobium of about 77% at 1% salt. - Halobacter alone obtained lowest efficiency - Mixed culture of activated sludge and Halobacter obtained the highest efficiency at 1% salt and COD removal of 70- 80% at 4 – 5% salt Woolard and SBR with moderate halophilic - 99% phenol removal was obtained at 15% salt Irvine (1995) bacteria Tellez et al. Biokinetic experiments for oil field - Maximum specific growth rate reduced from 0.14 to (1995) produced wastewater 0.05 h-1 when TDS was increased from 50 to 100 g/L Hinteregger and Using moderate halophilic - 0.1 g/L phenol was completely degraded after 13 h Streichsbier bacterium, Halomonas sp. to treat at 30 g/L salt (1997) phenolic wastewater Dincer and Kargi Using Halobacter halobium to - 97% COD removal was obtained at HRT of 30 hrs (1999) treat pickling wastewater and salt content of 3-5% Yang (2000) Aerobic-submerged biofilter - TOC of above 90% was obtained at VLR of 1.5 kg coupled with trickling filter TOC/m3.d at salt content of 3.4 %. cultured with salt-tolerant-bacteria

2.4 Yeasts

2.4.1 General

Yeasts are eucaryotic, heterotrophic, unicellular microorganisms with a variety of shapes ranging from spherical to egg-shaped (common shape) and ellipsoidal, and from cylindrical to considerably elongated and even filamentous (mycelium). Yeast have no flagella or other organs of locomotion. In general, yeast cells are larger than bacteria, ranging from 1 to 5 Pm in width and from 5 to 30 Pm or more in length. Yeasts have a complex internal structure as shown in Figure 2.9. The vegetative budding yeast cell, in the log growth phase, contains a very large vacuole and has rigid walls.

Yeasts multiply as single cells, which divide by budding or direct division (fission which is similar to that by bacteria reproduce), or sporulation (sexual reproduction takes place by means of ascospores) (Fig 2.10). In some unicellular varieties, large numbers of cells attach themselves after budding, to form a pseudomycelium. In other cases, true mycelia are formed by fission (Fig. 2.11). Plasma membrane Centrosome Centrochromatin Cell wall Cytoplasm

Mitochondrium Nuclear membrane Vacuole

Figure 2.9 Diagram of yeast cell (Salle, 1961)

22 Budding

Fission

Sporulation

Figure 2.10 Budding is a common reproductive process in yeasts

a. Pseudomycelium b. True mycelium Figure 2.11 True mycelium (formed by fission) and pseudomycelium (formed by budding)

The dissimilation of organics may occur anaerobically (fermentation) or aerobically (oxidation). The most typical yeast process applied in food or beverage industries is anaerobic, also known as alcoholic fermentation. The end products of a fermentation can be alcohols, acids, esters, glycerol and aldehydes. Prior to fermentation, polymeric substances (carbohydrates, lipids, proteins) are hydrolyzed by enzymes (hydrolases). A typical reaction of sugar fermentation by yeasts is shown in the following reaction:

yeasts, nutrients Carbohydrate C2H5OH + CO2 + new yeast cells

Under aerobic process (assimilation), complete oxidation of organics yields and water. Abundant supply of oxygen enhances considerable yeast growth. When yeast are supplied with both sugar and oxygen, the colonies grow up to 20 times faster through cell division than without oxygen. However, incomplete oxidation may generate acids and other intermediary products. There are differences in the compounds which can be assimilated by various species of yeasts. Some can degrade pentoses, polysaccharides (starch), sugars, alcohols, organic acids (lactic, acetic, citric) and other organic substrates.

yeasts, nutrients Organics + O2 CO2 + H2O + new yeast cells+ end products

23 Yeasts may utilize the nitrogen required in their metabolism for the synthesis of protein from organic (amino acids, urea, vitamins, peptone, aliphatic amines, etc.) and inorganic sources (ammonia, nitrite and nitrate). Most species can utilize the ammonium ion. Other nutrients required for yeast growth include phosphorous, sulfur (organic sulfur and sulphate), minerals (potassium, magnesium, sodium and calcium). Trace amounts of boron, copper, zinc, manganese, iron, iodine, molybdenum are required to obtain optimum yields in synthetic culture media. Basic components of Candida utilis are showed in Table 2.13.

Table 2.13 Basic composition of Candida utilis yeast biomass (Defrance, 1993)

Element C O N H P S Mg Ash Value (%) 43.7 32 10.2 6.7 2.4 0.6 0.2 7.4

Based on these components, the chemical composition of Candida utilis yeast strain is formulated as follows: C3.64H6.7N0.73P0.07S0.02.

The nutrient demands can also be found from this formula. The C:N:P ratio of Candida utilis biomass is 100:20:5, corresponding to BOD5:N:P ratio of 100:7.5:2. Therefore, nutrient demands of yeasts are higher than that of bacteria whose BOD5:N:P ratio is 100:5:1 (Defrance, 1996).

Yeasts can grow in ranging from 0 to 470C. The optimum for most yeasts is 20 to 30oC. It is noted here that osmophilic yeasts are cable of growing in high osmotic pressure habitats such as high concentrations of salt or sugar which restrict the availability of moisture. On the other hand, yeasts can grow in a wide pH range (from 2.2 to 8.0). In general, yeasts grow well on media with acid reactions (3.8-4.0), whereas optimum pH values for bacteria growth range from 7.5 to 8.5.

Fungi or yeasts may be found wherever nonliving organic matter exists. Unpolluted stream water generally has relatively large numbers of species. Therefore, because of the relation between fungi and yeasts densities and organic loading, it is suggested that fungi and yeasts may be useful indicators of pollution. A survey of yeast populations along the St Lawrence River that received domestic wastewater from Quebec Province (Simard 1971; Simard and Blackwood, 1971). The results indicated that the blooms of pink yeasts (Rhodotorula spp.) and black yeasts (Candida, Crytococcus, Torulopsis and Pullularia spp.) occurred after bacteria had utilized the easily degradable components of the raw . Their density can be used as indicator of pollution.

2.4.2 Applications of Yeasts for Wastewater Treatment a. Domestic and Industrial Wastewater Treatment

The use of yeasts in biological treatment of domestic and industrial waste has been studied since 1970. Thanh and Simard (1971) studied the biological treatment of domestic wastewater with different yeast strains. All the tests were carried out with shaken 500mL- flasks at 26-28oC for 3 days. The initial pH was adjusted to 5.0. The result indicated that the yeast strains which gave high ammonia-nitrogen and COD removal efficiency were Rhodotorula marina (85% NH3-N and 67% COD removals) and Candida krusei (91% NH3-N and 72% COD removals). Especially, yeast strain Rhodotorula glutinis and Trichothecium roseum could completely remove phosphorous compounds in domestic wastewater (Simard

24 and Thanh, 1973). However, COD reduction was not as high as had initially been expected. The authors analyzed the cause to be the result of rapid uptake of phosphorous and nitrogen compounds before the organics could be assimilated. Deocadiz (1977) studied yeast treatment of mixture of domestic and paper mill white wastewater. Two yeast strains Candida utilis and Rhodotorula glutinis were cultured in shaking flasks. Approximately 80% of COD, 50% of N and 62% of P were removed after 24h. Rhodotorula yeast strain also gave the highest removal efficiency for the biological treatment of potato chips wastewater. The COD, N and P removals were 80%, 96% and 57%, respectively (Simard et al., 1973). The yeast sludge contained high protein content (53%).

Henry and Thomson (1979) observed that Candida ingens yeast spontaneously grew and formed a thick film on the supernatant of anaerobic piggery waste digesters. Based on this observation, the authors investigated treatment ability of C. ingens for the effluent from these digesters. The yeast was cultured in the stirring batch reactor. The results indicates that C. ingens could utilize almost all the VFA up to a concentration of 0.09 mol/L after 24h growth period. C.ingens grew well at pH ranged from 4.8 to 5.0 and at temperature of 29-32oC.

Miskiewicz et al. (1982) developed further yeast treatment of fresh piggery wastes by adding carbon source (beet molasses or sucrose). Four yeast strains, Candida tropicalis, Candida tropicalis, Candida robusta and Candida utilis were cultured in a batch aerated reactor. The study shows that the use of raw piggery waste without carbon supplement leads to low biomass yield and low treatment efficiency, even though the nutrients (N, P) are high. It is found that molasses are the most appropriate carbon source. The culture of C. utilis on molasses-enriched piggery waste (5570 mg COD/L) could obtain high treatment efficiencies. 76% TKN, 60% COD, 84% total P were removed at HRT of 7 hours and F/M ratio of 1.73 g COD/g MLSS.d. The maximum specific growth rate of C. utilis was 0.19 h-1.

Anciaux et al. (1989) investigated the influence of DO, substrate concentration, type of VFA on the growth of C. ingens in the aerated and stirred batch reactor. The result shows that higher the DO concentration, the shorter the lag phase and Pmax increased with the DO concentration according to the trend of the Monod model curve. Thus DO becomes a rate- limiting factor at a very low concentration (Figure 2.12a). The effect of substrate concentration is shown in Figure 2.12b. This figure shows inhibition of growth by the substrate concentration. At VFA concentration of 0.25 g /L (approximately 270 mg COD/L), the best growth rate (P) and yields obtained were 7.5 d-1 and 0.6 g DS/ g acid consumed, respectively. 7.20 8.00 -1 -1 6.80 day day P  P  6.00 6.40

6.00 4.00

5.60 Specific Growth Rate Rate Growth Specific

5.20 2.00 010 2040608 30 50 70 0 00.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 % DO (% saturation) VFA concentration (g/L) a. Specific growth rate vs. DO b. Specific growth rate vs. VFA concentration

Figure 2.12 Specific growth rate of Candida ingens vs DO and VFA concentration (Anciaux et al., 1989)

25 Katayama-Hirayama et al. (1994) cultured the yeast strain of Rhodotorula glutinis with phenolic wastewater. The cultures were propagated in shaking flasks and incubated at 30oC. Phenol and monochlorophenols were completely degraded after 2 days. COD removal obtained ranged from 79% to 83%. When compared with cell yields on the glucose (0.66 g/g) and acetate (0.39 g/g) media, phenol is an excellent carbon source (y = 0.61 g/g). None of the studies reviewed above evaluated the settling ability of yeasts.

Hu (1989) used ten different yeast strains in cultures to treat vermicelli wastewater which contains high concentration of starch, lactic acid and protein with BOD ranging from 24,000 to 44,000 mg/L. Based on the ability of starch degradation, protein hydrolysis and lactic acid tolerance, these yeast strains were screened from 391 colonies isolated from soil samples. Most could grow well within pH range of 3.0-5.0, with pH 4.0 being the optimum. The results shows that the two strains could reduce soluble COD by 92% at HRT of 7 days, F/M ratio of 0.48 g COD/g MLSS.d and VLR of 1.03 kg COD/m3.d. The long HRT in this process is due to the poor settling ability of yeasts. The yeasts could not be flocculated or settled as in a conventional activated sludge process, and were easily washed out with the effluent. Therefore, the HRT and SRT were kept constant. The author postulated that the fungi contamination prevented the formation of yeast flocs.

Similarly, Chigusa et al. (1996) used nine different strains of yeasts capable of decomposing the oil to treat wastewater from oil manufacturing plants. A pilot scale yeast treatment system had been run for one year. The results showed that 10,000 mg/L of hexane extracts in the raw wastewater were reduced by the yeast mixture to about 100 mg/L.

Also, Elmaleh et al.(1996) investigated the yeast treatment of highly concentrated acidic wastewater from the food processing industry. The strain Candida utilis was cultured in continuously completed mixing reactors. This system did not have a separate settling tank; the SRT and HRT of the system are identical. The carbon source of feed wastewater was a mixture of acetic acid, propionic and butyric acid. The pH was maintained at 3.5 to prevent any bacterial contamination. The TOC removal obtained was 97% at high loading rates (30 kg TOC/m3.day). The growth yield and maximum specific growth rate of yeasts were similar to - those for conventional activated sludge (Pmax = 0.5 h ; Y = 0.85-1.05 kg SS/kg TOC for acetic acid). In this study, the authors only evaluated biokinetic constants, but did not focus on the settling ability of yeasts.

Olive mill wastewater normally contains high concentration of fats, sugars, phenols, volatile fatty acids which contribute to a very high COD concentration (100-200 g/L). Scioli and Vollaro (1997) reported that Yarrowia lipolytica cultured in the 3.5L-aerated fermenter was capable of reducing the COD level of olive oil processing wastewater by 80% in 24 hrs. The effluent had a pleasant smell due to the presence of methanol and ethanol, while fats and sugars were completely assimilated. The authors postulated that using membrane to filter effluent before discharging into the sewage system might be a feasible approach for pollution reduction in olive-oil-producing countries. Useful biomass (40% protein) and valuable lipase enzyme could also be obtained in this process.

Silage is produced by the controlled fermentation of a crop with high moisture content, such as grass or maize. This silage can be used as an animal feedstock. Its effluent is extremely polluting, having very high BOD (30 – 80 g/L) and low pH (3.0-4.5). Arnold et al. (2000) investigated the ability of selected yeast strains (C. utilis and Galactomyces geotrichum) to purify silage effluent on the shaker-flask scale. High removal efficiencies of COD (74-95%), VFA (85-99%) and phosphate (82-99%) were obtained after 24 hrs. Some

26 ammonia was also removed. pH rose during treatment to 8.5-9.0 from initial values of 3.7-5.8. This was presumably due to removal of lactic acid and VFAs. The dramatic decrease in P (resulting in extreme P removal) may be attributed to the shortage of phosphorus.

In general, carbon and nitrogen removal from high organic-strength wastewater can be conducted with different processes, namely, anaerobic and aerobic processes, nitrification and denitrification. Ortiz et al (1997) proposed an effective and economic alternative process in which it is possible to achieve both carbon and nitrogen removal in two stages: anaerobic bacterial treatment and yeast treatment process. The fermentative bacteria transforms the organic nitrogen and the carbonaceous substrates into ammonia and volatile fatty acids (VFA) which are degradable substrates for the yeast growth (Fig. 2.13). A comparison between yeast and anaerobic treatment process is presented in Table 2.14.

Anaerobic Nitrification Dinitrification process process process

a. Traditional coupling for carbon and nitrogen removals

Acidogenesis Yeast process process

b. Coupling for Anaerobic acidogenesis and Yeast treatment Figure 2.13 Traditional carbon and nitrogen removal system can be altered with anaerobic and yeast treatment system (Ortiz et al. 1997)

Table 2.14 A comparison between yeast and anaerobic treatment process (Defrance, 1993)

Yeast process Anaerobic process Cannot degrade complex organic compounds Degrades cellulose

High nutrient requirement (BOD5:N:P) Low nutrient requirement Need for oxygen and agitation Slight agitation Exothermal reaction Æ need for cooling Producing methane as valuable bio-fuel Sensitive to variation of temperature Can consume VFAs produced from Dependent on two phases: liquefaction and acidogenesis gasification High organic loadings Low organic loadings (< 3 kg COD/m3.d) Short HRT High HRT (minimum 10 days) Valuable biomass Poor sludge production

Generally, dairy industry effluents contain large quantity of milk constituents such as casein, lactose, fat and high inorganic salts. Marwaha et al.(1999) investigated the effect of nitrogen supplements (urea and yeast extract) on the treatment ability of two yeast strains Candida parapsilosis and Candida haemulonii isolated from the dairy effluents. All tests were conducted with shaker-flasks and incubated at 30oC for 24 hrs. The pH of the medium was adjusted to 5.5. The result indicated that maximum BOD (90%) and COD (82%)

27 removals could be obtained when 0.6% yeast extract was supplemented. The relation between biomass growth and organic removal was not determined in this study. b. High salinity wasterwater treatment

Choi and Park (1999) studied the treatment ability of an osmotolerant yeast, Pichia guilliermondii A9, for waste brine from a kim chi factory using a shaker-flask scale. The growth of Pichia guilliermondii A9 in waste brine was not inhibited by NaCl concentrations up to 100 g/L. However, it was affected at concentrations above 120 g/L. Approximately 90% of BOD was removed from the waste brine after 24 hrs. The maximum cell yield was 0.69 g of dry cells per liter, containing 40% of protein. Cell growth was highest at pH 4, and declined slightly when pH increased to 8.

Nishihara ESRC Ltd. (2001) studied the effect of the Yeast Cycle System on marine products processing wastewater. In this system, yeasts are used for wastewater, where the excess yeast from the treatment process is recovered and reused. The system consists of pretreatment by yeast and by activated sludge process (Fig. 2.14). Table 2.15 and Figure 2.15 give a comparison with conventional complete mixing activated sludge in terms of the operating conditions. Marine products processing wastewater has BOD5 and SS concentrations ranging from 3,550-8,850 mg O2/L and 680-940 mg/L respectively. The chloride concentration was 5,160 mg/L. Some yeast strains, Candida edax, Candida valdivana and Candida emobii, were predominantly grown during enrichment with this raw wastewater. The predominance of yeast strains with the enrichment culture technique is based on free competition among different organisms in real wastewater. It was found that the yeast treatment process can obtain high efficiency at a higher volumetric loading (5 – 6 times), F/M ratio (2 – 3 times) when compared with the AS process. Yeast reactor Settling tank I AS reactor Settling tank II

yeast sludge return AS sludge return

excess yeast sludge excess AS Figure 2.14 Schematic diagram of the Yeast Cycle System (YCS)

Table 2.15 Operating conditions of YCS (Nishihara ESRC Ltd., 2001)

Parameter Unit YCS AS(*) Range Mean Range

Influent BOD5 mg/L 3,550 –8,650 6,100 110-400 Salt content g/L NaCl 5-8 6 0.05-0.45 3 BOD5 volumetric loading kg/m .day 4.5 – 10.4 7.5 0.8 – 1.9 Yeast concentration mg/L 8,000 – 10,000 9,000 2,500 – 4,000 BOD5 sludge loading (F/M) kgBOD5 0.56 – 1.04 0.9 0.2 – 0.6 /kgVSS.day Water temperature qC 23 – 30 26 23 – 30 pH 4.3 – 5.2 4.8 6.5 – 8.5 DO mg/L 0.51 – 0.95 0.7 t 2 SVI ml/g 60 – 72 66 100 – 120 Y kgVSS/kgBOD5 - 0.16 0.4 – 0.8 (*) Complete mixed activated sludge (Metcalf and Eddy, 1991)

28 6

5 Activated sludge Yeast Cycle System 4 han value t 3 imes higher imes higher

t 2 of activated sludgeprocess No. No. 1

0 LMLSS/LF/MY SVI Note: 3 L = BOD5 volumetric loading (kg/m .day) MLSS/L = Concentration of microorganism (mg/L) F/M = BOD5 sludge loading (kgBOD5/kgSS.day) Y = Sludge yield (kg VSS/kg BOD5) SVI = Sludge Volume Index (ml/g)

Figure 2.15 Comparison between Yeast Cycle System (YCS) and complete mixed activated sludge (AS) (Nishihara ESRC Ltd., 2001)

Large flocs formed in the yeast treatment system were able to settle quickly. Thus the MLSS could be maintained at high concentration of about 10,000 mg/L. The yeast sludge has low SVI of 50-60 mL/gram, which was equal to half the SVI value for activated sludge. This makes reducing the size of the sedimentation tank, and thickening of yeast sludge or chemical conditioning for dewatering are not necessary. The efficiency of this system is presented in Table 2.15 and Figure 2.23.

Table 2.16 Quality of treated water and efficiency of the YCS for seafood processing wastewater treatment (Nishihara ESRC Ltd., 2001)

After pretreatment After Parameter Influent E% E, % by yeast activated sludge BOD5 , mg/L 5,450 150 97% 4 99% SS, mg/L 798 113 86% 15 87% T-N, mg/L 153 72 53% 10 86% T-P, mg/L 33 18 46% 15 17 % Cl-, mg/L 5,160 5,080 5.080

BOD5 and SS removal by yeast were more than 95% and 87% respectively. The nitrogen (52%) and phosphorous removal (46%) during the yeast treatment were equal to those found in the components of excess yeast sludge. The company postulated that the structure of yeast flocs facilitated oxygen diffusion. Therefore energy could be saved through the reduction of the supplied air flow. Moreover, the excess yeast with high protein, vitamins, and lipid content could be used for animal feedstuff, mushroom growing or fertilizer.

29 Table 2.17 Summary of studies on yeast treatment of high salinity wastewater

Authors Experiment Results

Nishihara ESRC Yeast treatment system for - BOD5 and SS removal were more than 95, 97% Ltd. (2001) marine products process respectively 3 wastewater - High BOD5 volumetric loading (7.5 kg/m .day) - High BOD5 sludge loading (0.9 kg BOD5/kgVSS.day) - Low excess yeast, 0.16 kg VSS/kg BOD5

Choi and Park Yeast treatment for Kim Chi - Pichia guilliermondii can tolerate NaCl up to 100 g/L (1999) waste brine - 90% BOD removal obtained for 24h c. Waste recycle

Single-cell protein production (SCP)

Linkage between biomass for food production and waste and wastewater treatment has been widely developed. A number of organisms are utilized for biomass and protein production. These include: (1) protein-rich algae, fish, duckweed and water hyacinth in oxidation/stabilization ponds; (2) bulrush, cattails and other plants in constructed ; (3) worms from composting waste and sludge and (4) yeasts and fungi cultured from carbohydrate-rich wastewater. Therefore, utilization of food-processing wastewater as substrates for biomass production or single-cell protein production (SCP). SCP results in purification of effluent. This application can also obtain savings from decrease in disposal and treatment costs. Single-cell protein production (SCP) is defined as microbial biomass produced by some biological process and it can be used as food or food additives. Industries that produce large volumes of carbohydrate-rich-containing wastewaters free of toxic materials are most promising substrates for SCP. Such industries include milk, cheese- processing, confectionery manufacturing, and food canning. Effluents from these industries which contain high concentrations of COD and nutrients are costly to treat. Thus their utilization for SCP is an attractive alternative.

Yeasts such as Saccharomyces cerevisiae, Candida utilis and most fungi, are quite acceptable to animals and man. Whereas algal and bacterial biomass are less pleasant and contain undesirable levels of certain cellular materials (such as high nucleic acid content, toxic or carcinogenic substances absorbed from the growth substrate). In addition, due to abundance of valuable nutritious substances such as proteins and vitamins, yeasts are the most feasible and acceptable microorganisms in the production of SCP. In their simplest processing, yeasts can be cultured in a suitable substrate, normally carbohydrates, such as molasses, whey or starch, and under suitable conditions. The yeast biomass is harvested from the fermentation or assimilation followed by separation (settling, filtration, centrifugation, membrane), washed and dried to produce a free-flowing powder, rich in protein (Gray, 1989). For example, 50 thousand tons of yeasts per year are produced in North America from sulphide liquor (paper mill waste) containing high pentoses and hexoses.

A novel SCP process developed by George Bassett Co. in Sheffield is shown in Fig. 2.16. In this process, Candida utilis is cultured with confectionery wastewater and then harvested by centrifuging and drying. The SCP is packed and sold as a high-protein additive for animal feed. The present output of this plant is about 140 tons/year. It is able to remove 65% COD of the wastewater. Therefore, the wastewater after the SCP process contains remaining COD concentration, which is low enough to be discharged directly to the sewer (Gray, 1989).

30 Sterilizer Centrifuger Spray dryer

Crude effluent

Inocula reactor Main reactor

Package

Equalization tank Figure 2.16 SCP from confectionery effluent (Gray, 1989)

The Symba process developed by Swedish Sugar Company is based on the symbiotic culture of yeasts Endomycopsis fibuliger and Candida utilis with potato processing waste and wastewater (Figure 2.17). The basic substrate for SCP production in wastewater is starch, which is not easily assimilated by Candida utilis. However, the starch can be hydrolyzed to low molecular weight sugars (glucose, maltose) by the enzyme amylase. This enzyme is produced in large quantity by E. fibuliger. These hydrolyzed products (sugars) are then easily degradable substrates for the growth of C. utilis which has high nutritional value. In the process, the wastewater is strained in order to remove any large particles and then sterilized by heating to destroy any microbial contamination. The sterilized substrate is introduced to a preliminary reactor, in which E. fibuliger is grown to provide its population in the main reactor, as its growth rate is much lower than that of C. utilis. These reactors are maintained in aerobic condition. A large quantity of heat is generated from yeast assimilation activity and removed by cooling towers or heat exchangers. Excess biomass is purified by sieving and then concentrated by centrifuge and dried by spray drier. The Symba process can achieve BOD removal of 90% (BOD of wastewater is reduced from 15,000 mg/L to 1,500 mg/L), both N and P removals of about 50%. The yeast contains about 45% protein with less nucleic acids but rich vitamins, especially vitamin B (Gray, 1989). Endomycopis reactor

Symbiosis reactor

Cooling tower Sterilizer Storage tank Air blower Spray drier

Storage tank Separater Separater

Sewer Sewer Packing Figure 2.17 The Symba process (Gray, 1989) 31 Simard and Cameron (1974) evaluated the growth of Candida utilis on dilutions of spent sulphite liquor (SSL) with addition of different nitrogen sources. These were urea, ammonia sulphate and ammonium hydroxide. The result shows that the dilution of SSL (2 water:1 SSL) increased significantly the dried biomass. Urea, ammonia sulphate gave high conversion efficiency (70%).

Candida utilis yeast can be used to purify and uptake effectively ammonia present in the anaerobic digester supernatant supplemented with molasses as a source of carbohydrate (Irgen and Clark, 1976). The result shows that 100 g of molasses could yield 41 g of dried Candida utilis yeast cells and 20 g of protein.

Likewise, Barker et al. (1982) cultured Hansenula anomala, Candida krusei and Geotrichum candidum with whisky distillery spent waste. The influent COD of this waste ranges from 15 g/L to 58 g/L. These yeast strains were isolated from whisky distillery effluent. The results indicated that the yeasts could give high yield of protein biomass and COD removal of 55% .

Rashad et al.( 1990) found that mango peel waste from drink processing industries can be used for SCP production in which Pichia pinus is cultivated under optimum conditions (pH = 4.8-5.0; temperature = 30oC). The maximum yield obtained after 3 days of growth was 6.2 g/ biomass/L of wastewater and dried yeast biomass contained high crude protein (62%) and low nucleic acid (12%).

Liquid waste (deproteinized leaf juices) is generated from vegetable protein production. Chanda and Chakrabarti (1996) reported that depended on type of vegetable, BOD5 and COD of the waste can range from 12.9 to 19.0 g/L and 20.2 to 28.5 g/L respectively. This liquid waste can be a good substrate for the cultivation of S. cerevisiae, T. utilis, C. lipolytica. The yeast biomass obtained was rich in protein and vitamins. BOD of wastewater reduced significantly (74 – 97%) by the growth of these yeasts. The shrimp shell waste could also be converted into proteins by using the yeast Scharomyces cerevisiae KIV-1116 (Ferrer et al., 1996).

Bio-fuel

Fuel-alcohol production by yeast/fungi fermentation of the industrial or agricultural wastes has received considerable attention in Brazil and India in early 1990. Development of such technologies could reduce the oil imports and a partial solution to disposal of wastes. Nigam (1999) reported that the culture of Saccharomyces cerevisiae on pineapple cannery waste had high ethanol productivity (0.98g ethanol /g yeast.h) and sugar uptake rate (2.3 g sugar/g of yeast. h).

Yu et al. (1987) used the yeast Candida shehatae to ferment the spent sulphite pulp and paper mill. The ethanol yield gained was 0.46 g ethanol/g initial sugar at HRT of 18 h. This corresponds to the sugar removal of 95.5%. In general, the municipal primary wastewater solids contains about 10% cellulose and 26% lignin. These cellulose components can be effectively converted to ethanol by fungal cellulase and yeast Sacharomyces cerevisiae (Cheung and Anderson, 1997).

Lark et al. (1997) studied on the reuse of recycled paper sludge. At least 72% of cellulose in the sludge was converted into ethanol by the yeast Kluyveromyces marxianus. The paper sludge volume was reduced to 30 –35% of the original volume after 72h of

32 fermentation. The ethanol production by yeasts from cassava grate waste was also studied (Agu et al.,1998) where 60% of cellulose and lignin materials was hydrolyzed and converted to ethanol.

The cheese waste normally contains very high content of lactose which can be suitable substrate for ethanol production. Its concentration can be up to 50 g lastose/L. Ghaly and El- Taweel (1997) reused this waste for continuous ethanol production by yeast Candida pseudotropicalis. The results shows that high ethanol concentration (58 g/L) could be achieved at HRT of 42 hours.

2.5 Theoretical Modeling Consideration

2.5.1 Growth without Inhibition a. Specific growth rate (P)

Jackson and Edwards (1975) estimated specific growth rates of microorganisms in a culture by the following expressions: dX PX (2-1) dt

P (tto ) X X oe (2-2)

ln X P(t  to )  ln X o (2-3) ln X  ln X P o (2-4) t  to Where X = Biomass concentration at the time t (mg/L) Xo = Biomass concentration at the time to (mg/L) P = Specific growth rate (1/h).

Ln(Xt)

P Biomass concentration Ln(Xo)

t t o Time Figure 2.18 Growth curve of microorganisms in a culture

Generally, Monod's model is used to estimate different biokinetic reactions between microorganisms and the substrate in a continuous culture (Metcalf and Eddy, 1991). According to this model, specific growth can be related to substrate by the following relations: S P P m (2-5) S  K S Where P = Specific growth rate of microorganism (d-1) 33 -1 Pm = Maximum specific growth rate (d ) S = Substrate concentration (mg/L) KS = Half-velocity constant or Monod constant (mg/L).

Pmax ) P e ( t Pmax h ra t -1 2 time Specific grow

K s Substrate concentration, mg/ L Figure 2.19 The effects of a limiting substrate on the specific growth rate (Monod model) b. Substrate Utilization rate (U)

Substrate utilization rate(U) vis-a-vis COD removal rate, can be expressed as: (S  S) U o (2-6) T.X Where U = Substrate utilization rate, (mg substrate removed/ mg MLSS.day) So = Initial substrate concentration (mg/L) X = Biomass concentration (mg/L) S = Final substrate concentration (mg/L) T = Hydraulic retention time (day). c. Growth Yield Coefficient (Y)

The relation between new cell production and soluble substrate consumption can be represented as follows: dX dS Y * (2-7) dt dt Where Y = true growth yield coefficient (g SS/ g substrate removed.day) X = Biomass concentration (mg/L) S = Substrate concentration (mg/L)

For a given microorganism and essential nutrient/substrate under the same environmental conditions, the weight of microbial cells produced per weight of nutrient/substrate consumed is constant. This relationship is expressed as:

Y = Weight of organisms produced/Weight of substrate utilized

34 2.5.2 Growth with Inhibition

Han and Levenspiel (1988) proposed generalization of the Monod expression which takes into account inhibition effects caused by high concentration of substrate, product or toxics, ammonia, ion strength (salts) and other inhibitory substances. n § I · S P P m ¨1  ¸ © K I ¹ § I · (2-8) S  K S ¨1  ¸ © K I ¹ Where I = concentration of inhibitor KI = the critical inhibitor concentration above which reaction stops n = constants

Some kinetic models for growth with inhibitory substances are shown in Table 2-18.

Table 2.18 Kinetic models for inhibition growth (Han and Levenspiel, 1988)

Equation Model name § I · S S ¨1 ¸ P P m ¨  ¸ P obs (2-9) Ghose and Tyagi © K I ¹ S  K S S  K S 0.5 § I · S S P Pm ¨1  ¸ Pobs (2-10) Bazua and Wilke © K I ¹ S  K S S  K S n § I · S S (2-11) P Pm ¨1  ¸ Pobs Han and Levenspiel © K I ¹ S  K S S  K S

Where Pm = Maximum specific growth rate at inhibitor concentration of zero (I = 0). Pobs = Observed maximum specific growth rate at certain inhibitor concentration (I).

Pmax e t hra t

-1 n = 0.3 ), time n = 1.0 n = 0.5 obs P ( Observed specificgrow

K Inhibitor concentration ( I) , mass/ vol I

Figure 2.20 Curves of inhibition growth models (n =1: Ghose and Tyagi; n= 0.5: Bazua and Wilke model)

Eq.2-11 is a generalized form of Eq.2-9 and Equ.2-10, in which the constant n is 1.0 and 0.5 respectively. . 35 a. Substrate Inhibition

Some authors have suggested models for the growth inhibition of Candida utilis on acetic acid as substrate. These are:

S § S · P Pm ¨1  ¸ (2-12) Defrance (1993) K S  S © Sc ¹ S P Pm § S · (2-13) Haldane (Ortiz et al. 1997) (K S  S)¨1  ¸ © Ki ¹ S P Pm S 2 (2-14) Andrew (Ortiz et al. 1997) K S  S  Ki

Where Sc = The critical substrate concentration above which reaction stops Ki = Inhibition constant

Defrance’s model is also a modification of Han & Levenspiel’s model (Eq. 2-11) in which KI =Sc and n = 1.

Pmax Monod e t hra t -1

Defrance

),time Andrew obs P ( Haldane Observed specific grow specific Observed

S Substrate concentration ( S) , mass/ vol c Figure 2.21 Curves of substrate inhibition growth models b. Salt/ions Strength Inhibition

Webb’s model presents ion-strength inhibition for microorganism growth:

S P Pmax exp(1.17V ) § V · (2-15) S  K S ¨1  ¸ © K I ¹ Where V = Ion strength

Dincer and Kargi (1999) proposed expression to estimate salt inhibition for nitrification and denitrification.

N o  N KTN RN RON (2-16) T KTN  Cs 3 Where RN = Rate of nitrification and denitrification, kg/m .h KTN = Salt inhibition constant for nitrification and denitrification, g/L 36 Cs = Salt content, g/L RON = Nitrification and denitrification rates for salt-free wastewater. c. Other Inhibition Factors

The effect of pH, ammonia on the aerobic growth of Candida utilis at constant temperature (30oC) are illustrated by the following models:

Effect of >@NH P P 3 ammonia m 2 >@NH (2-17) (Ortiz et al., 1997) K  >@NH  3 >@NH3 3 K i>@NH3 Effect of pH 1 P Pm K H  (2-18) (Jackson and Edards, 1975) 1  2   H K1 Where K1 , K2 = pH constants

Henze et al. (1997) described effects of pH, temperature and DO on aerobic heterotrophic micro-organisms by the following kinetic models:

Effect of pH: K pH P m ( pH ) Pm ( pH opt ) (2-19) K pH  I Effect of temperature: o o o o P m (t C) Pm (20 C) * exp[D(t C  20 C)] (2-20) Effect of DO: S SO P P 2 (2-21) m K S K S S  O2  O2 Where I = ( pHopt  pH ) 10 1 KpH = pH constant D = Constant SO2 = DO in the mixed liquor KS,O2 = Saturated constant for oxygen

P(DOopt) P pHopt) -1 -1 time (DO), time (DO), P Observed specific growth rate growth specific Observed Observed specific growth rate rate specificObserved growth

246810 02468

pH DO concentration, mg/L Figure 2.22 pH and DO models

37 2.6 Respirometric Method

The respirometry measurement technique is used to measure the biochemical oxygen uptake rate (OUR) under well-defined experimental conditions. The respirometers are based on measuring the rate at which biomass takes up dissolved oxygen from the liquid phase (Vanrolleghem et al., 1999). Assessment of wastewater components is often referred to as wastewater characterization. The procedures for characterization involve a combination of physic-chemical and biodegradation tests. Using this method, the biodegradable components in the wastewater can be quantified (Vanrolleghem et al., 1999).

2.6.1 Respirometer

In principle, the respirometer consists of an oxygen electrode, DO meter, recorder, respirometric reactor and water jacket vessel to maintain a constant temperature. It is placed on a magnetic mixer in order to obtain a complete mixing of the reactor volume. A ceiling of the respirometric cell is oblique, so that the air bubbles can easily escape from the cell. The expansion funnel is used for adding the substrate solution and for escaping air bubbles during periods of aeration. A cross-sectional area of the funnel stalk is small enough to minimize oxygen absorption during the measurement. (Fig.2.23).

Figure 2.23 Schematic diagram of respirometer 1. Respirometric cell 2. Water jacket 3. DO probe 4. Air diffuser 5. Magnetic bar 6. Expansion funnel 7. DO meter 8. Recorder

2.6.2 Experimental Procedure

An expected concentration of endogenous activated sludge is transferred into the respirometry and aerated to increase the dissolved oxygen concentration to 6-8 mg/L. When these concentrations are reached, the aeration is stopped. A slow decrease in oxygen concentration is due to heterotrophic endogenous respiration. A typical respirogram is shown in Fig. 2.24, and can be interpreted as follows (Cech et al., 1984):

38 Adding substrate A B C

OC OURx,e DO, mg/L D OURx,t E Endogenous phase

Time, minute Figure 2.24 Recorder chart with a typical respirogram (Cech et al., 1984)

During the endogenous phase of respiration, heterotrophic microorganisms utilize oxygen at a constant rate over a relatively long period of time, as demonstrated by the line A- B-C. At time B, a small volume of concentrated substrate solution is injected into the cell by means of a hypodermic syringe. Addition of a limited amount of substrate to the respirometry reactor causes a temporary increasing respiration rate, as shown by the line B-D. This line is a maximum-value tangent to the curve B-E. It represents the constant total respiration rate at the substrate concentration S added. When the substrate concentration decreases with time, the respiration rate also decreases. When the substrate has been removed (at point E) the respiration rate returns to a value (line D-E), which is equal to, or perhaps slightly different from, the original endogenous rate.

When the measurement of one concentration is finished, a new dose of substrate can be injected into the cell and a next respirogram is recorded (Cech et al.,1984). In order to evaluate a respirogram, the endogenous respiration rate (OURx,e), the total respiration rate (OURt) and net oxygen consumption (OC) are calculated. The line section CE is equal to net oxygen consumption (Fig. 2.25). If the OC value is higher than 4 mg/L O2, the determination of OC is conducted using Ekama et al. (1986) method (Fig. 2.25). The high OC value occurs when a high substrate concentration is introduced. This method is normally used for determination of COD fraction (i.e. biodegradable COD/total COD).

In this test a preselected volume of wastewater of known total COD is mixed with a preselected volume of mixed liquor of known MLVSS concentration in a batch reactor. After mixing, the OUR is measured approximately every 5 to 10 minutes until OUR attains to a constant value that is approximate or equal to OUR in the endogenous phase (Ekama et al. 1986). The respirogram is obtained by plotting the curve of OUR versus time (Fig. 2.25).

39 D g A

f B C OUR, mg/L.h e

Time, minute T Figure 2.25 OUR response in respirometer (Ekama, et al., 1986) Where

Area A: This area gives the concentration of Readily Biodegradable COD (RBCOD) oxidized by the biomass. This is useful for assessing the amount of volatile fatty acids (VFA) that needs to be added in a biological phosphorus removal plant.

Area B: This area represents the amount of less readily biodegradable material being oxidized.

Area C: This area shows the amount of oxygen being used to convert ammonia into oxidized nitrate (nitrification).

Area D: The area under the whole curve shows the total oxygen demand of the liquor. This is the total amount of oxygen which must be supplied to the sludge to achieve full treatment.

OUR at line e: The respiration rate at the end of the curve, when at least 95% of the organic waste has been treated, is the endogenous respiration rate. This rate is proportional to the activity of the biomass.

OUR at line f: This rate is termed the Average Viability, and it is the average respiration rate for the period where nitrification and the breakdown of less readily biodegradable substrates are occurring.

OUR at line g: This is the maximum respiration rate observed at the start-up of the respiration cycles. At this point all oxidative reactions take place, including the oxidation of carbon and nitrogen compounds and the uptake of phosphates.

Time T: The time for the sample to reach an endogenous respiration rate. This is a direct method to determine the minimum HRT required to achieve at least 95% treatment efficiency.

2.6.3 Determination of Kinetic Constants

Specific OUR of substrate oxidation at a substrate concentration S (OURx,ox) is given by:

40 OURX ,ox OURX ,t  OURX ,e (2-22) Where:

OURx,t = Total respiration rate (mg O2/mg VSS.h) OURx,e = Endogenous respiration rate (mg O2/mg VSS .h)

Further specific substrate removal rate at a substrate concentration S (RX) is given by:

OUR R X ,ox (2-23) X OC / S Where RX = Substrate removal rate (mg COD removed/mg VSS.h) OC = Net oxygen consumption (mg O2/L) S = Substrate concentration (mg COD/L)

OC is then equal to the area between the OUR curve and the second plateau level where the OUR decreases rapidly and levels off (OC = Area A+area B) (Figure 2.25)

Biomass yield coefficient (Y) is expressed as: 1 § OC · Y ¨1 ¸ (2-24) f © S ¹ and the specific growth rate (P ) as:

P Y.RX (2-25) Where P = Specific growth rate (h-1) f = COD/VSS ratio of the sludge (mg COD/mg VSS) Y = Yield coefficient (mg VSS/mg COD removed)

2.7 Membrane Bioreactor (MBR)

MBR is the combination of two basic processes: (1) biological degradation and (2) membrane seperation into a single process where suspended solids and microorganisms responsible for biodegradation are seperated from the treated water by a membrane filtration unit (Manem and Sanderson, 1996).

MBR systems have two principal configurations: (1) The submerged MBR (or integrated MBR) in which outer-skinned membrane is submerged inside the bioreator and permeate is extracted by suction or by pressuring the bioreactor; (2) The external circuit MBR (or recirculated MBR) in which the mixed liquor is recirculated at high pressure through a membrane module placed outside the bioreactor (Fig. 2.26). The permeate is extracted by high cross-flow velocity through the membrane and the concentrated mixed liquor at the feed side is returned to the bioreactor. Excess sludge is withdrawn in order to maintain constant sludge age and the membrane is regularly cleaned by air or water backwash and chemical cleaning (Visvanathan et al. 2000).

41 Recirculation

Inffluent

Bioreactor

Inffluent Air

Inffluent Membrane filtration Bioreactor

Air Membrane module

Effluent Air diffuser

a. Recirculated MBR b. Surbmerge MBR process

Figure 2.26 Diagram of membrane bioreactor processes

2.7.1 Advantage of the MBR Process

The main advantages of MBR process can be listed as follows:

ƒ High quality of treated water: Biological treatment using the MBR process can obtain extreme high removal efficiency of SS, COD, BOD and pathogen concentration. Therefore the treated water can be discharged directly into the surface water, or reused for cooling, flushing and lawn watering.

ƒ Flexible in the operation: SRT is independent on HRT and can be controlled completely. Long SRT can be maintained to allow the development of slow-growing microorganisms, such as nitrifying bacteria.

ƒ Compact plant size:Due to the high biomass concentration that can be maintained in MBR, a high volumetric loading rate can be applied which results in the reduced size of bioreactor. In addition, secondary settling tank, sludge thickener or post treatment for further BOD and SS removal are not necessary in the MBR process, and thus the plant becomes more compact.

ƒ Independence of settling ability: the selection of microorganisms present in the membrane bioreactor is no longer dependent on either their ability to form biological flocs or the settling characteristics (Manem and Sanderson, 1996).

ƒ Low sludge production: Maintaining a low F/M ratio results in minimum sludge waste.

ƒ High degradation rate: High tangential velocities limit floc size and lead to an increase in mass transfer rates of microorganisms.

2.7.2 Main Design Parameters

In order to have an optimal MBR process from the economic point of view, many parameters should be considered. These can involve membrane selection, membrane

42 performance (permeate flux, transmembrane pressure, viscosity) and biological performance (MLSS, SRT, HRT, F/M ratio) and economic considerations (energy consumption, sludge treatment and disposal cost). These parameters can influence each other, and are mutually dependent. For example, high MLSS can be maintained by controlling long SRT and thereby increasing the volumetric loading (reducing HRT) and reducing the investment costs. However, high MLSS requires high maintenance energy consumption due to increases in viscosity that results in flux decline, high oxygen demand for aerobic organic degradation and cell growth (Manem and Sanderson, 1996; Visvanathan et al. 2000).

Table 2.19 presents a comparison between biological performances of MBR and a conventional AS process. This table shows that the mixed-liquor volatile suspended solid concentrations (MLVSS) in aerobic MBR process are much higher than those in conventional AS processes, reaching concentrations of 30 g/L. Based on particle size distribution tests, Nazim et al. (1999) indicated the AS sludge contained large flocs, while the MBR sludge contained small flocs. The high mass loads applied to aerobic MBR can be explained by high biomass concentrations and high specific substrate removal rate (Manem and Sanderson, 1996). HRTs of 2-4 hrs and F/M ratio of 0.1 g COD/g MLSS.d are normally applied to domestic wastewater treatment (Table 2.19). A combination of carbonaceous BOD removal and nitrification can achieved high efficiency in MBRs. Muller et al. (1995) reported using MBR process for domestic wastewater treatment could obtain 90% carbon removal and 100% nitrification efficiency at COD loading rates of 0.9-2.0 kg/m3.d. (HRT of 2.0 h - 7.5 h). Thus slow-growing nitrifying bacteria are retained by the membrane in the reactor. The aerobic MBRs are capable of high-strength industrial wastewater treatment. High organic loading rates (1.7- 8.6 kg COD/m3.d) can also applied.

43 Table 2.19 Comparison between biological performances of MBR process and conventional AS process

Combined domestic Domestic Domestic Vegetable Oily Fermentation conventional Parameters wastewater+ Ice cream wastewater wastewater canning wastewater wastewater AS industrial wastewater Type of membrane MF 0.1Pm MF 0.1Pm MF 0.1Pm MF MF Tubular UF UF Completed mixing VLR, kg BOD/m3.d 1.35 5.4 - 12.8 kg COD/m3.d 1.5 1.2 0.47 - 8.6 6.2 0.8-1.7 0.8-1.9 HRT 4 h - 24 h - 13 h 5.8 d 2.7 d 6-8 h F/M ratio, g/g.d 0.1 0.1 0.2 0.5 0.6-0.8 - 0.4 0.2-0.6 Sludge concentration, g/L 14 11 2.5-3.0 11 15-25 16 10-15 2.5-4.0 SRT 50-100 50 25 5-10 COD removal, % >95% 96 96 99 97 97 92-98 99 DO - 0.5-1.5 > 3 6.3 - - 2.0-3.5 t 2.0 Y 0.2 0.2 - Source Yamamoto Buisson et Trouve et al. Krauth Fuchs and Scott and Lu et al. Metcalf and et al. (1989) al. (1998) (1994) and Staab Scholz Smith, 1977 (1999) Eddy (1991) (1993) (2000)

44 2.7.3 Membrane Fouling

Membrane fouling or flux decline which leads to high-energy consumption and a large cleaning chemical requirement is a major problem hindering the widespread application of biomembrane reactor process. Membrane clogging in the MBR process might be the result of (a) the biofilm growth, or adsorption or deposition of foulants on the top surface of the membrane (external fouling) and (b) at the pore entrances or within the internal pore structure of the membrane (internal fouling). Fig. 2.27 schematises the fouling mechanisms.

Adsorption is used here to mean an interaction between foulants and membrane. The adsorption arises from physical forces which involve electrostatic forces (surface electric charges), Van Der Waals forces (attractive forces in close proximity), solvation forces (hydrogen bonds) and steric forces (attachment of polymers on the surfaces). particle macromolecule

Fluid flow Concentrate

membrane pore Permeate Figure 2.27 Diagram of fouling mechanisms (adsorption and deposition)

The phenomenon of fouling is very complex and depends on physical chemical parameters such as concentration, pH, ion strength and surface properties of particles and membrane such as electrical charges, hydrophilicity or hydrophobicity. For example, a more hydrophilic membrane can decrease the ability of adsorption and fouling rate. Macromolecules (proteins, EPS) which are normally hydrophobic adsorb easily on hydrophobic membranes. The adsorption layer is also more difficult to wash out from a hydrophobic surface than from a hydrophilic one. Surface charges can also have an effect on fouling. If there is electrostatic repulsion between the membrane surface and bioflocs or macromolecules, fouling is decreased. Stability of macromolecules is influenced by the pH of mix-liquor. Macromolecules are more compact at their isoelectric point, pI, where intramolecular electrostatics repulsion is minimum. Thus rapid flux decline occurs at this pH value due to an increase in the amount of deposited macromolecules. a. Foulants

Three main types of foulant can be differentiated (Mulder, 1996): x Organic precipitates (biological substances, macromolecules, etc.): Macromolecules can be protein molecules in wastewater, extracellular polymers (ECP) or long chain organic by-products generated from biodegradation process. x Inorganic precipitates (metal hydroxides, calcium salts, etc.): Changes in environmental conditions (pH, solute or ion strength) due to microorganism actions in MBR can form precipitates. Gelatinous precipitates (such as hydrated complex of calcium phosphate and citrate) can seriously foul membranes (Howell and Nystrom, 1993).

45 x Particulates (cells, debris, microbial flocs, etc.): Particulates in the mixed liquor build-up the solid cake on the surface of membrane, which results in a decline in flux. b. Biofouling

Biofouling can be defined as adsorption/adhesion and growth of microorganisms which forms biofilm on the membrane surfaces. Adhesion can be due to bonding interactions between the membrane surface and adhesive structures such as flangella, fimbria, or macromolecules (proteins, extracellular polymers) on the cell surface. Once attached, cells may grow and multiply by using substrates and nutrients from the bulk solution (Fig. 2.28). Harry et al.(1996) postulated flux decline can be significantly attributed to extracellular polymers (EPS) rather than to the colloidal nature of bacterial cells.

cell # 1

cell # 1

secondary adhesion surface EPS charges primary EPS adhesion Growth Growth

membrane

Figure 2.28 Schematic illustration of membrane biofouling process (Ridgway and Flemming, 1996).

Extracellular polymers (EPS)

The production of EPS is a general property of micro-organisms in natural environments, and occurs in bacteria, algae, yeast and fungi (Flemming and Wingender, 2001). EPS are major component of activated sludge (AS) matrix and biofilms. They play an important role in bioflocculation, settling and dewatering of AS process and in biofilms development in attached-growth process. They consist largely of proteins, polysaccharides and humid substances. EPS act as a bridge between cell surface and therefore initiate floc or biofilm formation (Bura et al. 1998). However, the presence of high EPS concentration may result in poor settling (bulking phenomenon) or dewatering condition (i.e. increasing sludge volume index SVI, or capillary suction time CST) in conventional AS process. In addition, high EPS concentration can increase the specific hydraulic resistance (R) of the filtration cake in MBR process (Manem and Sanderson, 1996). Fig. 2.29 presents the schematic diagram of the matrix of biofloc or biofilm.

46 Divalent

cation 3- PO4

COO-

bacteria cell

EPS

Figure 2.29 Schematic diagram of biofloc or biofilm

Various experimental studies have demonstrated the important role of macromolecules in fouling and flux decline. Hodgson et al.(1993) investigated the role of the bacterial EPS in cake resistance of MF system (0.2 Pm membranes at 100 kPa in batch filtration cells). The gram-negative marine bacterium SW8 was used in this study. The role of the EPS in resistance was confirmed by changes in flux through treated and untreated bacterial cakes. The treated bacterial suspensions here means bacterial cells whose EPS were removed by proteolytic enzyme and chelating agent (EDTA). Whereas the untreated cake consists of particular cells with the void spaces filled with EPS proteins and polysaccharide The authors found that the untreated cake had more higher resistance than the treated cakes. This confirmed that the major cause of resistance was not the bacterial particles themselves, but the EPS associated with those bacteria.

Likewise, Nagaoka et al. (1996) carried out a study on the influence of bacterial EPS on the membrane separation AS process. Loop-type hollow fiber membrane modules with pore size of 0.1 Pm were used. The experiments were intermittently operated with a cycle of 10- minutes-run and 5-minutes-off. Feed wastewater was a mixture of acetic acid (as carbon source) and necessary nutrients. The results indicate that EPS which was accumulated in the aerations and also on the membrane caused an increase of viscosity of the mixed liquor and an increase in the filtration resistance. There was a linear relationship between the filtration resistance and viscosity of the mixed liquor, which is caused by rapid attachment of the suspended EPS.

Mukai et al.(2000) estimated flux decline of ultrafiltration membrane in different cultural growth phases, different EPS and metabolic product concentrations in AS process. The authors reported that flux decline was effected by protein to sugar ratio of EPS and metabolic products. Lower permeate flux occurred with higher retention of protein and greater amounts of retained protein during filtration.

The other influences

Autolysis occurring at high SRT or starving conditions (low F/M ratio) can lead to increase in concentrations of cell debris and soluble microbial by-products (such as humid substances and proteins). Absorption of these substances on the membrane surface can boost biofouling (mainly internal fouling). In addition, high hydraulic stresses may also enhance cell and floc breakage that releases metabolites and cell debris.

47 The effects of MLSS, soluble COD and viscosity on membrane fouling was estimated by Sato and Ishii’s model as follows (Manem and Sanderson, 1996): R 842.7 * 'P * (MLSS)0.926 * (COD)1.368 * (P)0.326 (2-26) Where R = Filtration resistance, m-1 'P = Transmembrane pressure, Pa P = Viscosity, Pa.s MLSS = mixed liquor suspended solid, mg/L COD = Soluble chemical oxygen demand, mg/L -1 Rt = Total resistance for filtration, m

It can be seen that soluble COD concentration can contribute significantly to increase in filtration resistance (Eq. 2.26).

48 Chapter 3 3 Methodology

This research comprises four main studies: (1) biokinetic study; (2) parametric study (optimization of operating conditions); (3) biomembrane study, and (4) sludge characterization study. Flowchart of the different phases of the experimental studies is shown in Fig. 3.1.

Aclimatized yeast and bacterial sludge

DO Glucose (1) Biokinetic experiments SRT Protein extract

(2) Parametric study pH (Optimization of operating conditions)

(4) Sludge characteristics (3) MBR study

High influent COD Low influent COD 5,000 mg/L 1,000 mg/L

VLR SRT (4) Sludge characteristics

Figure 3.1 Flowchart of different phases of experimental study

3.1 Biokinetic Study

The objective of this study was to evaluate the biokinetic coefficients of mixed yeast and mixed bacterial treatment of high salinity wastewater by means of respirometric techniques. Two feed synthetic wastewaters were used, namely: glucose-feed wastewater and protein-feed wastewater. In the protein-feed wastewater, commercial tuna fish protein extract (T.C. Union Agrotech Co., Thailand) was mixed with tap water to obtain wastewater whose composition was similar to that of tuna fish processing wastewater. The composition of two feed wastewaters is presented in Tables 3.1 and 3.2. The flowchart of biokinetic study is shown in Fig. 3.2.

49 Table 3.1 Composition of glucose-feed wastewater (Defrance, 1993)

Component Concentration (*) (mg/L) Glucose 4,673 (**) (NH4)2SO4 1,870 Yeast extract 94 KH2PO4 235 MgSO4.7H2O 467 ZnSO4.7H2O 0.5 CaCl2 1.0 MnCl2 1.0 FeCl2 1.0 (NH4)6Mo24.4H2O 0.2 CuSO4 0.2 CoCl2 0.2 (*) Composition of synthetic wastewater used for bacterial system was similar to that for yeast culture, but the concentration of each component was five times lower than that of the yeast culture. (**) Corresponding to COD concentration of 5,000 mg/L and BOD20 concentration of 4,440 mg/L (Biodegradability of glucose is 0.95 g BOD20/g glucose (Henze et al., 1997).

Table 3.2 Composition of protein-feed wastewater

Parameters Concentration (mg/L) COD 5,000 BOD5 3,850 Organic-nitrogen 690 Ammonia-nitrogen 26 Total phosphorous 45

Seed yeast sludge Seed bacterial sludge

Enrichment

Acclimation of - Glucose as substrate high salinity - up to 45 g/L NaCl

Acclimation of protein extract

Biokinetic experiments

Glucose Protein extract

50 Figure 3.2 Flowchart of biokinetic experiments

3.1.1 Seed Sludge a. Yeast sludge and enrichment

The term mixed yeast sludge implies the mixture of all wild yeasts which exist in the raw wastewater and then quantitatively propagate under proper enrichment conditions. The procedure of enrichment for yeasts was carried out according to the Standard Methods for the examination of water and wastewater (APHA et al., 1995). The yeast strains were selected by the enrichment culture technique based on free competition among different organisms in wastewater (Nishihara Ltd., 2001). Figure 3.3 shows the procedure of the enrichment process.

The osmotolerant yeast sludge was enriched from the bottom sediments of an equalization tank of a fish sauce factory located in Rayong province, Thailand. This tank received wastewater containing high salt and organic content (30.2 g/L NaCl and 800 mg/L COD). The enrichment was conducted using two-liter container and the fill-and-draw operation. In the first batch, the raw sediment was added to two containers containing two liters of feed wastewater with 20 g/L and 32 g/L NaCl. MLSS concentration of mixed liquors obtained were around 1,000 mg/L. The feed wastewater (glucose as substrate) was mixed using a diffused aeration system, and pH was adjusted to 3.5 in order to optimize yeast growth and limit the bacterial contamination (Pelczar and Reid, 1972; Elmaleh, et al., 1996). After eight hours of aeration, the biomass suspension was allowed to settle for 12 hours. Yeast cells, normally, settle to the bottom; acid-tolerant bacteria and filamentous fungi remain in suspension. The bacteria and fungi are removed by decanting supernatant. 1.5 liters of supernatant was decanted and a fresh medium was added for next batch. When yeast biomass (MLSS) exceeded 3,000 mg/L, the enrichment process was stopped.

Seed yeast sludge (sediments)

Feed-glucose- Filling Drawing wastewater

Aeration Settling (32 h) (10 h)

no MLSS>3000 mg/L

yes

Completion

Figure 3.3 Schematic diagram of enrichment procedure

51 b. Bacterial sludge

The bacterial seed sludge was obtained from the activated sludge process of the same fish sauce processing wastewater treatment plant. This plant treats combined wastewater with salt content of approximate to 1g/L NaCl and mean COD concentration of 540 mg/L.

3.1.2 Acclimation

Acclimation was carried out to obtain mixed bacterial and yeast sludges that can tolerate salt contents (32 and 45 g /L NaCl). The two-litre-batch-reactors with fill-and-draw operation were used in the acclimatization stage. Table 3.3 shows the operating conditions. In order to obtain operating conditions similar to saline seafood processing wastewater treatment using yeast treatment followed by mixed bacterial system, the initial COD concentrations of 5,000 mg/L for yeast and 1000 mg/L for mixed bacterial culture were selected.

Table 3.3 Operating conditions for high salinity acclimation

Operating conditions Yeast sludge Bacterial sludge Initial COD (mg/L) 5,000 1,000 pH 3.5 7.5 Temperature ( oC) 25 – 32 25 – 32 MLSS (mg/L) 5,000 4,000 HRT (h) 36 24

After aeration (24 h for bacterial sludge and 32 h for yeast sludge), the biomass suspension was allowed to settle for 12 hours, and the supernatant was sampled and centrifuged at 4,000 rpm for 15 min. COD of the supernatant was analyzed. When the COD removal efficiency was less than 80%, the experiment was repeated at the same operating conditions. When the COD removal exceeded 80%, the NaCl concentration was increased by 3 g/L. Acclimation to high salt contents (32 and 45 g NaCl/L) was assumed to be completed when 80% COD removal was attained.

3.1.3 Biokinetic Experiments

The kinetic coefficients of the acclimated yeast and bacterial cultures at different salt contents with two substrates (glucose and protein extract) were assessed using a closed 0.9 liter batch respirometer, equipped with a recorder, DO meter and water jacket vessel to maintain a constant temperature as shown in Figure 3.4. Table 3.4 presents the operating conditions of the respirometric experiments for yeast and bacterial culture. Initially, glucose was used as a substrate complemented with necessary nutrients. The So/Xo ratio (initial substrate concentration/biomass concentration) governs the quality of the batch respirometric tests (Cech et al., 1984). In order to obtain So/Xo ratio ranging from 0.01 to 0.20 (Mathieu and Etienne, 2000), the Xo and So values should be ranged from 1,500 to 2,000 mg VSS/L and 20- 500 mg COD/L, respectively.

52 7

8 2 4 3 1 5

9

6

1. Respirometric cell 2. Water jacket 3. Air diffuser 4. DO probe 5. Magnetic bar 6. Magnetic mixer 7. Expansion funnel 8. DO meter 9. Recorder Figure 3.4 Respirometer set-up

Table 3.4 Operating conditions for the respirometric experiments

Operating conditions Yeast sludge Bacterial culture Initial pH 3.5 7.5 Temperature (oC) 30 r 0.5 30 r 0.5 (1) Xo (mg MLSS/L) 1,500 1,500 Substrate (mg COD/L) 20 – 500 20 – 200 (2) So/Xo ratio 0.01 – 0.35 0.01 – 0.15 Suppressing nitrification None Adding 70 mg N-ammonia/L (3) Sources: (1) Cech et al., (1984) (2) Chudoba. et al., (1992) (3) Liebeskind, (1999)

The experimental procedure for OUR determination are summarized below:

1) Obtaining endogenous sludge: The respirometer was filled with fresh sludge without substrate and aerated at least for 2 hours.

2) Suppressing nitrification: NH4Cl was used with the concentration of 70 mg N- ammonia/L. Liebeskind (1999) postulated that if ammonia was present in wastewater, organic oxidation and nitrification simultaneously occurred. At high enough ammonia concentration (70 mg/L N), OUR of nitrification is constant during organic oxidation. When this ammonia dose is added to the endogenous sludge, nitrification OUR will be determined. Thus, OUR of organic oxidation will be the difference between total OUR and the sum of endogenous OUR and nitrification OUR.

3) Recording endogenous OUR: After suppressing the nitrification process, the mixture was aerated at least half an hour before measuring endogenous OUR.

53 4) Adding substrate: An accurate amount of substrate was added to the respirometer and total OUR was recorded by respirogram. New reaeration was necessary when the dissolved oxygen concentration dropped below 2 mg/L.

The results of the respirometric experiments provided OUR data that are used for calculating specific growth rates (P) using Equations 2.22 to 2.25 in Chapter II. By using OUR values and specific growth rates (P) with respect to corresponding substrate concentrations (S), maximum oxygen utilization rate (OURmax), observed specific growth rate (Pobs) and half velocity constant (Ks) at the selected salt content were determined by regression analysis based on Monod kinetics.

By using observed maximum specific growth rates (Pobs) at the corresponding salt contents (I), the critical salt content above which reaction stops (KI) and maximum specific growth rate of free-salt solution (d-1) were evaluated by linear regression analysis based on the Ghose and Tyagi model (2-9). The regression analysis was done by Grapher sofware.

3.2 Parametric Study

3.2.1 pH values

The effects of pH values on bacterial and yeast treatment systems were also evaluated in terms of OUR by respirometer. The protein-feed wastewater with 32 g salt/L was used. The experiments were done at constant temperature of 30r0.5 oC. The operating conditions are presented in Table 3.5.

Table 3.5 Operating conditions for the pH effect experiments

Operating Condition Units Values COD mg/L 50 MLSS mg/L 2,000 Temperature oC 30 r 0.5 Salt g/L NaCl 32 pH values: + For mixed yeast sludge 2.5 – 9.0 + For mixed bacterial sludge 4.0 – 10.5

The main procedure of the experiment can be described as follows: x Obtaining endogenous sludge: The respirometer was filled with fresh sludge without substrate and then aerated for at least 2 hours. x Suppressing nitrification (for mixed bacterial sludge): NH4Cl was used with a concentration of 70 mg N-ammonia/L x Adjusting pH value: H2SO4 solution 0.1N or NaOH 0.1N solution was added into the mixture until the desired pH value. x Recording endogenous OUR: After 10 minutes of aeration, the endogenous OUR was recorded. x Adding substrate: Stock solution of fish extract (25,000 mg/L) had been prepared and adjusted to the desire pH value before adding into the mixture. An accurate amount of 54 substrate (300 mg/L COD) was injected into the respirometer and then total OUR was recorded. New reaeration was necessary when the DO dropped below 2 mg/L.

3.2.2 Sludge Retention Time (SRT)

Each of the SRT variation experiments was conducted in a two-liter batch reactor using the fill-and-draw operation. Here, the MLSS variation was monitored for a minimum period of 3 weeks. The steady state condition was reached when the MLSS values remain constant for at least 5 days. The experimental operating conditions are presented in Table 3.6. To avoid the effects of protein precipitation (at low pH) on the nitrogen uptake ability of yeast sludge, removal of protein precipitates prior to feeding is necessary. Raw wastewater was acidified to pH 3.5 and left to settle at least for 12 hours at 40C.

Table 3.6 Operating conditions of the experiments on SRT effect

Operating conditions Unit Value COD mg/L 5,000 pH 3.5-4.0 MLSS mg/L 7,000 HRT h 24 Salt g/L 0.5, 15, 32 and 42 SRT d 5, 7, 10, 20 and 45

Sludge retention time is defined as the average time for which a unit of biomass remains in the system. For a completely mixed process with a sludge return arrangement, SRT can be expressed mathematically as follows:

Vr X T c (3-1) Qw X  (Q  Qw )X e Where

Tc = Sludge retention time, d Qw = Sludge waste rate, L/d Q = Influent flow rate, L/d Vr = Volume of aeration tank, L Xe = Volatile suspended solids in effluent X = Mixed liquor volatile suspended solids in the aeration tank, mg/L

To facilitate operation of lab-scale experiment, it is assumed that solids lost in the effluent can be neglected (Xe = 0). Equation 3-1 can be rewritten as:

Vr T c Qw

Vr Qw (3-2) T c

Equation 3-2 provides the calculation for sludge volume to be wasted daily from the reactors. After 24 hours of aeration, waste sludge was withdrawn. Due to poor settling ability of mixed yeast sludge, settling step in a typical batch process was replaced with

55 centrifugation. The remaining of suspended biomass was centrifuged at 3,000 rpm for 15 min. The centrifuged sludge was returned to the reactor for the next batch.

3.3 Biomembrane Study

The biomembrane study was conducted with the fish-protein-feed wastewater at 32 g/L NaCl. This study consisted of two phases, namely: (1) high COD loading, and (2) low COD loading. The difference between the two phases is shown in Table 3.7.

Table 3.7 Difference between the high COD loading and low COD loading

Parameter High COD loading Low COD loading Influent COD (mg/L) 5,000 1,000 Experimental YMBR YMBR set-ups Feed wastewater Feed wastewater (5,000 mg/L COD) (1,000 mg/L COD) Yeast BMBR BMBR reactor SRT 50 d for both YMBR and BMBR; 50 d and 10 d were 15 d for yeast reactor investigated for both YMBR and BMBR

3.3.1 High COD loading

The fish-protein-feed wastewater used in this phase was similar to that used for biokinetic study (Table 3.2). Both yeast and bacterial sludges were excess sludges obtained from the biokinetic study. Two parallel experimental set-ups were conducted, namely: (1) yeast pretreatment followed by the Bacterial Membrane Reactor (BMBR) as schematized in Figure 3.5, and (2) the Yeast Membrane Reactor (YMBR).

Both YMBR and BMBR tanks were made of transparent acrylic tube of 10 cm in diameter with working volumes of 8 L and 3.6 L respectively. In order to provide enough effluent volume to BMBR at different HRTs, the working volume of the yeast reactor (YR) was made adjustable by changing outlet points installed along the height of the column. The total volume of the yeast reactor was 21 L. These reactors were continuously aerated through the stone diffusers placed at the bottom, and equipped to monitor pH and DO. For the YMBR system, through an external pH dosing pump, the reactor pH values were maintained at the required level. In each reactor, a polyethylene 0.1 Pm hollow fiber membrane module with a surface area of 0.42 m2 was fixed on the upper end.

Speed-controlled roller pumps were used to withdraw the permeate from these membrane modules. Both bioreactors were operated with periodic air backwashing (20 minutes filtration and 2 minutes of air backwashing at a pressure of 400 kPa arrangement. The alternative operations of filtration and air injection was controlled by an intermittent controller and solenoid valves. The transmembrane pressure was measured using a mercury manometer.

For the BMBR system the feed wastewater was pretreated with the yeast reactor with continuous aeration through diffusers with a mean HRT of 32 h. The pH was maintained at 3.5. The treated effluent from this reactor was continuously withdrawn and sent to the sedimentation unit where the yeast sludge is separated then returned to the yeast reactor. The hydraulic retention time of the settling tank was 5 h, which corresponds to the shortest HRT of BMBR (4.5 h). From the yeast reactor, excess sludge was periodically withdrawn to

56 maintain a mean sludge retention time (SRT) of 15 days and mean biomass concentration of 4,500 mg/L MLSS. The settled effluent was stored in an intermittent storage “effluent tank” and fed into the BMBR reactor system. In the BMBR system, the pH was maintained at above 7.0, whereas in the YMR, the feed water did not undergo this pre-treatment, but was fed directly into the YMBR tank where the pH was maintained at 3.5.

The investigations were carried out by step-wise increase in volumetric loading. The different loading steps used is summarized in Table 3.8. Each of volumetric loading rate (VLR) variation was maintained for at least 7 days. The mean influent COD concentration fed into the BMBR was the effluent COD from the yeast reactor.

Table 3.8 Experimental operating conditions of YMBR and BMBR systems

YMBR BMBR Mean Mean influent Mean Mean Stage Time VLR Time VLR influent kg COD HRT kg (*) HRT days 3 days 3 COD COD/m .d mg/L COD/m .d h mg/L h I 1-22 5.0 5,000 24.0 1–11 2.1 1,200 13.7 II 22-29 3.4 5,000 36.0 11-21 3.4 1,280 9.1 III 30-40 6.6 5,000 18.2 22-31 7.9 1,450 4.5 IV 41-51 9.9 5,000 12.2 32-41 5.3 1,080 4.9 V 52-62 16.3 5,000 7.1 42-51 1.7 1,170 16.1 VI 63-78 23.0 5,000 5.1 52-75 2.4 1,140 11.7 VII 77-90 3.6 1.450 3.6

(*) Effluent from the yeast reactor

57 Timer Hg-manometer Timer Hg-manometer Air backwash Air backwash

Level water Level water Outlet point tank tank

NaOH sol. (to pH 6.8-7.5) YEAST REACTOR Overflow Overflow

to feed tank Sulphuric acid sol. BMR effluent tank Yeast effluent tank (pH 3.5-3.8) Sulphuric acid sol. Bacterial (pH 3.5) Membrane reactor Effluent tank 1 Feed tank Compressed air Compressed air Compressed air Yeast excess sludge Yeast excess sludge

Yeast excess sludge Bacterial excess sludge YEAST MEMBRANE REACTOR BACTERIAL MEMBRANE REACTOR SYSTEM

Figure 3.5 Membrane reactor systems in the high COD loading

58 3.3.2 Low COD loading

This phase was a sequence of the high COD loading. Therefore, yeast and bacterial sludges were acclimated. Both YMBR and BMBR had the same working volume and influent wastewater with COD of 1,000 mg/L. The experimental set-up is schematized in Fig. 3.6. Both YMBR and BMBR tanks were made of transparent acrylic tube of 15 cm diameter with working volume of 10 L. Likewise, in the high COD loading, the reactor pH values were maintained to 3.5 in the YMBR set-up. In each reactor a 0.1 Pm hollow fiber membrane module with a surface area of 0.42 m2 was fixed on the upper end. The air-backwash operation of both set-ups in this phase was similar to that of the high COD loading.

The composition of the low COD wastewater is presented in Table 3.9. The experiments were conducted using a step-wise increase in the flux rate (i.e. increase in the volumetric loading) at sludge retention times of 10 and 50 d. During the transition period (between SRT of 10d and 50 d), no sludge was wasted from the reactors except for sampling. The operating conditions for this phase is shown in Table 3.10.

Table 3.9 Composition of the low COD wastewater

Parameters Unit Concentration COD mg/L 1,000 BOD5 mg/L 780 Organic-nitrogen mg/L 120 Ammonia-nitrogen mg/L 25 Total phosphorous mg/L 12 Salt content g/L NaCl 32

Timer Air backwash Hg manometer

Feed tank

Bioreactor Level water tank Membrane Effluent tank

(H2 SO4 solution only for YMR)

Compressed air

Excess sludge

Figure 3.6 Schematic diagram of membrane bioreactor

59 Table 3.10 Effects of different HRTs and SRTs on yeast and bacterial membrane reactors

YMBR BMBR SRT VLR Mean HRT SRT VLR Mean HRT 3 days kg COD/m3.d h days kg COD/m .d h 10 2.66 8.8 10 2.97 8.1 10 2.95 7.7 10 3.57 6.3 10 3.66 6.1 10 4.30 5.2 10 4.59 5.0 Transition 3.63 7.1 Transition 3.58 7.2 Transition 4.08 6.0 Transition 4.28 6.1 50 5.56 4.7 50 4.93 5.3 50 6.35 4.0 50 6.55 4.0

3.4 Sludge Characterization Study

To investigate variation of sludge characteristics with salt contents and simultaneously obtain a comparison between membrane bioreactor and batch systems, yeast and bacterial batch reactors were operated at different salt contents (0.5, 15, 32 and 45 g/L). Two-liter- batch reactors with fill-and-draw operation were used. The initial mixed yeast and bacterial sludges were withdrawn from the YMBR and BMBR run at SRT of 10 d and 32 g salt/L. These sludges were later acclimatized at salt contents by gradual increases or decreases. Table 3.11 presents the experimental operating conditions. The sludge was sampled when each batch reached the steady state condition (i.e. COD removal was above 80% with a stable MLSS value). The sludge was examined for ECP content, dewatering property (CST) and sludge settleability (SVI). In addition, N and P contents of both yeast an bacterial sludge were evaluated.

Table 3.11 Operating conditions for the sludge characterization study

Operating conditions Value Initial COD (mg/L) 1,000 pH: - Mixed yeast sludge 3.5-4.0 - Mixed bacterial sludge 7.0-7.5 Initial MLSS (mg/L) 7,000 SRT (d) 10 HRT (h) 8 Salt (g/L) 0.5, 15, 32 and 42

3.5 Analytical Methods

All analyses were conducted according to Standard Methods (APHA et al., 1995). COD was analyzed by the potassium dichromate close reflux method with correction for chloride interference.

The extraction of extracellular polymers (ECP) is based on the thermal extraction and ethanol precipitation method (Brown and Lester, 1980). The sludge was separated by centrifugation (2,000 g for 15 minutes), then washed and resuspended in distilled water. A portion was taken for measurement of suspended solids and the remaining part was heated at

60 800C for 1h. The extracted polymers were collected by removing the sludge by centrifugation (9,500 g for 15 minutes). The extracellular polymers in supernatant were precipitated by adding two volumes of solvent mixture (1:1 acetone and ethyl alcohol) to one volume of supernatant. It was then left overnight at 4oC. The ECP content was measured by means of suspended solid analysis. Viscosity of the mixed liquor was measured using a rotating torque cylinder. Table 3.12 listed parameters and their analytical methods used in this study.

Table 3.12 Parameters and their analytical method

Parameter Analytical method Analytical Interference Treatment Source equipment of salt pH pH meter pH meter None APHA et al., 1995 DO DO meter DO meter None APHA et al., 1995

COD Dichromate Reflux Yes Adding HgSO4 APHA et al., 1995 according to the 10:1 ratio of HgSO4: Cl. Ammonia Distillation UV-vis None APHA et al., 1995 Spectro. Nitrite Colorimetric UV-vis None APHA et al., 1995 Spectro. Nitrate Cadmium reduction UV-vis None APHA et al., 1995 Spectro.

TKN Macro-Kjeldahl Titration Yes Adding conc. H2SO4 APHA et al., 1995 Phosphate Ascorbic acid UV-vis None APHA et al., 1995 Spectro. TS Dried at 103-105oC Oven None APHA et al., 1995 VS Ignited at 550oC Furnace None APHA et al., 1995 CST Capillary time CST None APHA et al., 1995 apparatus SVI Settled sludge 1000mL None APHA et al., 1995 volume after 30 cylinder minutes Viscosity Rotating torque None cylinder EPS Thermal and None Brown and Lester, 1980 centrifugation method SS Dryed at 103-105oC Filter/Oven None APHA et al., 1995

61 Chapter 4 4 Results and Discussions

4.1 Biokinetic Study

Respirometric experiments were used in this study to evaluate biokinetic coefficients in yeast and mixed bacterial treatment of saline wastewaters containing 20, 32 and 45 g/L NaCl. Two carbon sources were investigated, namely (1) Glucose-feed wastewater (glucose as carbon source) and (2) Fish-protein-feed wastewater (fish protein extract as carbon and nitrogen sources).

4.1.1 Enrichment and Acclimation of Yeast and Mixed Bacterial Sludge

Prior to the biokinetic study, enrichment and acclimation were carried out to obtain a mixed yeast sludge and mixed bacterial sludge able to tolerate high salt contents (32 and 45 g NaCl/L). In order to propagate all wild yeasts present in the raw sediments taken from a fish sauce factory, the enrichment technique was applied prior to the acclimation. pH was adjusted to 3.5 in order to limit bacterial contamination. The enrichment was completed when the yeast concentration reached to 3000 mg/L. The enrichment and acclimation were conducted with two-litre batch reactors using the fill-and-draw operationes. a. Yeast Enrichment with Glucose-feed Wastewater

During enrichment and acclimation of the yeast culture, it was found that the color of the sludge changed from black to brown and finally to white. A change in color is a typical indication of the change in the proportion of different species in any microbiological culture. Further, microscopic observations revealed that the yeast sludge contained predominantly spherical yeast cells with few egg-shaped cells and few hyphal filaments. However round cells budding multilaterally, bipolarly and unipolarly could be easily recognized in the culture (Fig. 4.1).

Cultural characteristics were estimated by differences of yeast colonies in terms of shape, texture and margin. To evaluate cultural characteristics, isolation was done for three yeast mixtures. Isolation was carried out on the yeast-glucose-peptone agar (APHA et al., 1995). The results of isolation for yeast mixtures at different salt contents show that there were two predominant colony types. The majority of colonies were round shape, smooth surface, opaque in color and round edge. The second colony had irregular shape, rough surface and curled edges (Appendix A).

20 Pm x 500

10 Pm x 1500

Figure 4.1 Appearance of yeast cells predominantly grown in glucose-feed wastewater

62 b. Acclimation of Mixed Yeast and bacterial sludges to High Salt

The acclimation of mixed yeast and bacterial sludges to high salt was conducted with the glucose-feed and fish-protein wastewaters. In order to obtain feed wastewater composition similar to saline seafood processing wastewater using yeast treatment followed by mixed bacterial system, the initial COD of 5,000 mg/L for yeast and 1000 mg/L for mixed bacterial culture were used. When COD removal reached more than 80% (after 24 hours of aeration), the NaCl concentration was increased by 3 g/L.

Biomass

The time required for yeast acclimation was about 16 days for an initial F/M ratio of 1.12 g COD/g MLSS.day compared to 26 days for bacterial culture for an initial F/M ratio of 0.5 g COD/g MLSS.day, at 45 g salt/L. Acclimation was assumed to be complete when COD removal exceeded 80 %. Fig. 4.2 shows variation in COD removal with time at 45 g salt /L for mixed yeast culture. Similar trend curves were obtained for the mixed bacterial culture (Fig. 4.3). The asymptotic nature of the curves indicates that COD removal efficiency was stable after a certain time, which marks the completion of acclimation.

16000

12000

8000

MLS S,/L mg 4000

60

90

50

80

70 40 COD removal ( % ) MLSS Salt concentration ( g/ L NaCl) 60 COD% 30 Salt concentration

50 0 1020304050 Time ( days )

Figure 4.2 Acclimation of yeast sludge cultured with glucose at high salt contents

During the acclimation, the biomass increased 4.5 times that of the initial biomass concentration for yeast, as compared to 1.7 times for bacterial culture. Yeast biomass concentration increased 10,700 mg/L after 40 days, whereas bacterial sludge concentration increased from 2,040 to 3,400 mg/L. Differences in biomass concentration can be attributed to the application of higher volumetric loading for yeast sludge (3.3 kg COD/m3.d for yeast and 1.0 kg COD/m3.d for bacterial sludge). Therefore, a large quantity of substrate consumed was converted to yeast biomass. Even at a higher volumetric loading, the time required for yeast acclimation was about 60 % that of the bacterial sludge showing a far better adaptability of yeast at high salt. A better acclimation influences the start-up time of a wastewater treatment

63 plant, and also indicates the tolerance of the culture to occasional salt variation in the glucose- feed wastewater.

3600

3200

2800

2400

MLSS,/L mg 2000

50

90 40

80 30 ion (g/L NaCl) ion t ra t 70

COD removal ( %) 20 concen

MLSS t

60 COD% Sal Salt concentration 10

50 0 10203040 Time ( days ) Figure 4.3 Acclimation of microbial mixed culture with glucose-feed wastewater as function of salt

Organic removals

In order to estimate organic removal rates, the COD profiles of acclimatized yeast and bacteria batches were examined. The COD profile is defined as the quantity of COD varies with aeration time in a culture batch. The typical COD profiles of a mixed yeast batch at 32 g salt /L is shown in Fig 4.4. All COD profile data of yeast and bacterial batches are presented in Appendix B. Table 4.1 summarizes operating conditions and COD removal of yeast and bacterial sludges acclimatized to glucose-feed wastewater at high salt contents. Optimum HRT referred to aeration time at which COD removal exceeded 90%.

Table 4.1 Performance of mixed yeast and bacterial batches adapted to glucose-feed wastewater with high salt

Yeast batch Bacterial batch Parameters Unit Salt content Salt content 20 g/L 32 g/L 45 g/L 20 g/L 32 g/L 45 g/L Mean MLSS mg/L 8700 9500 9750 3050 3650 3200 Optimum HRT h 5 9 13 2.5 8 17 F/M g/g.d 2.77 1.39 0.96 3.27 0.84 0.44 Effluent COD mg/L 220 255 290 20 30 70 %COD % 95.6 94.9 94.2 98 97 93 COD removal rate g /g.d 2.65 1.32 0.90 3.20 0.81 0.41

64 5000 100 COD%

COD 4000 80

3000 60

COD (mg/L) 2000 40 CO D removal( % )

1000 20

0 02468101214HRTopt Time ( days ) Figure 4.4 Typical COD and COD removal profile of mixed yeast batch in glucose-feed wastewater at 32 g salt/L

The COD removal efficiency for acclimated yeast and bacterial cultures at 25, 32 and 45 g salt/L was studied with respect to substrate utilization rate U as shown in Figure 4.5. Since the F/M ratio for the two cultures differs significantly, the effect of the variation has to be taken into account for determining COD removal efficiency. COD removal efficiency is therefore expressed by Equation (4-1) (Metcalf and Eddy, 1991).

F / M * E U * 24 (4-1) HRTopt .X Where

U = Substrate utilization rate (g COD removed/g MLSS.day) F/M = Food:microorganism ratio (g COD applied/g MLSS.day) COD F / M inf * 24 HRTopt .X

CODinf = Initial COD concentration (mg/L) HRTopt = Optimum hydraulic retention time (h) X = Biomass concentration (mg/L MLSS) E = COD removal efficiency (%)

It could be observed that, while increasing the salt content, U is significantly decreased for both cultures due to salt inhibition. However, the rate of decrease is found to be much higher for bacterial culture than for yeast, indicating that the bacterial culture is much more sensitive to changes in high salt content. When salt content increased from 20 to 45 g/L, U decreased from 3.26 to 0.40 g COD/g MLSS.d for the bacterial culture, while U decreased from 2.65 to 0.88 g COD/g MLSS.d for the yeast culture.

65 4.0

Mixed bacterial sludge

Mixed yeast sludge 3.0

X Y 6.890u e0.0485 2.0 2

e ( COD/ g MLSS.d) g R = 0.978 t

1.0 X Y 25.48 u e 0.104 CO D removal ra R2 = 0.989

0.0 20 30 40 50 NaCl (g/L)

Figure 4.5 Variation in COD removal rate versus salt contents in acclimatized yeast and bacterial mixed cultures

It can be concluded that wastewater containing high salt is better for yeast culture, it may be better to opt for yeast culture while at low salt bacterial culture is preferred. The intersection point was found to be at 25 g salt/L, which indicates that below this value bacterial culture may be a better solution and vice versa.

As indicated in Section 4.1.1.b, the yeast culture is subjected to a higher F/M ratio than the mixed bacterial culture in order to obtain a comparable substrate removal efficiency. This can be considered a specific advantage of the mixed yeast culture over bacterial culture. Normally, for aerobic treatment systems, higher F/M ratio vis-à-vis higher organic loading puts greater stress on the system. This generally results in lower efficiency of substrate removal and oxygen utilization. Therefore, in practice, aerobic systems are not subject to volumetric loading exceeding 1.2 kg COD/m3.d. The normal range is 0.3 to 0.8 kg COD/m3.d, which avoids low efficiency and higher O2 requirement (because of low utilization efficiency). Thus, by allowing a higher F/M ratio on the yeast culture without sacrificing efficiency, the problem of higher organic loading on an aerobic treatment system is to some degree addressed.

However, higher organic loading allows downsizing the treatment reactors, which improves the overall economy of the system. In this study, the mixed bacterial culture was loaded at 1.0 kg COD/m3.d (corresponding to a F/M ratio of 0.50 g COD/g MLSS.d) as compared to 5.0 kg COD/m3.d (F/M ratio of 1.12 g COD/g MLSS.d) for yeast culture. This range of loadings (for yeast) is generally acceptable for anaerobic treatment systems having a low efficiencies normally in the range of 60 - 70% at best. Moreover, it has also been found (Feijoo et al., 1995) that anaerobic microorganisms are highly sensitive to salt and total inhibition could be noted for some treatment systems at a salt exceeding 20 g/L, whereas a COD removal efficiency of more than 80 % (for 45 g/L) and 90 % (for 20 g/L) can be easily obtained for yeast culture at HRT of 24 h. Thus, the yeast system is more useful than an anaerobic system at high salt, and can be considered a better substitute for anaerobic systems in terms of COD removal efficiency.

66 Protein-feed wastewater

Prior to the estimation of biokinetic constants for protein-feed wastewater, acclimation of yeast and bacteria sludges to this new substrate was necessary. Both yeast and bacterial sludges were the ones that had been adapted previously to glucose-feed wastewater at high salt contents (20, 32 and 45 g/L). This acclimation lasted two weeks. The COD removal of yeast batches at high salt contents was higher than 80% after 12 days of acclimation (Fig. 4.6). Whereas, COD removal of bacterial batches was higher than 90% after a few days. Thus, the mixed bacterial sludge was able to acclimatize faster to the substrate with high protein content. Unlike composition of glucose feed wastewater, the protein-feed wastewater contains a large complex of organics such as proteins, colloidals and polysaccharides. These substance can be slowly degraded by most yeast strains grown predominantly in the previous glucose culture (Defrance, 1993). During acclimation to protein-feed wastewater, yeast strains could be inhibited due to limitation of glucose, while other strains which are able to degrade the complex organics grow rapidly and become predominant after acclimation. In general, complex organics must be hydrolyzed by enzymes (hydrolases) before yeasts can degrade them. The type of hydrolases produced by yeasts are dependent on the species (Pelczar and Reid, 1972). This reveals that utilization of mixed yeast culture, based on a symbiotic process, for treatment of wastewater having complex composition is more efficient than pure yeast culture.

10000

8000

6000

MLS S,/L mg 4000

2000

90

80

70 COD removal ( %) Yeast sludge

60 Bacterial sludge

50 024681012141618 Time ( days )

Figure 4.6 Acclimation of yeast and bacterial sludges to fish-protein-feed wastewater containing 32 g/L salt

Sludge characteristics

The change in color of the sludge and microscopic observations strengthen the fact that acclimation of yeast and bacterial culture is completed. The yeast sludge that was fed with glucose wastewater was milky white and became dark brown to black often acclimation to protein wastewater. Microscopic observation detailed the changes by predominant yeast

67 strains when substrate was changed. The yeast sludge fed with glucose mainly contained spherical yeast cells with multilateral or bipolar budding, whereas mycelia (hypha filament) and large size egg-shaped cells with monopolar budding were predominant in the yeast mixture fed with protein wastewater (Fig. 4.7).

20 Pm x 500

a. Predominance of egg-shaped cells with monopolar budding in suspension

20 Pm x 800

b. Predominance of mycelial yeast (hypha filament) in settled sludge

Figure 4.7 Predominance of wild yeast strains in the cultures fed with fish-protein wastewater (at 32 g/L salt)

Similarly, the bacterial sludge changed in color and settling properties. Its color changed from light brown or orange to brown or dark brown when glucose-feed wastewater was altered with protein-feed wastewater. Its flocs were larger and easily trapped fine particles during settling. Therefore, supernatants of bacterial batches fed with protein wastewater were clearer than those from glucose wastewater after 30 minutes of settling. SS of supernatant and SVI in the batch fed with glucose wastewater (at 32g/L salt) were 275 mg/L and 16 mL/g, respectively, while SS and SVI in the protein-feed wastewater were 230 mg/L and 81 mL/g. The result revealed that there is a large difference in SVI but not in supernatant SS. Thus, the bacterial sludge fed with protein wastewater may have poor thickening or dewatering ability; details are discussed in Section 4.4. These can be explained by proteins in the wastewater enhancing formation of extracellular polymers (ECP) which have a great influence on bacterial floc structure, settling and dewatering ability (Dignac et al. 1998).

68 Organic removals

All COD profile data of yeast and bacterial cultures fed with protein wastewater were presented in Appendix B. Table 4.2 summarizes operating conditions and COD removal efficiency of yeast and bacteria acclimatized to protein-feed wastewater at high salt contents. Here, optimum HRT refers to aeration time at which COD removal efficiency obtained was higher 80%.

Table 4.2 Performance of mixed yeast and bacterial sludges adapted to protein-feed wastewater with high salt contents (Initial COD cof 5,000 mg/L).

Yeast Bacteria Parameters Unit Salt content Salt content 20 g/L 32 g/L 45 g/L 20 g/L 32 g/L 45 g/L Mean MLSS mg/L 6050 5810 6430 4300 3720 4120 HRT h 31 34 37 9 21 28 F/M g/g.d 0.64 0.61 0.51 0.64 0.31 0.21 CODeff mg/L 790 830 950 40 50 90 %COD % 84.2 83.4 81 96 95 91 COD removal rate (U): g /g.d + Protein ww 0.54 0.51 0.41 0.61 0.29 0.19 + Glucose ww 2.65 1.32 0.90 3.20 0.81 0.41

The COD removal rate of both acclimatized yeast and bacterial sludges were significantly reduced when glucose-feed wastewater was added protein-feed wastewater as shown in Table 4.2. However, the COD removal rates of mixed yeast sludge at higher salt (32 and 45 g/L NaCl) were still higher than those of bacterial sludge when protein wastewater was used. The difference in U of yeast sludge with salt increases was relatively minor. In addition, in order to obtain equivalent COD removal efficiency, lower F/M ratios were required at higher salt contents for both yeast and bacterial sludges. However, F/M reduction for the mixed yeast sludge was small. This enhanced the advantages of the yeast system for seafood processing wastewater having high organic strength and high salinity.

4.1.2 Evaluation and Comparison of Biokinetic Coefficients

Specific growth rates (P) were obtained through OUR measurement by respirometric method. The P values of different initial CODs (20 to 500 mg/L) at 20, 32 and 45 g salt/L were determined. Observed maximum specific growth rate (Pobs) and the half-velocity constant (KS) were determined from regression analysis.

Figures 4.8 and 4.9 show typical OUR curves of yeast and bacterial sludges fed with glucose and protein wastewater at 32 g salt/L. OUR curves for a specific COD concentration can represent maximum oxygen uptake rate of microorganisms for a given substrate, knowing its biodegradability. For example, Fig. 4.8 indicates that for glucose-feed wastewater, OURs of the mixed yeast sludge was higher than that of the bacterial sludge. Thus, yeast culture was able to degrade glucose more efficiently at 50 mg/L COD and high salinity (32 g/L salt), while the degradation ability of the mixed bacterial culture fed with protein-feed wastewater was better at COD lower than 100 mg/L. Discussion is presented in Section 4.3.2.a (Fig. 4.32). The fraction of readily biodegradable matter is represented by the area A in these figures. By comparison between the area A of the two substrates, it is noted that glucose-feed wastewater mainly contains readily biodegradable matter, whereas the fraction of readily biodegradable matter was lower for protein-feed wastewater. 69 50

40 A

30 /mg VSS.h) /mg 2

bacterial sludge 20 A yeast sludge OUR (mg O (mg OUR 10

0 0 5 10 15 20 25 30 Time ( minutes) Figure 4.8 OUR curves of mixed yeast and bacterial sludges feed with 50 mg/L COD and 32 g/L salt (glucose-feed wastewater)

30

25

20 A /mg VSS.h) /mg 2 15 bacterial sludge

10 A yeast sludge OUR (mg O (mg OUR B 5 B

0 0 20 40 60 80 100 120 Time ( days ) Figure 4.9 OUR curves of mixed yeast and bacterial sludges feed with 100 mg/L COD and 32 g/L salt (protein-feed wastewater)

A given microorganism will survive in the system if it is able to reproduce at a faster rate than the rate at which it was removed from the system. The mechanisms to remove the microorganism can involve predation or wash-out in the effluent. Therefore the growth rate is important in the biological treatment process. It is normally used to estimate the effects of toxic substances, inhibitors or overloads on performance of the process. Figures 4.10 and 4.11 show the variation in specific growth rate with COD concentration (glucose-feed wastewater) at different salt contents, for yeast and bacterial cultures, respectively. It can be seen that the specific growth rate progresses in according to the Monod’s model to reach a maximum value, then this value decreases as the salt content increases. Table 4.3 summarizes values of Pobs, KS, and Y of yeast and bacterial sludges at different salt contents for glucose and protein- feed wastewater.

70 5.0 20 g NaCl/L

32 g NaCl/L

4.0 45 g NaCl/L -1 day P 3.0

2.0

Specific G rowthRate 1.0

0.0 0 100 200 300 400 500 COD (mg/L) 20 g/L NaCl: 32 g/L NaCl: 45 g/L NaCl: S S S P 5.60 P 4.74 P 2.70 158  S 118  S 129  S R2 = 0.971 R2 = 0.975 R2 = 0.967

Figure 4.10 Variation in specific growth rate of yeast sludge as function of COD concentration at different salt contents for glucose-feed wastewater

20 g NaCl/L

8.0 32 g NaCl/L

45 g NaCl/L -1

day 6.0 P

4.0

Specific G rowthRate 2.0

0.0 0 40 80 120 160 200 COD (mg/L) 20 g/L NaCl: 32 g/L NaCl: 45 g/L NaCl: S S S P 9.95 P 2.80 P 1.15 45  S 55  S 53  S R2 = 0.965 R2 = 0.974 R2 = 0.950 Figure 4.11 Variation in specific growth rate of bacterial culture as function of COD concentration at different salt contents for glucose-feed wastewater

For both substrates, the specific growth rate of mixed yeast culture is higher than the bacterial one at high salt contents, while the opposite is observed for lower salt contents. This observation is in line with the nature of the COD removal rate (Fig. 4.5). It was also found that irrespective of the salt content, the yield constant Y of mixed yeast batch fed with glucose is lower than that of the bacterial culture, whereas there was no considerable difference between the Y constants of yeast and bacterial systems fed with protein at any salt content. This may be due to change in the predominant species or changes in the carbon assimilation metabolism as substrates change. The yield constants for both yeasts and bacteria grown on

71 protein-feed wastewater were slightly lower than for glucose-feed wastewater. The yield constant estimated by the respirometric method is considered as the maximum yield coefficent (Ymax) under certain environmental conditions such as temperature and type of wastewater. In a biological treatment system, the observed yield constant Yobs may vary from 0 and upto Ymax. The observed yield constant Yobs values depend on design of the system such as F/M ratio and sludge age (Henze et al., 1997). Therefore, unlike specific growth rate, Y constant should not be used to evaluate the effects of inhibitors or to compare performance of two systems with different operating conditions.

Table 4.3 Biokinetic coefficients of the yeast and bacterial sludges at different salt contents for glucose and protein-feed wastewaters

S Pobs YKS g /L Substrate day-1 g VSS/g COD mg COD/L NaCl Yeasts Bacteria Yeasts Bacteria Yeasts Bacteria Glucose feed wastewater 20 5.60 9.95 0.46 0.57 158 45 32 4.74 2.80 0.48 0.58 118 55 45 2.70 1.15 0.41 0.53 130 53 Protein feed wastewater 15 4.69 5.65 0.43 0.40 201 54 32 3.66 1.95 0.44 0.47 329 93 45 2.46 1.11 0.40 0.40 396 96 Candida ingens in culture using VFAs (Anciaux et 6.8-7.2 0.56 N/A al., 1989) C.utilis in culture using acetic acid 7.2-9.6 0.38 N/A Jackson and Edward, 1975 Domestic wastewater <1.0 N/A 4-8 N/A 0.35-0.50 N/A 5-30 (Henze et al., 1997)

Ks values of bacterial cultures (both substrates) at high salt contents were found to be higher than Ks of normal activated sludge. This indicates that heterotrophic aerobic microorganisms living at high salinity show lower affinity for the substrate than those at low salinity, probably because of reduced functioning and multiplication. Whereas Ks values of yeasts were found to be 3-6 times higher than those of bacteria. Thus maximum growth rate of yeast culture is only obtained at high organic substrate concentration.

Figures 4.12 and 4.13 compare maximum specific growth rate of mixed yeast and bacterial cultures with glucose and protein-feed wastewaters at different salt contents. At higher salt contents, the bacterial growth is severely inhibited, while the growth rate of yeast mixture is sustained. The inhibition effect of high salt contents on yeasts and bacteria based on the Ghose and Tyagi model is also shown. Salt inhibition constants (KI) have been calculated from the linear relationship. It was 70 g/L (80g/L) for yeast and 46 g/L (51g/L) for the bacterial culture with glucose (protein). This indicates that the inhibitory salt content is much lower for bacterial culture compared to the yeast which is in line with the previous observations. However, actual critical salt limits may be higher than the values derived, found from the Ghose and Tyagi model. This can be recognized from studies concerning osmotolerant microorganisms by Choi and Park (1999). They reported that growth of Pichia guilliermondii on Kimchi brine waste is still sustained at up to 120 g NaCl/L. Similarly, Dincher and Kargi (2000) also reported that Halobacter sp. in activated sludge culture could

72 continue removing COD even at 50 g salt/L. Therefore, the limits obtained in this study may only be used to compare the relative performance.

16.0 Bacterial sludge § I · P 15.9*¨1 ¸ Yeast sludge © 46 ¹ 12.0 R2 = 0.959 ) -1 8.0 day P § I · P 8.1*¨1 ¸ © 70 ¹ 4.0 2

Observed Specific Growth Rate Observed Specific Rate Growth R = 0.870

0.0 0 1020304050607080

Salt (g/L NaCl) Figure 4.12 Inhibition effect of salt contents on mixed yeast and bacterial cultures on glucose-feed wastewater 16.0 Bacterial sludge

Yeast sludge e t 12.0 h Ra t § I ·

) P 7.65*¨1 ¸ -1 © 49 ¹ 8.0 day R2 = 0.928 P § I · P 5.86*¨1 ¸ © 80 ¹ 4.0 R2 = 0.985 Observed SpecificGrow

0.0 0 1020304050607080 Salt (g/L NaCl) Figure 4.13 Inhibition effect of salt contents on mixed yeast and bacterial cultures on protein-feed wastewater

4.2 Parametric Study

This study focused on several operating parameters such as pH, DO, SRT and nitrogen content for the acclimatized mixed yeast and bacterial cultures. In fact, the effect of pH on bacterial and yeast systems were evaluated in terms of OUR by respirometric assays. The optimum pH values for bacterial and yeast growth under high salinity conditions were evaluated. Variation in DO and nutrient components in the yeast and bacterial batches was monitored for the two feed wastewaters (glucose and fish-protein) with 32 g/L salt. The SRT variation experiments were conducted in mixed yeast cultures using the fill-and-draw operation. Based on nutrient and COD removals, a suitable SRT value for yeast treatment was determined.

73 4.2.1 DO and pH a. DO

Figures 4.14 and 4.15 compare DO profiles of mixed yeast and bacterial cultures fed with glucose and protein wastewaters at 32 g salt/L. These figures also show that there is a link between DO of mixed liquor and remaining COD (COD profile). DO of the mixed-liquor in the yeast batch fed with glucose was low (0.7 mg/L) in the first 5 hours. DO then sharply increased to saturated value (6.3 mg/L), whereas low DO level of 0.7 mg/L in the batch fed with protein was steady for longer duration (28 h of aeration). DO then was also sharply raised to 5.6 mg/L. DO values were lower for both substrates during the first hours of aeration. This can be attributed to the presence of high initial concentration of organics (5,000 mg/L COD) after filling. The oxygen uptake rate of yeasts exceeded oxygen diffusion rate from aeration at the first hours. Based on COD profiles, DO increased to saturated value when most of the organic matters in the system was completely degraded.

5000 COD-protein-yeast

COD-glucose-yeast 6.0 4000 DO-protein-yeast DO-glucose-yeast

3000 4.0 DO (mg/L) DO COD (mg/L) 2000

2.0 1000

0 0.0 0 10203040 Time ( h)

Figure 4.14 DO and COD changes of yeast batch fed with glucose and protein wastewater at 32 g salt/L

Similarly, DO in mixed bacterial cultures for both glucose and protein wastewater was low during the first hour of aeration. It then increased sharply to the saturated concentration of 6.3 mg/L. This suggests that COD loading (influent COD of 1000 mg/L) applied to bacterial batches was not as high as for the yeast batch. Oxygen consumption rate of bacterial sludge did not exceed oxygen supply rate through aeration.

74 1000 6.0

800

4.0 600

COD-protein-bacteria DO (mg/L) DO COD (mg/L) 400 DO-protein-bacteria 2.0 COD-glucose-bacteria

200 DO-glucose-bacteria

0 0.0 0 5 10 15 20 25 30 Time ( h)

Figure 4.15 DO and COD changes of mixed bacterial batch fed with glucose and protein wastewater at salt content of 32 g/L b. pH

A comparison of pH profiles of mixed yeast and bacterial batches fed with glucose and protein wastewater (at 32 g/L salt) is presented in Figures 4.16 and 4.17. In the yeast batch fed with protein, pH increased from 3.5 to above 4.0 after 2 hours of aeration. In order to maintain pH at 3.5, 0.1N H2SO4 was added after every two or three hours. Inversely, pH of yeast fed with glucose decreased pH to 2.6 after 8 h of aeration. Then it slightly increased to 2.9. Based on the COD profile of the yeast batch fed with glucose-feed wastewater (Fig. 4.14), pH started to raise when carbon supply was limited (about 250 mg/L COD).

5.0 Adjust pH to 3.5

4.0 pH

3.0 yeast-protein

yeast-glucose

2.0 010203040 Time ( h)

Figure 4.16 pH changes of yeast culturefed with glucose and protein wastewater at 32 g salt/L

Thus, there is a significant difference in pH variation between glucose and protein-feed wastewaters. This may be attributed to difference in substrates involving carbon and nitrogen sources. The main components of glucose wastewater are the glucose as carbon source and inorganic nitrogen (ammonium sulphate) as nitrogen source, while organic acids or complex organics such as lipids and polysaccharides may be predominant carbon sources in protein-

75 feed wastewater. The amount of organic nitrogen (such as protein, amino acids) is very high in the protein-feed wastewater (690 mg /L organic-N; 26 mg/L ammonia-N).

The increase in pH for the protein wastewater has been previously observed by Lu (1983) who used yeast mixture to treat vermicelli wastewater. pH increased from 4.0 to 8.5. Arnold et al. (2000) examined silage wastewater treatment using Candida utilis and filamentous yeast Galactomycetes geotrichum. The initial pH was 3.65, rising to 8.8 after treatment. The authors postulated that the increase in pH was due to lactic acids removal, VFAs or consumption of H+ during oxidation of organic N into ammonia. Lu (1983) also suggested that the degradation of protein and release of ammonia caused increase of pH.

By contrasts, the decrease in pH for the yeast fed with glucose wastewater can be due to the production of organic acids or the use of basic compounds such as ammonia by the cells or the absorption by the medium of CO2 produced by yeasts (Thanh and Simard, 1973).

9.0

8.5

8.0

pH 7.5

7.0 Bacteria-glucose

6.5 Bacteria-protein

6.0 0 5 10 15 20 25 30 Time ( h)

Figure 4.17 pH changes of mixed bacterial batch fed with glucose and protein wastewaters at 32 g salt /L

Unlike yeast growth, variation in pH in the mixed bacterial batch was not much dependent on type of substrates. Both wastewaters had same initial pH of 7.5 and after treatment, pH of both batches were increased. The difference in pH variation between yeast and bacteria culture on glucose substrate may be explained by the difference in pathways of nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism (Vicente, et al. 1998). Yeasts liberate H+ ion during ammonia uptake. In general, bacteria produce alkalinity during ammonification and consume alkalinity in nitrification. However at high salinity, nitrification is inhibited. Nitrite and nitrate concentrations of treated wastewater in bacterial batches were lower than 2.5 mg/L N at 32 and 45 g/L salt. Fig. 4.17 shows that pH of the mixed bacteria fed with protein wastewater (8.5) was higher than that fed with glucose (7.8). This might be due to the increase of alkalinity during conversion of protein to ammonia. c. Evaluation of optimum pH

Optimum pH of mixed yeast and bacterial cultures were evaluated in terms of OUR using respirometric experiments. These were conducted with the protein-feed wastewater at 50 mg/L COD and 32 g salt /L. The bacterial and yeast sludges used in these experiments were from the biomembrane reactors operated with the protein-feed wastewater at 32 g salt/L.

76 Figures 4.18 and 4.19 show suitable range of pH values for yeast and bacteria growth in high salinity. 18

16

14

12 /gVSS.h) 2 10 Total OUR

(mgO 8 Endogenous OUR

max 6 OUR 4

2

0 2.04.06.08.010.0

pH Figure 4.18 Variation in OUR as funtion of initial pHs for mixed yeast fed with protein wastewater at 32 g salt/L 25 Total OUR

Oxidation OUR 20

/gVSS.h) 15 2 (mgO 10 max OUR 5

0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 pH Figure 4.19 Variation in OUR as function of initial pHs for mixed bacterial fed with glucose wastewater at 32 g salt/L

OUR obtained in the mixed yeast culture was highest at pH values of 5.0 - 5.5 and declined slightly as pH increased to 8.0 or decreased to 3.0. The respiration rate of yeasts was inhibited at pH 2.5 and above 9.0, whereas bacterial culture attained the highest OUR at pH range of 7.5-9.0 where OUR declined slightly when pH was increased to 9.7, or decreased to 6.3. The respiration rate of bacteria was inhibited at pH below 5.3 or pH above 10.0. Thus, the osmotolerant yeasts were able to tolerate a wider pH range than bacterial culture. Choi and Park (1999) obtained similar results for Pichia guilliermondii, an osmotolerant yeast used to treat kim chi waste brine with 80 g/L salt. Cell growth was not affected at pH ranging 4.0 to 8.0. By scanning electron micrographs, they showed that yeast cells only shrunk in size, but did not rupture at the high osmotic shock pressure or high ion strength. This appears to be a typical advantage of using yeasts to treat industrial effluents having large pH fluctuation. However, at neutral pH (6.5 - 8.5), bacteria do multiply in significant numbers. Bacterial growth should be inhibited by pH control in the yeast treatment process for the following reasons:

77 a. If the yeast biomass is used as animal feed, large numbers of bacteria or pathogens will reduce the quality of the yeast biomass product; b. Excessive bacterial growth leads to operating problems, especially membrane clogging.

Previous studies have reported that bacterial contamination could be inhibited in pH range of 3.5 - 3.8 (Peczar and Reid, 1972; Elmaleh, et al., 1966). The results of this study shows that there was not a significant difference between OUR at 3.5 and OUR at the optimum pH (5.0 - 5.5). The difference was 9%. Therefore, maintenance of pH 3.5 in the reactor cannot reduce considerably COD removal rate.

4.2.2 Nitrogen Variation in Mixed Yeast and Bacterial Cultures

Figure 4.20 shows the variation in nitrogen components in the mixed yeast cultures fed with glucose and protein wastewater. In the yeast fed with glucose wastewater, nitrogen source was an inorganic salt (NH4)2SO4 with initial concentration of 365 mg N/L (corresponding to COD:N = 100:7.2). Ammonia-nitrogen removal of 65% was obtained after eight hours. Total nitrite and nitrate-N concentration of treated wastewater was very low (around 2.0 mg N/L). This indicated that nitrification did not occur in the yeast reactor. Likewise, total nitrite and nitrate concentration was not important in the yeast batch fed with protein wastewater. The initial total nitrogen of the protein-feed wastewater was 790 mg N/L mostly organic-N (745 mg/L organic-N). The total-N concentration was reduced to 446 mg/L after 32 h, whereas ammonia-N increased from 45 mg/L to 420 mg/L. In comparison to the yeast culture with glucose-feed wastewater, ammonia content and ammonia removal was dependent to the availability of nitrogen sources (e.g. proteins, amino acids or ammonium salt) in the feed wastewater and the BOD:N ratio. Due to the high BOD:N ratio (100:18) of the protein-feed wastewater, the total nitrogen concentration of treated wastewater was still high and mainly in the form of ammonia. If reuse of yeast biomass for single-cell-protein production vis-à-vis further removal of nitrogen is considered, the combination with carbohydrate-rich wastes (lack of nitrogen) such as molasses or pulp and paper wastewater would be possible.

Moreover, the increase in ammonia may be related to the increase in the accumulated acid volume used for adjusting pH to 3.5 as shown in Fig. 4.21. The link between H+ ion consumption and ammonia release was discussed in Section 4.2. The amount of acid consumed per gram ammonia-N released was about 26 meq H+.

78 800 18 Total N (protein ww) Accumulated acid volume

NH4-N (protein ww) NH4-N (glucose ww) 16 600 14 volume (mL) volume 4 SO 2 400 12 N (mg /L N) /L N (mg 10 200 8 Accumulated 0.1N H

0 6 0 10203040 Time ( hours ) Figure 4.20 Variation in nitrogen components as funtion of time in the mixed yeast at 32 g salt/L NaCl (Nitrite and nitrate concentration of both feed wastewaters were not dectected)

The total-N removal obtained in the batch fed with protein wastewater after 32 h was 45%. Because nitrification did not occur in the yeast treatment, all nitrogen removed was uptaken in the yeast sludge. Based on biomass produced in the batches and the amount of nitrogen removed, the nitrogen content of the yeast sludge was estimated to be about 7.5% of dried solids for glucose wastewater, and 13.8% for protein wastewater. The high nitrogen content of the yeast sludge fed with protein wastewater can be attributed to precipitation of protein at low pH (3.5). This can be confirmed through the sudden decrease in total nitrogen concentration during the first hour of aeration (Fig. 4.20). The nitrogen uptake ability of yeasts will be further discussed in Section 4.4.

200

160

120

Total-N (protein ww)

Ammonia (protein ww) 80 NO2+NO3 (protein ww) Nitrogen (mg /L N) /L (mg Nitrogen Ammonia (glucose ww)

40 NO2+NO3 (glucose ww) < 1.5 mg/L N

0 0102030 Time ( hours ) Figure 4.21 Variation in nitrogen components vs. time in the mixed bacterial culture at 32 g salt/L NaCl

In the bacterial culture fed with protein wastewater, 24% of total nitrogen was removed at HRT of 21 hrs. Fig. 4.21 shows that the oxidation of organic-N (ammonification) results in an increase from 49 to 137 mg N/L after 21 hrs, while in the culture fed with glucose wastewater, the initial ammonia concentration of 53 mg N/L was reduced to 14 mg/L (about 72% N removal) after 9 hrs. Thus the variation of ammonia during the bacterial cultures was similar to that of mixed yeast cultures. Nitrite+nitrate-N concentration was lower than 2.5 79 mg/L and 1.2 mg/L for the cultures fed with protein and glucose, respectively. Whereas nitrite and nitrate-N (fed with protein) at 15 g salt/L was 12.3 mg/L after 21 hours (Appendix A). Thus nitrification was inhibited at 32 g salt/L. Lower reduction at higher salt (32g/L) is consistent with previous studies (Dincer and Kargi, 1999; Panswad and Anan, 1999). In fact, Dincer and Kargi (1999) reported that nitrification efficiency dropped quite sharply at salt content above 3%. Depending on nitrogen balance, nitrogen uptake in the mixed bacterial sludge for both feed wastewaters was reached to 4.5 % of dry solids. This result will be confirmed by nutrient analysis of sludge in the sludge characterization study.

4.2.3 Effect of SRT on COD and Nitrogen Removal

The optimum SRT was evaluated on the basis of COD and nitrogen removal. Five mixed yeast batch experiments corresponding to SRT of 5, 7, 10, 20 and 45 days were conducted at the same organic loading of 5 kg COD/m3.d (HRT of 24 h) for 25 days. The seed sludge used was taken from the yeast membrane bioreactor (YMBR) operated with SRT of 50 days. As the degradation of protein and the release of ammonia caused an increase in pH during the culture, pH was adjusted to 3.5 - 4.0 using 0.1 N H2SO4 during aeration. Figure 4.22 shows that all the runs reached steady state after 15 days.

The mixed yeast culture run at higher SRT reached higher biomass concentration. At the VLR of 5.0 kg COD/m3.d, when SRT increased from 5 d to 45 d, the MLSS increased from 2,400 to 10,300 mg/L. Thus a long SRT implies low F/M ratio. This resulted in high organic removal efficiency at long SRT. The results are shown in Table 4.4 and Fig 4.23. COD removal increased from 43 to 85% when SRT increased from 5 d to 45 d. Furthermore, the sludge production (yield constant) was also minimized at long SRT (Table 4.4). Therefore, SRT has a mutual relationship with the net specific growth rate of the mixed yeast sludge. The net specific growth rate (P’) was the difference between the specific growth rate (P) and the endogenous decay rate (kd), which includes endogenous respiration, death and subsequent lysis. At long SRT, the system is operated in the endogenous phase. Thus kd will have a significant effect on the net amount of biomass produced. This means that large fraction of the substrate removal is oxidized for energy required for cell maintenance rather than for synthesis of new cells.

12000

10000

8000

6000 MLSS ( mg/ L)

4000

2000 0 5 10 15 20 25

Time ( days )

45 d 10 d 5 d

20 d 7 d

Figure 4.22 Variation in MLSS as funtion of SRT 80 Table 4.4 Variation of parameters during various SRTs (Initial COD of 5000 mg/L)

Biomass COD N Mean production Yield SRT CODeff TKNeff (*) removal removal MLSS (g SS/g COD (d) (mg/L) (mg/L) rate (%) (%) (mg/L) (mg SS removed) produced/d) 5 2850 524 43 7.3 3245 657 0.305 7 1950 515 61 8.9 5351 765 0.251 10 1050 509 79 9.8 8150 810 0.205 20 900 535 82 5.2 9455 475 0.116 45 950 550 85 2.7 10335 228 0.053 (*) Influent TKN concentration was 565 mg/L N. The data are average values of at least three steady state batches.

Figure 4.23 indicates that if the highest COD removal was obtained at SRT of 45 d, maximum nitrogen removal was achieved at SRT of 10 d. Uptake of nitrogen by the biomass is a major nitrogen reduction mechanism in yeast culture as discussed in Section 4.2.2. Hence, the nitrogen removal efficiency will depend on the biomass production rate. Table 4.4 shows that the highest biomass production was obtained at SRT of 10 d. It can be suggested that selection of optimum SRT should be based on the purpose of yeast application. SRT of 10 d is optimum for single-cell-protein production, while longer SRT is suitable for enhancing treatment efficiency.

100 14000

12000 80 )

  10000 60

8000 COD removal 40 MLSS ( mg/ L) Nitrogen removal 6000

Removal efficiency Removal MLSS 20 4000

0 2000 4 5 6 7 8 9 10 20 30 40 50

SRT ( days) Figure 4.23 Variation in COD, nitrogen removal and MLSS in funtion of SRT in mixed yeast culture at VLR of 5 kg COD/m3.d (32 g salt/L)

4.3 Biomembrane Study

The objective of this study was to examine the potential for development of membrane bioreactor systems using wild salt-tolerant yeast mixture and bacteria mixture to treat high salinity wastewater (32 g/L NaCl). Based on COD of the feed-wastewater, this study was divided into two phases, namely (1) high COD loading with 5000 mg COD/L and (2) low COD loading with 1000 mg COD/L. The process efficiency was investigated in terms of organic removal and membrane filtration flux for various volumetric loading rates, F/M ratio and SRT values.

81 Two membrane modules having 0.1 Pm pore size and 0.42 m2 area were used for YMBR and BMBR. When the pressure reached a value of 70 kPa, the membrane was removed from the reactor, and chemical cleaning was conducted. During the chemical cleaning, the external sludge cake layer was initially washed with water and the attached biomass (on the membrane surface) was collected and analysed. After removal of the sludge cake, the membrane was washed with tap water and backwashed with 2.5% sodium hydroxide for 15 minutes followed by 1% nitric acid for 5 minutes before reuse.

After every chemical cleaning, the initial membrane resistance was measured, to verify the cleaning efficiency. The initial Rm were determined by filtering the tap water through a new or chemically cleaned membrane. Here, linear flux variation with applied pressure was obtained. This variation for two fresh membrane modules is presented in Fig. 4.24. The 11 obtained initial Rm of two cleaned membrane modules were quasi equivalent (7.11x10 and 7.18x1011 m-1).

5 5

4 4

3 3 P ( kPa) 2 (P kPa) 2

1 1 Y = 0.172 * X - 0.1372 Y = 0.174* X - 0.1472

0 0 0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 J (L/m2.h) J (L/m2.h) a. First membrane b. Second membrane Figure 4.24 Variation in flux as function of membrane transmembrane pressure (Viscosity of water at 26oC = 8.70 x 10-4 kg/m.sec)

4.3.1 High COD loading

In this phase, fish-protein wastewater with 5,000 mg COD/L and 32 g salt/L was used. Two experimental set-ups were investigated: (1) Yeast pretreatment followed by BMBR and (2) YMBR. These were run at different HRTs at SRT of 50 d (as presented in Fig. 3.5). a. Organic removal

The performance of YMBM and BMBR systems for various volumetric loading are shown in Figures 4.25 and 4.26. Here it can be note that after 11 days of acclimation (stage I), yeast biomass increased from 3,700 to 14,500 mg/L at a volumetric loading of 5.0 kg COD/m3.d (average HRT of 24 h). COD and BOD removal obtained were above 76% and 85%, respectively.

In contrast, the BMBR system which involved the yeast reactor (YR) and BMBR reached the steady state after 11 days. The YR reached the mean biomass of 6,500 mg/L and COD removal of 76% at SRT of 15 days and average HRT of 36 h. The bacterial biomass in BMBR increased from 4,000 to 11,000 mg/L at a volumetric loading of 2.1 kg COD/m3.d (HRT of 13.7 h). COD and BOD removal obtained was above 85% and 97%, respectively. These results demonstrate the rapid adaptability of the mixed yeast and bacterial cultures to degrade the high salinity-organic wastewater.

82 40 80

30 60

20 40 Mean HRT Mean HRT ( h) Transmembrane Pressure 20

10 Transmembrane ( kPa)

6000 20000

Stage I Stage II Stage III Stage IV Stage V Stage VI

16000

4000 12000

CO D ( mg/ L) 8000 MLSS ( mg/ L) 2000

Effluent COD 4000 Influent COD

MLSS 0 0 1020304050607080 Time (days) Figure 4.25 Variation in COD, biomass and transmembrane pressure in the YMBR as function of volumetric loading

In the YMBR, as the loading rate was progressively increased through different stages (3.4 - 16.3 kg/m3.d), the COD removal efficiency decreased from 85 to 60%, with the COD in the effluent increasing from 870 to 2,300 mg/L. For the BMBR process, when the VLR was increased from 2.1 to 7.9 kg COD/m3.d (F/M of 0.08 - 0.41), COD removal efficiency decreased from 91 to 76 % (Fig. 4.7). Effluent BOD5 of the BMBR ranged from 45 - 60 mg/L at low F/M ratio. Thus, the low BOD5:COD ratio (0.12 - 0.17) indicates that BMBR effluent also contains a high proportion of non-degradable organic compounds due to the presence of these products from yeast pretreatment.

YMBR could attain a COD removal efficiency higher than 60% at VLR ranging from 5 - 15 kg COD/m3.d as shown in Figure 4.27. These VLRs are generally within the acceptable range for anaerobic treatment systems, which normally have low efficiency in the range of 60 - 70%. Moreover, it has also been found (Feijoo et al. 1995) that anaerobic microorganisms are highly sensitive to salt content and total inhibition could be noted for some treatment systems at a salt content above 20 g/L. Thus the yeast system is more useful than an anaerobic system at a high salt content, and can be considered a better substitute for anaerobic systems in terms of COD removal efficiency.

In addition, YMBR attained a lower COD removal rate at F/M ratios lower than 0.34 g/g.d (the corresponding average VLRs less than 7 kg/m3.d), compared to BMBR as shown in Fig.4.28. However, YMBR achieved higher specific COD removal rate at F/M ratios higher than 0.34 g/g.d. Thus, it can be concluded that the YMBR is subjected to a higher F/M ratio and higher VLR than the BMBR to obtain a comparable COD removal efficiency. This can be considered as an advantage for the yeast sludge compared to bacterial sludge.

83 20 Mean HRT 80 Pressure 15 60

10 40 Mean HRT ( h) 5 20 Transmembrane Pressure ( Pressure kPa) Transmembrane 1600 Stage I Stage II Stage IV Stage V Stage VI

Stage III 30000 1200 Stage VII

20000 800 CO D ( mg/ L) MLSS ( mg/ L) Effluent COD

400 Influent COD 10000

MLSS

0 0 0 102030405060708090

Time ( days ) Figure 4.26 Variation in COD, biomass and transmembrane pressure in the BMBR as function of volumetric loading

The mean biomass concentration of the yeast reactor was 6,500 mg/L after steady state. 3 77% of BOD5 removal was achieved at VLR of 2.6 - 3.1 kg BOD5/m .d and F/M ratio of 0.39 - 0.47 d-1, while the Yeast Cycle System (YCS) for seafood processing wastewater treatment 3 (with 8 g salt/L) reached 97% BOD removal at higher VLR (4.5-10.4 kg BOD5/m .d) and higher F/M ratio (0.6 - 1.0 d-1) (Nishihara ESRC Ltd., 2001). This may be due to yeast ability to grow in relative low salinity environment with higher specific degradation rate. Similar results were found in the biokenetic study (Section 4.1). In comparison to the yeast reactor, the YMBR that was run at higher biomass concentration (11,000 mg SS/L) enables the 3 enhancement of volumetric loading to 5.0 kg BOD5/m .d with higher BOD removal efficiency (81%).

Table 4.5 compares operating conditions between YMBR, BMBR and few yeast treatments, MBR process treating different wastewaters. Krauth and Staab (1993) found that using BMBR for treatment of vegetable canning wastewater could achieve COD removal efficiency exceeding 99% at high F/M ratio (0.5 g/g.d) and high VLR (5.4 kg/m3.d). BMBR could also efficiently treat oily wastewater at mean F/M ratio of 0.7 g/g.d and VLR between 8.6 and 12.9 kg/m3.d (Scholz and Fuchs, 2000). Whereas the BMBR in this experiment can only be subjected to lower VLR (3.4 - 5.0 kg/m3.d) and lower F/M ratio (0.1 - 0.3 g/g.d) to obtain a comparable COD efficiency. It is important to note that all the above mentioned BMBR systems were operated with salt contents lower than 1.0 g/L NaCl. However, this current work was carried out at 32 g/L, the high salinity could be the major cause of the drop in specific organic removal rate of bacterial sludge. Thus high salinity also reduces the specific organic removal rate of bacterial sludge. However, compared to conventional activated sludge systems for low salinity wastewater treatment (maximum VLR of 1.2 kg/m3.d), the BMBR could be operated efficiently at higher VLRs (3.4 - 6.0 kg/m3.d).

84 Table 4.5 Operating parameters of the YMBR, BMBR, some yeast treatments, MBR processes treating different wastewaters and conventional AS system

Parameter Yeast Bacterial membrane bioreactor process Conventional AS Protein Seafood Protein Vegetable Oily Fermentation Domestic Wastewater extract Vermicelli Glucose processing extract canning wastewater wastewater wastewater YCS MBR shaking MBR MBR with MBR with Continuous Completed Process (Continuous MBR with UF completed with MF completed culture with MF MF MF mixing mixing mixing) Mixture of Yeast Yeast Microorganisms 10 yeast AS AS AS AS AS AS mixture mixture strains VLR, kgCOD/m3.d 4.9-6.5 4.5 – 10.4(*) 1.03 3.4-6.0 5.4 8.6-12.9 1.73 - 0.8-1.9 0.3-0.6 F/M ratio, g/g.d 0.4 0.6 – 1.0 (*) 0.5 0.1-0.3 0.5 0.6-0.8 - 0.2-0.6 (0.8-2.1) MLSS, g/L 15 8 – 10 2.6 16-20 11 15-25 10 2.5-4.0 COD removal, % 86-91 97 92 91-97 99 97 92 99 99 pH 3.5-3.8 4.3 –5.2 3.0-4.0 7.5-8.0 >6.5 7.0-7.8 6.8-7.2 6.5-8.5 DO, mg/L 1.5 0.5-0.9 2.1 6.3 - 2.0-3.5 > 2.0 t 2.0 30 Salt, g/L 32 8 <1 32 <1 <1 <1 <1 (salt free) Reference This study Nishihara ESRC, Hu (1989) This study Krauth and Scholz and Lu et al. (1999) Hamoda and Metcalf and Ltd. (2001) Staab (1993) Fuchs (2000) Al-Attar Eddy (1991) (1995)

3 Note: (*) : kg BOD5/m .d MBR : Membrane Bioreactor MF : Microfiltration UF : Ultrafiltration YCS : Yeast Cycle System AS : Activated sludge

85 This can be explained by high sludge concentration and high substrate removal rate. Similar results have been reported by Manem and Sanderson (1996), who found that VLR for dairy wastewater was six times greater than for conventional activated sludge process, although the biomass concentration was only twice as high. Moreover, the effluent suspended solids of both membrane reactors were less than 5 mg/L and was almost constant throughout all experiments. 100

YMBR 90 BMBR 80

70

60

50 CO D removal( %) YMBR: Y = -0.044*X2-1.045*X+88.0 40 R2 = 0.929 BMBR: Y = -0.572*X2 + 3.568*X + 82.7 30 R2 = 0.836

20 0 2 4 6 8 1012141618202224 Volumetric Loading (kg COD/m3.d) Figure 4.27 Variation in COD removal in function of volumetric loading rate

1.0

0.8

0.6 e (e COD/ g MLSS.d) g t 0.4 YMBR: Y = -0.337*X2 + 1.156*X– 0.1041*X R2 = 0.951 BMBR: Y = -1.285*X2 + 1.331*X - 0.0323 R2 = 0.938 0.2 BMBR CO D removalra YMBR

0.0 0.0 0.5 1.0 1.5 2.0 2.5 F/M ratio ( d-1) Figure 4.28 Variation in COD removal rate in function of F/M ratio (initial COD = 5,000 mg/L) b. Transmembrane pressure and membrane clogging

Variation in transmembrane pressure in YMBR and BMBR at different operating stages is shown in Figures 4.25 and 4.26. The pressure ('P) in YMBR was almost constant throughout the various stages of VLR for a total duration of 72 days. It then increased rapidly after the 76th day (63 kPa), indicating rapid membrane clogging. This may be due to the increase in high filtration flux (89.6 L/d.m2), corresponding to HRT of 5 h in the last stage. However, when YMBR was run at short HRT (5h), uncompleted biodegradation by yeast results in high soluble COD and accumulation of fine particles (from the influent) retained in the reactor which may cause a rapid fouling rate. The high soluble COD and fine particles in the reactor could increase the filtration resistance (Manem and Sanderson, 1996). Whereas 'P in BMBR sharply increased from 2 to 60 kPa after 12d, 6 d and 2 d at hydraulic retention time

86 (HRT) of 14h, 9 h and 4h, with average biomass concentrations of 6.1, 15 and 20 g MLSS/L in stage I, II and III, respectively.

Values of different parameters during YMBR and BMBR filtration cycle in both phases are presented in Table 4.7. As noted, increasing biomass concentration promotes the membrane clogging, and difference between bacterial and yeast sludge results in different filtration performances. In fact, characteristics of yeast mixture in the YMBR could prolong the filtration cycle period. These characteristics are responsible for reducing membrane clogging rate and can result in large yeast cells, low operating pH, poor adhesion capacity, inhibiting biofilm formation, low net negative surface charge, low viscosity and low production of the adhesive extracellular polymers (ECP) that play an important role in floc or biofilm formation. These characteristics will be discussed in the next section (low COD loading) and in the sludge characteristics study (Section 4.4).

4.3.2 Low COD loading

Fish-protein wastewater with 1,000 mg COD/L and 32 g salt/L was used in this phase. Two experiments were conducted: (1) YMBR and (2) BMBR. The objective of this phase was to obtain a comparative evaluation of treatment performance of YMBR and BMBR at different HRTs and SRTs of 10 and 50 days (as presented in Fig. 3.6). a. COD removals

Figures 4.29 and 4.30 present the overall performance of YMBR and BMBR process for various HRTs in this phase. The mean influent COD concentration was maintained at 1,000 mg/L, and the VLR was increased from 2.7 - 6.5 kg COD/m3.d with a corresponding decrease in HRT from 9 to 5 hours. The MLSS concentration for SRT of 10 days ranged from 4,500 - 5,100 mg/L and 5200 - 5500 mg/L for yeast and bacterial membrane reactors respectively. By contrast, when the SRT was increased to 50 days, the MLSS concentration increased to within the range of 13,600 - 15,200 mg/L in the YMBR and 15,200 - 16,300 mg/L in the BMBR. In all experiments, the DO was maintained above 2.0 mg/L with salt content of 32 g/L. The effect of changing VLR (vis-à-vis HRT) on COD removal at SRT of 10 days and 50 days is shown in Fig. 4.31.

It was observed that at SRT of 10 days, the COD removal efficiency of the YMBR remained low (about 76%) at lower HRTs (5h), but increased to 94% with the increase in HRT (> 8 h). Whereas for the BMBR, the COD removal efficiency remained constant within the range of 92 - 97% when HRT ranged from 5 to 8 h. Thus the COD removal efficiency of the YMBR is lower than that of the BMBR at short HRTs, but converged at longer HRTs. In general, the removal efficiency of a biological system increases with HRT (until a certain limit), this was also observed for both systems.

87 10

9 Mean HRT 60 Tran.. pressure 8

7 40

6

Mean HRT ( h) 5 20

4 Transmembrane Pressure ( Pressure kPa) Transmembrane 3 20000 1200

1000 16000

800 SRT = 10 d 12000 600 Effluent COD CO D ( mg/ L) Influent COD MLSS ( mg/ L) 8000 400 MLSS SRT = 50 d 200 4000

0 0 102030405060708090

Time ( days )

Figure 4.29 Variation in COD, biomass and transmembrane pressure in the YMBR as function of volumetric loading

However, due to lower MLSS vis-à-vis the higher F/M ratio (1.02 g/g.d) in the YMBR or possibly lower specific growth rate of yeast, the efficiency was low at lower HRT. Indeed, the difference in the specific growth rates of yeast and bacteria at 32 g salt/L can be found in the biokinetic study. Figure 4.32 reveals that although yeasts had higher maximum specific growth rate (Pmax) at 32 g salt/L. Its specific growth rate (P) at low substrate concentrations (less than 180 mg/L) was lower than that of bacteria. Thus, the yeast growth was more inhibited at low COD in the membrane reactor.

Higher HRT vis-à-vis low F/M ratio (0.5 d-1) enabled better conversion of organic matters with higher yeast mass available. The MLSS concentration in BMBR was relatively high at SRT of 10 d. This might be due to higher specific growth rate of bacteria in a substrate-limiting condition (COD < 200 mg/L) as shown in Fig. 4.9. Therefore, the COD removal efficiency of BMBR remained high through out HRTs. A peak efficiency of 97% was obtained for BMBR at a HRT of 7 - 8 hours, which represents the best range of operating conditions.

88 9 100

8 80

7 60 6 40

Mean HRT ( HRT Mean h) 5

20 4 Mean HRT Transmembrane Pressure ( Pressure KPa) Transmembrane Trans.Pressure 3 20000 1200

16000

800 12000 Effluent COD

SRT = 10 d Influent COD CO D ( mg/ L) MLSS ( mg/ L) MLSS 8000 400 SRT = 50 d

4000

0 0 20406080 Time ( days )

Figure 4.30 Variation in COD, biomass and transmembrane pressure in the BMBR as function of volumetric loading 100

90

80 YMBR: Y = -0.683 * X2 + 10. 824 * X + 53.67 R2 = 0.885 70 BMBR: Y = -0.502 * X2 + 7.815*X + 67.01 R2 = 0.701 COD removal (%) removal COD 60 SRT of 50 d YMBR BMBR 100

90

80 YMBR: Y = -1.491 * X2 + 26.0218 * X - 18.294 R2 = 0.885 70 BMBR: Y = -1.149 * X2 + 16.744*X + 36.636 R2 = 0.767 CO D removal( %) YMBR 60 SRT of 10 d BMBR

50 4.05.06.07.08.09.010.0

HRT (h)

Figure 4.31 Variation in COD removal as function of HRTs in YMBR and BMBR

89 2.50 )

-1 2.00 day P 1.50

1.00

0.50 Specific Growth Rate Bacterial sludge

Mixed yeast sludge

0.00 0 50 100 150 200 250 300 350 400 450 500

COD (mg/L)

Figure 4.32 Variation in specific growth rate of yeast and bacteria at 32 g salt/L in function of COD

At SRT of 50 d, there was no significant difference between the COD removal efficiency of YMBR (86-91%), and BMBR (91–93%) probably due to the lower F/M ratio (0.35 - 0.40) in both reactors. As mentioned earlier, yeast growth is limited at low substrate concentration. Thus, this result shows that maintenance of high MLSS (long SRT) in the MBR can significantly enhance treatment efficiency for substrate limited growth. Moreover, there was no appreciable change in the COD removal efficiency in the transition phase (Table 4.6). In this phase, low F/M ratios were maintained within the range of 0.35 - 0.55 by controlling high HRT. High efficiency (95%) could be obtained at these low F/M ratios for both YMBR and BMBR.

However, a conventional activated sludge system can be operated at a maximum VLR of 1.2 kg/m3.d and F/M of 0.6 d-1, and degradation rates reduced considerably with an increase in salinity. Therefore, this high salinity wastewater should be treated at lower F/M ratios (Kargi and Dincer, 2000). Three-fold-lower F/M ratios were applied in conventional activated sludge at 30 g salt/L compared to those applied in salt-free wastewater (at the same SRT) in order to obtain equivalent substrate removal.

In comparison, a comparable performance is obtained from the membrane bioreactors at a very high salinity and VLR (3.0 - 5.0 kg/m3.d). Similar results have been reported by Manem and Sanderson (1996) who found that a six-fold higher VLR could be applied for dairy wastewater compared to conventional activated sludge system without deteriorating performance. The results show that both YMBR and BMBR can be effectively used to treat high salinity wastewater such as pickling and seafood processing wastewater to conform to effluent standards of COD lower than 120 mg/L and BOD lower 20 mg/L.

90 Table 4.6 Operating parameters and performance of YMBR and BMBR in high COD loading phase

YMBR BMBR SRT VLR Mean MLSS F/M COD Effluent Effluent SRT VLR Mean MLSS F/M COD Effluent Effluent (days) (kg HRT (mg/L) (g/g.d) removal COD BOD (days) (kg HRT (mg/L) (g/g.d) removal COD BOD COD/m3.d) (h) (%) (mg/L) (mg/L) COD/m3.d) (h) (%) (mg/L) (mg/L) 10 2.66 8.8 5050 0.49 94 50 10 10 2.97 8.1 4800 0.38 97 25 < 5 10 2.95 7.7 5030 0.56 94 50 15 10 3.57 6.3 4600 0.78 97 25 < 5 10 3.66 6.1 4440 0.83 84 150 90 10 4.30 5.2 5730 0.76 92 70 30 10 4.59 5.0 4530 1.02 76 230 150 10-50(*) 3.63 7.1 6430 0.57 97 30 5 (*) 10-50 3.58 7.2 5300 0.58 96 45 15 10-50(*) 4.08 6.0 10600 0.39 95 55 15 10-50(*) 4.28 6.1 11500 0.37 95 50 15 50 5.56 4.7 13100 0.43 93 70 30 50 4.93 5.3 14500 0.35 91 90 40 50 6.35 4.0 16300 0.39 91 100 55 50 6.55 4.0 15000 0.44 86 150 90 (*) Transition phase

91 b. Membrane clogging

Membrane systems are often subjected to clogging, and this poses serious problems for operation and maintenance. In order to investigate membrane clogging, experiments were carried out in a continuous operational mode. Figures 4.29 and 4.30 show variations in transmembrane pressure with time for YMBR and BMBR. The trend of pressure variation in the YMBR and BMBR was similar to that in the high COD loading. Table 4.7 shows the mean flux, transmembrane pressure and accumulated permeate volume during the filtration cycle of the BMBR and the YMBR in both phases. In the high COD loading, it was observed that the transmembrane pressure ('P) of the YMBR remained almost constant for approximately 72 days before rising sharply. Whereas, the 'P increased sharply after 32 days for the low COD loading in which the mean flux was three times higher than that in the high COD loading. Total operating time (before 'P reaching to 70 kPa) for a SRT of 10 days is higher than 50 days. For YMBR, there was no significant difference in the volume of the permeate collected, although mean MLSS concentration at SRT of 50 days was 2.5 times that at SRT of 10 days. This indicates that the clogging rate of the membrane is not entirely dependent on the concentration of MLSS.

Table 4.7 Values of different parameters during YMBR and BMBR filtration cycle

SRT Reactor Influent Mean Mean flux Accumulated Filtration (days) COD MLSS (L/m2.d) permeate cycle (mg/L) (L/cycle) (d) High COD loading: 50 YMBR 5,000 11000 34 2550 76 BMBR 1,200 20000 60 63 1.0

Low COD loading: 10 YMBR 1,000 4,500 95 3,100 38 BMBR 1,000 4,600 91 320 3.5 50 YMBR 1,000 11,000 98 2,910 32 BMBR 1,000 10,600 95 120 1.5

Membrane clogging in the BMBR was much more severe than in the YMBR. The average filtration time for the BMBR, at SRT of 10 days was 3.5 days. This decreased to 1.5 days for SRT of 50 days, requiring frequent membrane washing. The rapid development of biofilm for BMBR was in relation to the MLSS concentration in the reactor. At higher MLSS concentrations (10,600 mg/L) for 50-day SRT, the rate of clogging was much higher than in the YMBR. The difference in the performance between both YMBR and the BMBR is probably due to the mechanism by which the biofilm develops on the membrane surface.

For bacterial system, biofilm develops by two mechanisms; first, by attachment of cells on the membrane surface, which promotes further growth of bacteria; secondly by capturing more cells (from the mixed liquor) in the already developed matrix formed by the first process. The formation of biofilm on the membrane surface could be related to the production of adhesive extracellular polymers (ECP) by bacteria. ECP is partly soluble in water and goes into a colloidal suspension after production, which results in ECP accumulation in the mixed liquor. During filtration, the macromolecular ECP compounds accumulate on the membrane surface, and play the key role of binding the cells on the membrane surface and entrapping larger organic particles in the slimy matrix. The availability of organic substance in the ECP matrix promotes growth of new bacteria. This is further aided by the entrapment of more cells

92 from the mixed liquor, which seriously impairs membrane performance and module life. Thus, at higher concentration of microorganisms in the mixed liquor, production of ECP increases and biofilm develops at a much higher rate, which leads to rapid development of transmembrane pressure.

The mechanism of biofilm development in the YMBR is different to that in the BMBR. In the YMBR the yeasts attached physically to the membrane surface during filtration instead of getting trapped in a matrix. Though ECP is produced in the YMBR, the quantity is much less than that of the BMBR where a dense gel matrix is formed. The yeast cells are attached together by physical interwinding of mycelia or pseudomycelia (Nishihara ESRC Ltd., 2001). In addition larger yeast cells (2.5 - 3.9 Pm) in the YMBR also forms a secondary layer on the membrane surface that acts like a second barrier to fouling particles and aggregates (Guell et al. 1998). However, with increase in thickness of the cake, it gets heavier and starts sloughing off by uplifting air flow or air-backwash, as soon as the cake is unable to sustain its own weight. Thus the cake thickness cannot enlarge indefinitely and thus reduce the problem of frequent membrane clogging. Mechanisms of flux enhancement by yeast sludge are shown in Fig. 4.33.

Hydrogel Partial detachment (high resistance)

biofilm (ECP matrix)

membrane

Porous cake Complete detachment (secondary filter layer)

Yeast cells

Air bubble (from aeration) Filtration Backwash Figure 4.33 Possible mechanisms for flux enhancement by yeast cells

The membrane clogging depends upon the MLSS concentration of bacteria in the BMBR, but to a much lesser extent for YMBR, primarily due to the difference in the mechanisms of clogging. Due to this difference between BMBR and YMBR, a prolonged filtration cycle (about 10 times higher compared to BMBR) could be obtained for the YMBR without much problem with membrane clogging.

4.4 Sludge Characterization Study

In the membrane bioreactor system, fouling problem can be linked to the sludge characteristics as discussed in Section 4.3.2.b. In order to investigate the difference in fouling phenomenon in both YMBR and BMBR, sludge characterization was carried out. Series of yeast and bacterial cultures were run at various salt contents. The sludge was examined for ECP content, dewatering property (CST), viscosity as well as sludge settleability (SVI). These results were also compared with sludge obtained from YMBR and BMBR systems operated at 32 g salt/L. Furthermore, in both these bioreactors, prior to chemical cleaning, the characteristics of the sludge cake formed on the membrane surface was also analyzed. 93 4.4.1 Culture Study

Results of ECP, CST and viscosity of the mixed yeast and bacterial sludges at different salt contents are presented in Table 4.8 and Figure 4.34. ECP content in the mixed yeast sludge was very low at all salt contents compared to that in bacterial sludge. While the ECP concentration of bacterial sludge rises considerably with salt content. Similar phenomena have been reported for on flocculation of marine bacteria (Watanabe et al., 1998). They suggested that the ECPs did not originate from autolysis, but seem to be excreted by living marine bacterial cells. Unlike ECP production of bacteria living in salt-free water, which only occurs during the stationary phase, marine bacteria can produce large amount of ECP in the exponential growth phase (Flemming and Wingender, 2001). This leads to significant accumulation of ECP in a high salinity environment. In addition, due to alteration of genetic structure under high osmotic stress by NaCl concentration, bacteria can synthesize more specific proteins that may contribute to increase in the ECP production (Vijaranakul et al., 1997). Eikelboom (2000) found that Zoogloea bulking problems were due to high production of ECP in the bacterial flocs. This phenomenon could explain the acceleration of biofouling problem in BMBR.

In contrast, the production of ECP in yeasts is much lower and its concentration remained low. Reeslev et al. (1996) reported that Aureobasidium pullulans, a mycelial yeast, can only synthesized ECP when growth was nitrogen-limited while no ECP was produced when the culture was carbon-substrate-limited. The feed wastewater was rich nitrogen (COD:N = 100:15), and therefore growth of yeast is carbon-substrate-limited. This may result in less ECP production, saving it from frequent clogging.

Table 4.8 Yeast and bacterial sludges characterization

Salt MLSS SS (*) SVI ECP CST Viscosity Sludge (g/L) (mg/L) (mg/L) (mL/g) (mg/g) (s/g) (**) centipoises

Bacteria 0.5 3320 80 72 28.4 1.48 5.04 15 3860 152 63 29.0 1.22 5.14 32 3200 232 81 34.6 8.41 5.60 45 3840 220 108 48 14.9 7.14 Bacterial MBR 32 6320 N/A 159 57.8 9.8 7.26

Yeast 0.5 3260 1373 N/A < 1 1.19 5.01 15 4020 1767 N/A l<1 1.12 5.28 32 4480 1440 N/A 5 0.94 6.00 45 4580 1530 N/A 7.5 0.94 6.34 Yeast MBR 32 4300 N/A N/A 11.4 1.44 4.86 (*) Suspended solids concentration of supernatant after 2 h of settling (**) s/g = second/g MLSS

94 50 16 Mixed yeast sludge

40 Mixed bacterial sludge 12

30 8 20 ECP (ECP g / mg) CST (CST s/ g MLSS) 4 10

0 0 0 1020304050 0 1020304050 Salt (g/L) Salt (g/L) a) ECP vs. salt b) CST vs. salt

Figure 4.34 Variation in ECP and CST in function of salt content

Sludge dewaterability is measured by CST, which is used as a relative indicator to characterize the performance of sludge dewatering. While SVI is used as a field measure of sludge settleability. CST of bacterial sludge increased from 1.5 to 15 s/g with the increase of salt content from 15 to 45 g/L. Similarly, SVI of the bacterial sludge also increased with an increase in salt content, although the SVI values remained lower than 150 (a value corresponding to bulky sludge) even for a salt content of 45 g/L. However, the bacterial sludge at high salinity (above 30 g/L) was not compacted and remained fluffy compared to the sludge at salt contents of 15 g/L or lower. Similarly, it was that increasing viscosity trend with salt content was comparable to CST and ECP. In general, viscosity is dependent on MLSS. MLSSs of cultures at different salt contents were maintained relatively stable in this study. Thus, increase in viscosity with salt increase may be due to the ECP content of the sludge.

Microscopic examination did not show any filamentous bacteria or fungi in the bacterial sludge even at high salt contents, and relationships could be established between the increase in the SVI, CST and ECP with salt content for the bacterial sludge. The linear correlation between SVI and ECP was found in some previous studies (Goodwin and Foster, 1985; Urbain et al., 1993). The increase in CST and SVI as function of salt content in bacterial sludge may be attributed to excess sodium which can cause a deterioration in the floc structure, and an increase in ECP. This results in weak flocs, poor settling (increase in SVI) and dewatering (increase in CST). Likewise, Urbain et al. (1993) reported that ECP content was correlated linearly with SVI, high polysaccharides and proteins resulting in a worsening of sludge settleablity. In addition, the increase of ECP can be explained by the structure of the three-dimensional ECP matrix kept together by divalent cations such as Ca2+, Mg2+. An exchange of divalent cations (Ca2+, Mg2+) with Na+ will take place when the ratio of Na+ to divalent cation (Na+:M2+) exceeds two. In general, the Na+:M2+ ratio is very high in saline environment. In such conditions, large amount of polymers produced resulted in weaken the floc strength (Bruus et al., 1992). These ECP molecules extend out from cell surfaces and form a dense gel that retains water in gel pores (Liao et al., 2001). Liss et al. (1996) also reported that these complexes and hydrated ECPs within the floc matrix have a large capacity to retain water, which decreases dewaterability. Furthermore, suspended solids in the supernatant of bacterial batches appear to increase when the CST or SVI (vis-à-vis ECP) increases (Fig. 4.35). This may be due to disruption of weakened floc structure at high salinity (by shearing forces from aeration) which forms pin-point flocs and thus results in high effluent suspended solids.

95 SVI ( mL/g) ECP ( mg/ g) Viscosity (centipois) SS of supernatant (mg/L) 50 8.0 250 SVI SS of supernatant 140 ECP Viscosity 200 40 120 7.0 150 100 30 6.0 100 80 20 50 5.0 60 10 0 0 1020304050 0 1020304050 Salt (g/L) Salt (g/L)

Figure 4.35 Variation in SVI, SS, ECP and viscosity with salt content in mixed bacterial cultures

For mixed yeast sludge, CST remained relatively constant with salt increases. This can be understood in terms of the negligeable amount of ECP produced and indicates that the yeast sludge has better dewatering or thickening ability than bacterial sludge at high salinity, which is a specific advantage of the yeast system. The viscosity of yeast mix liquor, was slightly higher than that of bacteria. This can be attributed to the difference between MLSS of mixed yeast (4500 mg/L) and bacterial sludges (3500 mg/L). Thus, unlike bacterial sludge, viscosity of yeast sludge is mainly effected by biomass concentration, but not by ECP.

Table 4.8 shows that SS in the supernatant of the mixed yeast sludge was high even after 2 hours of settling. SS removal efficiency was only around 65%. The SS in supernatant were mainly dispersed as small yeast cells, which had not been captured by mycelia or pseudomycelial yeast matrix as shown by microscopic examination. Settled solids after two hours of settling were found to be compacted, and mainly consisted of yeasts interwind with each other and large yeast cells. Unlike activated sludge flocs, yeast flocs could not trap all fine yeast cells or fine particles, which led to high SS in the supernatant even after prolonged settling time. Conflicting results were obtained by different studies concerning the floc characteristic of yeast sludge. Nishihara ESRC Ltd. (2001) obtained large yeast flocs that settled quickly while other authors (Hu,1989; Arnold et al., 2000) obtained poor settling sludge when dealing with different yeast strains. This suggests that the predominance of mycelial yeasts may depend on the free competition among different yeast strains in wastewater.

4.4.2 YMBR and BMBR

Large differences of ECP obtained from both YMBR and BMBR and the batch reactors (run at 32 g salt/L) are shown in Figure 4.36. This may be due to of washing-out of macromolecules (ECPs and proteins) with fine particles in supernatant during the decantation stage, while most of these substances were retained by membrane module with spore size of 0.1 Pm. However, in comparison between YMBR and BMBR, the ECP concentration of mixed bacterial sludge was higher than that of yeast sludge at both SRTs. This observation was similar to that obtained in batch studies.

96 600

SRT of 10 d 490 500 SRT of 50 d

Sludge cake (attached to the membrane surface) 400

300 232 200 147 mg ECP/g driedbiomass

100 58 39 11 0 YMBR BMBR

Figure 4.36 ECP contents of mixed yeast and bacterial sludges in YMBR and BMBR

Meanwhile, visual observation of the clogged membrane revealed that large amount of extremely viscous and gelatinous sludge cakes was attached to the BMBR (Appendix A). However, the YMBR has a very thin layer of sludge cake, which could be easily washed out with tap water. This difference in the nature of the attached sludge contributes to the improvement of the membrane performance in the YMBR process.

In both the YMBR and BMBR, the ECP content seems to vary with the SRT, as shown in Fig. 4.36. It was found that ECP concentration increased with increase in SRT for both BMBR and YMBR. This can be explained by the variation in ECP production rate at different growth phases. Low SRT corresponds to the stationery phase, while long SRT (50 d) corresponds to the decay phase in which cell lysis takes place. Similarly, Pavoni et al. (1972) reported that during the early decay phase, the rate of ECP production was maximum. Sheintuch et al. (1986) reported that ECP content is a function of SRT in continuous bioreactors and it increased linearly with SRT.

4.4.3 Microscopic Observations of Mixed Yeast Sludge

From microscopic observations, it was noted that there were changes in predominant yeast strains and presence of other microorganisms (protozoa, rotifers) when operating conditions was changed. The operating conditions consisted of influent COD concentration, organic loading, SRT and the type of substrate (glucose and protein extract as protein source) and salt content. Photographs of mixed yeasts and bacteria flocs are shown in Appendix A.

Most yeast cells in the cultures were larger than those in the YMBR, even though both reactors was run at the same operating conditions (Protein-feed wastewater, SRT of 50 days, COD of 5,000 mg/L). Mycelia (hypha filament) and large size egg-shaped cells with monopolar budding were predominant in the cultures. While round or smaller egg-shaped cells, which may be different yeast strains from the cultures, were predominant in the YMBR (mean size of 2.4 Pm). The mean size of the mother cells was 3.9 Pm. This might be due to membrane with pore sizes of 0.1 Pm retaining most fine size cells among which minority of acid-tolerant bacteria was involved. Fine cells suspended in supernatant were washed out by a decanting step after more than 2 hours of settling in batch operation.

97 For substrate as glucose, most yeast cells are round with multilateral budding and mean size of 5.5 Pm. In the YMBR, predominant yeast strains were also changed as organic loading rate varied. The white or light brown color of yeast mixture gradually changed to dark brown when the high COD loading (5,000 mg COD/L) changed to low COD loading (1,000 mg COD/L). The majority of yeast colonies were black, orange and yellow growing on the yeast- glucose-peptone agar when growing in the low COD loading (influent COD of 100mg/L). Most yeast cells were egg-shaped and mycelia. It could be easily observed that free-living ciliates (protozoa) grow well in this yeast mixture. These ciliates move freely and rapidly in the mixed liquor. This change may be due to the increase in DO concentration. DO of all runs in the low COD loading is above 4.0 mg/L, whereas DO in that with COD of 5,000 mg/L was less than 2.0 mg/L.

4.4.4 Nutrient Uptake

Nutrients of mixed yeast and bacterial sludges are presented in Table 4.9. The volatile solids of both yeast and bacterial sludges were not significantly different, ranging from 89 to 95% of dried solids. The nitrogen content of the mixed yeasts sludge fed with glucose wastewater was found to be 7.1% on average compared to 3.2 % for bacterial sludge. Similarly, the phosphorus uptake ability of the yeasts was approximately twice as high as that of bacterial sludge. For fish-protein wastewater, nitrogen content of the mixed yeast cultures was more than 15%. In general, nitrogen content of the yeast and fungi biomasses was in the range of 7-12% (Westhuizen and Pretorius, 1998; Defrance, 1993). Thus the amount of nitrogen uptaken for protein-feed wastewater is too high (above 15%) compared to normal values or values of yeast sludge fed with glucose wastewater (7.1%). This may be attributed to the precipitation of a part of influent protein at low pH (3.5). The nitrogen content of yeast sludge in the YMBR was not excessive (7.64%) and was in the normal range. Thus, it is postulated that in the YMBR process with a low F/M ratio, yeast can produce enzyme to hydrolyze protein aggregates and can assimilate them.

Table 4.9 Composition of mixed bacterial and mixed yeast sludge

Volatile solids Nitrogen content Phosphorous content Microorganisms (%) (% of dries solids) (% of dried solids) Glucose Protein Glucose Protein Glucose Protein Yeast sludge: + 20 g /L: 94.1 90.3 6.79 15.2 2.23 3.61 + 32 g /L: 92.3 93.4 7.28 17.1 1.79 3.43 + 45 g /L: 95.0 90.5 7.19 15.7 1.70 3.53

YMBR sludge 91.5 7.64 3.52 (low-COD loading)

Bacterial sludge + 20 g /L: 89.5 91.6 2.70 5.61 0.61 1.56 + 32 g /L: 92.0 92.9 3.05 5.02 0.86 2.37 + 45 g /L: 94.2 90.2 3.74 5.32 0.90 2.23

BMBR sludge 89.7 5.21 1.51 (High COD loading)

The nitrogen and phosphorous contents of the bacterial biomass fed with protein-feed wastewater were approximately twice higher than that fed with glucose wastewater as shown in Table 4.9. By exception of nutrient uptake into real bacterial cells, the remaining of these 98 nutrients may entered into composition of floc structure such as ECP and phosphate bonds which can enhance ECP production.

Even when different wastewaters were used, the mixed yeast sludge from the cultures or the YMBR contained higher nutrients than the bacterial sludge. The average crude protein content obtained was 45% (corresponding to 7.2% N). This value was similar to the protein content obtained from the well-known “Symba” process which is a single-cell-protein production (SCP) for human food consumption by using potato processing waste to culture yeasts Endomycopsis fibuliger and Candida utilis. In general, algal and bacterial biomasses are less pleasant to taste because they contain undesirable levels of certain cellular materials such as high nucleic acid content, toxic or carcinogenic substances absorbed from the growth substrate. By contrast, yeasts and most fungi are quite acceptable to animals and man due to the abundance of valuable nutritious substances such as proteins and vitamins. Thus, it is suggested that a combination of yeast treatment and SCP production can be a cost-effective approach for seafood processing industries which, at present, face difficulties in treatment efficiency and high costs.

99 Chapter 5 5 Conclusions and Recommendations

This study investigated biological processes in using wild salt-tolerant yeast and bacteria for treatment of saline seafood processing wastewater. Basic studies on biokinetic coefficients and optimum operating parameters of yeast and bacterial treatment were conducted. The effects of high salt contents (20, 32 and 45 g/L NaCl) on the biokinetic coefficients were evaluated using respirometric method. Then the optimum operating parameters for the yeast and bacterial treatments were found from the parametric study using the acclimatized mixed yeast and bacterial cultures.

The main part of this study focused on the membrane bioreactor. The potential for developing membrane bioreactor systems using salt-tolerant yeast and bacteria to treat saline seafood processing wastewater was examined. A comparative evaluation of treatment performance of both systems was done. The last section focused on sludge characteristics concerning membrane clogging. The relationship between sludge properties and membrane flux decline was investigated. The conclusions drawn from these results are presented below.

5.1 Conclusions

From the biokinetic study, it can be concluded that the yeast is more efficient for treating wastewater containing high organic load and high salt content. It can be reasoned that they would be more suitable for varying salt loads because they require lower acclimation time than does bacterial culture. This was attributed to their better osmotolerant properties. The salt inhibition was found to be much higher for yeast culture. The maximum specific growth rate for yeast is higher than for bacterial culture at high salt contents. However, yeast growth was more inhibited at low CODs.

The results of the parametric study indicate that the osmotolerant yeasts were able to tolerate wider pH range than bacterial culture. Total OUR of yeast sludge was highest for pHs 5.0 –5.5. The respiration rate of yeasts was inhibited at pH 2.5 or pH above 9.0. The OUR of yeast at pH 3.5 was slightly lower than that at pH 5.0 - 5.5. However, pH of YMBR was maintained at pH 3.5 in order to limit bacterial contamination. The results of SRT study show that the highest nitrogen removal by uptake into yeast biomass was obtained at SRT of 10 d, whereas the maximum COD removal efficiency was obtained at SRT above 45 d.

In the high COD loading of membrane bioreactor study, the COD removal rate for BMBR was lower than the YMBR at high VLRs at high salt contents (32 g/L). Thus, the mixed yeast system could be subjected to higher F/M ratio. Even though DO concentration of yeast mixed liquor was lower than 1.0 mg/L at the high F/M ratio, the treatment efficiency of the yeast system does not decline. This may be due to the structure of yeast flocs facilitating oxygen diffusion. It is suggested that the yeast system represent a better substitute for an anaerobic system in terms of COD removal rate at high salinity.

The low COD loading phase revealed that both YMBR and BMBR give high COD removal efficiency (>90%) at high salt content, low F/M ratio and high SRT. Both reactors generated good effluent quality (COD < 120 mg/L, BOD < 20 mg/L and SS < 5 mg/L).

100 Yeast sludge in fact achieve significantly better reduction in the membrane clogging rate than bacterial sludge. BMBR is highly prone to membrane clogging, whereas YMBR can be operated at a relatively low pressure for prolonged filtration cycle. The filtrate cycle of YMBR was approximately 10 times higher than BMBR. Thus using yeasts in the biomembrane reactor can enhance membrane performance and has the potential to improve the economics of treatment system due to reducing operating and maintenance costs.

Several factors such as mechanisms of biofilm formation, concentration of ECP and size of cells contributed to the better filtration cycle of the YMBR. Reduction of problems associated with membrane clogging supports the use of YMBR in practice. Variation of ECP content that is responsible for biofilm as well as floc formation was found to vary with salt content for bacterial sludge, whereas for yeast sludge, ECP concentration remained practically constant even at high salt contents. Along with increases in ECP, CST of bacterial sludge was increased at high salinity, while CST of yeast sludge remained practically constant, indicating that dewatering would be easy for high salinity wastewater. Thus, using yeasts in membrane bioreactor can enhance membrane performance and reduce the operational problems associated with sludge dewatering and disposal.

Nitrogen and phosphorous contents of the yeast sludge were approximately twice higher than those of bacterial sludge. This suggests that yeasts have high nutritional values. In addition, yeasts and most fungi, are quite acceptable to animals and man due to the abundance of other valuable nutritious substances (vitamins). Thus, a combination of yeast treatment and SCP production can be a cost-effective approach for seafood processing industries.

5.2 Recommendations

Based on the extensive experimental data obtained, several recommendations for future studies can be outlined:

High Salinity Wastewater

1. Due to flux-enhancing ability of yeast sludge, operation modes for yeast membrane bioreactor can be examined to shorten backwash time. This may results in reduction of operation costs and increase in total permeate flux.

2. This study has not evaluated in depth the yeast sludge properties at different salt contents which may be related to membrane flux. These properties can consist of specific filtration resistance, hydrophobicity, surface charge, bound water, cell size and composition of ECP. In order to understand thoroughly the fouling inhibition mechanism of yeast sludge, a detailed study of the sludge properties at various salt content should be undertaken.

3. ECP production of mixed yeast sludge or activated sludge under varying nutrient compositions or cation concentrations of influent wastewater may be effected. A balance of nutrient contents or cations (divalent cation: monovalent cation) may lead to significant decrease in the membrane clogging rate. A study on the effects of nutrient and cation contents on ECP production would perhaps be useful.

4. Since seafood factories process a large range of products with important seasonal variations, changing in pollution characteristics vary significantly from plant to plant, and even within the same plant. Large variations in salt content can therefore be expected. Thus, a study on the effects of salt shock loading on yeast sludge may be 101 necessary. The shock salt loading experiment for bacterial sludge may be conducted in parallel to obtain a comparative evaluation.

5. In order to confirm the effectiveness of yeast treatment, pilot-scale yeast membrane bioreactors for long-term treatment of real saline seafood processing wastewater should be developed.

Biomembrane process and Membrane clogging

1. The wastewater pH of the environment influences surface charges, protein deposition and deflocculation. Low pH can cause an increase in surface charges, protein aggregates and deflocculation, which may enhance membrane filtration water flux. However, low pH values will also inhibit bacterial growth. Therefore, optimum pH values to control membrane clogging in the bacterial system should be considered.

2. A low pH environment may result in predominance of acid-tolerant microorganisms such as fungi, yeasts and acidogenic bacteria. Thus, under suitable operating conditions (such as pH, DO, organic loading and SRT) there may be a symbiotic relationship between these acid-tolerant microorganisms. For example, acidogenic bacteria enable to hydrolyze and convert quickly organic complexes (such as protein, lipids, and polysaccharides) to lower molecular-weight intermediate compounds (such as VFAs, amino acids and short-chain carbohydrates) which may be suitable substrates for yeast and fungal growth in a low pH environment. Therefore, using a mixture of acid-tolerant microorganisms in the biomembrane process for treatment of certain wastewaters can be investigated.

3. In this study, it was observed that ECP production increases with salt content. Thus, combination of activated carbon (AC) adsorption and the bacterial membrane process should be examined. Activated carbon addition to the BMR process can enhance permeate flux by forming porous cakes and can remove refractory organic matters by AC adsorption. Therefore, this method may be applied successfully for TOC removal as a pre-treatment of in the RO process.

Using Yeasts and Fungi for treatment of toxic wastewaters

Based on tolerance ability of yeasts or fungi in extremely strict conditions, further investigation are proposed:

1. A comparative study of bacterial and yeast sludge response to acute and chronic heavy metal stress using respirometric method is recommended. The results from such a study might provide information on quantitative toxicity evaluation of the for both yeast and bacterial treatment systems. Resting and growing cells (biomass) can demonstrate completely different mechanisms of resistance to acute and chronic heavy metal stress. Thus, mechanisms of heavy metal biosorption by yeasts and bacterial sludge may also be investigated.

2. Using yeast or fungi biomembrane process with low F/M ratio for treatment of high strength hazardous organic wastewater may be a feasible biological approach. Toxic wastes or chemicals suggested include pesticides, herbicides, phenolic derivatives, aromatic compounds, cyanides and tannery wastewater.

3. Identification of the various yeast or bacteria present at high salt conditions. 102 References

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109 Fig. A-1 Yeast colonies cultured with glucose-feed wastewater containing high salt concentrations

Round, smooth colony

Irregular, rough colony

Fig. A-2 Two predominant yeast colonies cultured with glucose-feed wastewater

10 Pm

10 Pm

Fig. A-3 redominantP yeasts in the batch culture with glucose-feed wastewater containing 32 g/L salt (x 1500)

A-1 20 Pm

Fig. A-4 redominantP yeasts in the batch culture with protein-feed wastewater containing 32 g/L salt (x 500)

20 Pm

Fig. A-5 redominantP yeasts in the batch culture with protein-feed wastewater containing 45 g/L salt (Growth of mycelia yeasts occurred) ( x 500)

A-2 20 Pm

Fig. A-6 Mycelia yeasts cultured( with fish-protein wastewater) ( x 800)

20 Pm

Fig. A-7 eastY flocs formed by interwinding of mycelia yeasts settled( sludge in theYMBR) ( x 500)

A-3 20 Pm

Fig. A-8 Fine yeast cells suspended in supernatant of MBRY

10 Pm

Protozoa

Fig. A-9 Free-living ciliates (protozoa) grew well in the yeast mixture of YMBR in( COD loading) (x 250)

A-4 10 Pm

Fig. A-10 Bacterial flocs in the batch culture with protein-feed wastewater at 15 g/L salt rounded( and compacted sludge flocs) (x250)

10 Pm

Fig. A-11 Bacterial flocs in the batch culture with protein-feed wastewater at 32 g/L salt Open( and weak flocs) ( x 250)

10 Pm

Fig. A-12 Bacterial flocs in the BMBR Fine( and weak flocs) (x 250)

A-5 Fig. A-13 Respirometer system (Respirometer, recorder, DO meter and thermostat)

Fig. A-14 Respirometer

A-6 Fig. A-15 Respirogram (low COD dose)

Fig. A-16 Respirogram (high COD dose)

A-7

Fig. A-17 Yeast Y(MBR) and bacterial membrane Fig. A-18 Hg-U tube pressure( measurement) Fig. A-19 eastY reactor and settling tank BMBR)( reactors

A-8

Fig. A-20 Full clogged membrane of YMBR Fig. A-21 Fu ll clogged membrane of BMBR Fig. A-22 Clean membrane Fig. A-23 Membrane clogging

A-9 Table B-1 Acclimation of mixed bacterial sludge to 20 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT CODin CODeff Ecod MLSS pHo pHt SVI day g/L NaCl g/L Cl h mg/L mg/L % mg/L mL/g 2 20.5 24 952 175 81.6 2,116 6.79 6.79 88 4 19.0 23 931 143 84.6 2,120 7.17 7.47 83 5 18.9 22 940 143 84.8 2,132 6.76 6.35 81 7 19.6 24 930 154 83.4 2,274 6.99 6.11 71 9 20.0 24 930 136 85.4 2,488 7.61 7.01 70 11 20.9 29 1,010 32 96.8 2,640 7.80 7.10 80 13 20.6 28 870 51 94.1 2,516 7.75 6.85 87 15 21.3 25 915 35 96.2 2,668 7.84 6.40 47 17 21.3 12.9 25 915 60 93.4 2,672 7.69 6.33 45 19 21.3 12.9 26 915 55 94.0 2,888 7.32 6.22 43 28 20.0 12.2 24 1,090 52 95.2 2,750 7.80 6.26 36 40 20.5 12.4 22 1,070 102 90.5 2,724 7.47 6.28 34 42 20.5 12.4 15 1,070 127 88.1 2,760 7.75 6.52 35 44 20.5 12.0 14 1,070 24 97.8 2,837 6.97 6.16 34 46 20.5 12.4 17 1,070 71 93.4 2,993 7.64 6.53 32

Table B-2 Acclimation of mixed bacterial sludge to 32 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT CODin CODeff Ecod MLSS pHo pHt SVI day g/L NaCl g/L Cl h mg/L mg/L % mg/L mL/g 0 20.5 24 952 181 81.0 2,072 6.79 6.88 91 2 19.8 23 931 145 84.4 1,948 7.11 7.65 79 4 20.9 22 940 134 85.7 2,152 6.89 6.34 81 5 24.9 24 930 185 80.1 2,324 6.86 6.06 62 7 25.0 24 930 121 87.0 2,292 7.44 6.64 71 9 26 24 950 145 84.7 2,208 7.51 7.33 76 11 26.6 24 950 95 90.0 2,190 7.5 6.88 78 13 29 26 950 100 89.5 2,176 7.85 7.67 82 15 31.8 26 870 120 86.2 2,328 7.73 7.61 82 17 32.6 25 870 31 96.4 2,576 7.84 6.75 74 19 34 25 915 12 98.7 2,852 7.85 7.19 49 28 33.5 20.3 25 915 88 90.4 3,260 7.28 7.93 40 30 33.9 20.6 26 915 61 93.3 3,000 7.31 7.93 43 38 32.6 19.8 24 1,090 169 84.5 2,300 7.42 7.24 24 40 33.6 20.4 24 1,090 118 89.2 2,164 7.43 7.41 25 42 32.2 19.6 15 1,090 125 88.5 2,253 7.44 7.72 23 44 32.2 19.6 14 1,090 23 97.9 2,337 7.22 7.23 29 46 32 20.0 17 1,090 29 97.3 2,501 7.46 7.99 16

B-1 Table B-3 Acclimation of mixed bacterial sludge to 45 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT CODin CODeff Ecod MLSS pHo pHt SVI day g/L NaCl g/L Cl h mg/L mg/L % mg/L mL/g 0 20.5 24.0 952 415 56 2,040 6.8 6.81 86.5 2 19.3 23.0 931 420 55 1,888 6.93 7.5 79.4 4 21.4 22.0 940 431 54 2,100 6.78 6.37 71.8 5 24.8 24.0 930 445 52 1,738 6.89 6.47 81.7 7 24.9 24.0 930 395 58 2,408 7.51 7.71 62.3 9 25.5 24.0 1,010 375 63 2,344 7.33 7.51 64.0 11 27.4 24.0 1,010 310 65 2,143 7.2 6.68 70.0 13 30.5 24.0 1,010 325 70 2,048 7.7 6.64 75.0 15 33.5 25.0 870 215 67 2,200 7.85 7.72 70.5 17 33.5 25.0 870 201 77 2,412 7.87 6.89 71.3 19 37.4 26.0 875 215 75 2,792 7.85 7.31 58.7 21 40.0 25.0 915 197 78 2,748 7.85 7.41 62.2 23 42.0 26.0 1,031 220 79 2,848 7.14 7.86 54.1 25 42.2 25.0 1,031 231 81 2,936 7.85 7.2 48.7 26 46.2 24.0 1,031 205 80 3,252 7.26 7.75 44.9 28 45.3 27.5 24.0 1,031 157 85 3,270 7.38 7.93 35.9 30 43.5 26.4 24.0 1,031 120 88 3,310 7.42 7.92 33.1 38 44.3 26.9 24.5 1,090 135 88 3,100 7.48 7.79 21.0 40 47.0 28.6 24.0 1,090 148 86 3,400 7.54 7.98 18.0 42 44.4 27.0 24.0 1,090 167 89 3,230 7.56 7.98 18.0

Table B-4 Acclimation of mixed yeast at 20 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT CODin CODeff Ecod MLSS pHo pHt day g/L NaCl g/L Cl h mg/L mg/L % mg/L 0 20.5 24 5,100 325 93.6 3,700 5.61 3.05 2 21.5 24 5,100 305 94.0 5,420 5.87 2.86 4 20.8 26 5,100 370 92.7 6,732 5.97 2.92 6 21.1 26 4,980 272 94.5 7,916 5.54 2.91 8 20.6 25 7,400 363 95.1 6,676 5.64 2.54 10 20.1 15.9 27 7,400 1,230 83.4 6,416 5.72 3.25 21 20.1 15.9 26 7,400 1,270 82.8 5,028 5.66 4.22 23 20.9 14.5 27 5,350 447 91.6 6,596 3.50 2.93 33 20.2 23.1 22 7,490 352 95.3 7,756 3.57 2.37 35 20.0 14.5 15 7,490 352 95.3 9,340 5.00 2.40 37 20.0 14.6 21 7,490 331 95.6 10,367 4.79 2.51 39 20.6 15.5 15 7,490 444 94.1 12,216 4.07 2.32

B-2 Table B-5 Acclimation of mixed yeast at 32 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT CODin CODeff Ecod MLSS pHo pHt day g/L NaCl g/L Cl h mg/L mg/L % mg/L 0 32.8 24 4,980 920 81.5 4,679 4.09 2.78 2 33.0 24 4,980 1,010 79.7 4,710 5.52 2.83 4 32.3 25 4,980 415 91.7 5,704 5.32 2.83 5 32.3 24 4,980 428 91.4 5,828 5.51 3.13 7 31.3 24 4,980 351 93.0 3,804 5.49 3.07 9 32.6 24 5,100 368 92.8 4,242 5.84 2.86 11 33.5 27 4,980 413 91.7 6,596 5.92 2.98 13 32.2 27 4,980 409 91.8 8,040 5.56 2.84 15 32.6 25 7,400 355 95.2 7,496 5.64 2.50 17 33.8 26 7,400 382 94.8 10,168 5.46 3.34 28 31.9 23.0 24 7,400 478 93.5 9,312 5.78 2.78 30 31.7 23.5 24 7,400 423 94.3 10,152 5.08 2.52 38 32.0 22.5 24 7,500 369 95.1 13,944 4.46 2.64 40 31.7 21.7 23 7,400 449 93.9 14,324 5.14 2.65 42 32.0 22.5 24 7,400 396 94.6 14,713 4.56 2.59 44 33.0 22.5 21 7,400 278 96.2 14,557 5.36 2.58 46 31.8 21.1 24 7,400 411 94.4 15,550 4.57 2.53

Table B-6 Acllimation of mixed yeast to 45 g salt/L with the glucose-feed wastewater

Time Salt Cl HRT F/M CODin CODeff Ecod MLSS pHo pHt day g/L NaCl g/L Cl h d mg/L mg/L % mg/L 0 32.0 23 1.12 5,100 1,720 66.3 3,768 5.54 3.12 2 32.0 24 1.15 5,100 1,560 69.4 3,984 5.88 2.92 4 32.0 24 0.82 4,980 1,150 76.9 6,100 5.97 2.96 5 35.0 26 0.63 4,980 1,095 78.0 7,920 5.66 2.92 7 38.0 25 0.58 4,980 950 80.9 8,652 5.84 2.77 9 40.8 24 0.56 5,050 765 84.9 8,950 5.68 2.87 11 43.3 23 0.55 5,100 680 86.7 9,240 5.66 2.83 13 45.3 24 0.51 5,100 719 85.9 10,060 5.43 2.73 15 44.1 31.7 24 0.50 5,100 539 89.4 10,144 5.79 2.84 17 43.1 31.6 25 0.46 5,050 471 90.7 10,984 5.26 2.53 30 44.9 29.1 27 0.38 5,100 369 92.8 13,480 4.99 2.54 40 45.0 30.7 24 0.36 5,200 345 93.4 14,456 5.00 2.4 42 44.1 30.1 26 0.32 5,200 362 93.0 16,047 5.23 2.53 44 45.0 31.7 24 0.33 5,200 411 92.1 15,733 5.37 2.49 46 45.0 32.2 24 0.32 5,100 342 93.3 15,967 4.88 2.54

B-3 Table B-7 Acclimation of mixed yeasts to protein-feed wastewater at 20 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 4930 5450 2 5120 1587 69 5400 0.63 4 5210 1667 68 5870 0.59 6 5020 1456 71 6780 0.49 8 4970 1243 75 7920 0.42 10 4760 809 83 9250 0.34 12 5010 752 85 9350 0.36 14 5150 567 89 10850 0.32 16 5090 458 91 10750 0.32

Table B-8 Acclimation of mixed yeasts to protein-feed wastewater at 32 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 5010 5700 2 4850 1795 63 5400 0.60 4 5120 1640 68 5990 0.57 6 4950 1730 65 5930 0.56 8 4760 1190 75 6950 0.46 10 5230 1200 77 7050 0.49 12 5100 870 83 8345 0.41 14 5010 701 86 9050 0.37 16 5120 770 85 9710 0.35

Table B-9 Acclimation of mixed yeasts to protein-feed wastewater at 45 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 4790 5500 2 5100 2091 59 5200 0.65 4 5020 1640 61 5350 0.63 6 4970 1730 61 5320 0.62 8 5120 1587 69 6240 0.55 10 5070 1200 72 8705 0.39 12 5025 870 83 8956 0.37 14 4790 701 84 9310 0.34 16 4990 770 83 9420 0.35

B-4 Table B-10 Acclimation of mixed bacterial sludge to protein-feed wastewater at 20 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 3120 2 1020 92 91 3650 0.28 4 990 79 92 4270 0.23 6 1070 86 92 4750 0.23 8 1120 56 95 5120 0.22 10 1010 51 95 5250 0.19 12 980 29 97 5430 0.18 14 990 50 95 5910 0.17 16 1130 45 96 5670 0.20

Table B-11 Acclimation of mixed bacterial sludge to protein-feed wastewater at 32 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 3560 2 1100 165 85 3750 0.29 4 980 78 92 4340 0.23 6 995 90 91 4950 0.20 8 1020 51 95 5120 0.20 10 1050 73 93 5010 0.21 12 950 38 96 5700 0.17 14 970 78 92 5600 0.17 16 1105 55 95 6310 0.18

Table B-12 Acclimation of mixed bacterial sludge to protein-feed wastewater at 45 g/L salt

Time CODin CODeff COD% MLSS F/M days mg/L mg/L mg/L d-1 0 3950 2 1010 222 78 3650 0.28 4 1200 228 81 3750 0.32 6 1105 221 80 4210 0.26 8 1030 175 83 4760 0.22 10 970 175 82 4790 0.20 12 990 109 89 5700 0.17 14 1025 113 89 5970 0.17 16 990 89 91 6105 0.16

B-5 Table B-13 COD and DO profile data of the mixed yeast batch culture with glucose-feed wastewater at 20g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 5020 8250 2.5 1250 1.2 75 4.15 5.0 220 6.4 95.6 2.65 7.5 230 6.3 95.4 1.76 9.0 205 6.4 95.9 1.48 10.5 220 6.4 95.6 1.26 9130 Average 8700

Table B-14 COD and DO profile data of the mixed yeast batch culture with glucose-feed wastewater at 32 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 4950 9350 2.5 2750 0.7 45 2.25 5.0 1505 0.9 69.9 1.75 8 540 4.2 89.2 1.49 9.0 255 6.3 94.9 1.32 10.5 210 6.2 95.8 1.14 9645 13 245 6.3 95.1 0.91 Average 9500

Table B-15 COD and DO profile data of the mixed yeast batch culture with glucose-feed wastewater at 45 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 5050 9360 2.5 4200 0.7 16 0.80 5.0 2740 0.9 45 1.12 7.5 2000 4.2 60 0.99 9.0 1560 6.3 69 0.95 10.5 790 6.2 84 1.00 10130 13 290 6.3 94 0.90 Average 9750

B-6 Table B-16 COD and DO profile data of the mixed bacterial batch culture with glucose-feed wastewater at 20 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0 1036 2752 0.25 821 1.97 18 5.84 1.25 146 6.3 85 5.57 2.5 20 6.3 98 3.20 3.5 26 6.5 97 2.27 6.5 45 6.4 96 1.20 3351 Average 3050

Table B-17 COD and DO profile data of the mixed bacterial batch culture with glucose-feed wastewater at 32 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0 1020 3245 0.25 760 2.1 24 6.44 1.25 320 4.95 68 3.65 2.25 235 5.7 77 2.28 3.5 160 6.2 84 1.61 6.5 45 6.3 96 0.99 8 30 6.4 97 0.81 4053 11.5 35 6.3 97 0.56 14.5 25 6.4 98 0.45 Average 3650

Table B-18 COD and DO profile data of the mixed bacterial batch culture with glucose-feed wastewater at 45 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.25 945 0.9 6 1.65 1.25 836 1.2 16 0.98 2.25 769 2.3 23 0.77 3.5 578 3.4 42 0.90 6.5 405 4.7 60 0.69 8.5 259 5.4 74 0.65 3450 11.5 218 5.9 78 0.51 14.5 113 6.1 89 0.46 17 70 6.2 93 0.41 Average 3200

B-7 Table B-19 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed wastewater at 20 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 5010 5432 2.5 0.6 5.5 3180 0.7 36.5 1.32 7.5 2800 0.7 44.1 1.17 10.5 2490 0.6 50.3 0.95 16.0 2191 0.7 56.3 0.70 18.5 1650 0.6 67.1 0.72 20.5 1500 0.6 70.1 0.68 26.0 0.6 29.0 1010 2.2 79.8 0.55 31.0 790 4.5 84.2 0.54 34.0 680 4.9 86.4 0.51 6670 37.0 490 5.1 90.2 0.48 42.0 540 5.6 89.2 0.42 Average 6050

Table B-20 COD and DO profile data of the mixed yeast batch culture with protein-feed wastewater at 32 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 5010 5325 5.5 3234 0.7 35.4 1.33 7.5 2707 0.7 46.0 1.27 10.5 2392 0.6 52.3 1.03 16.0 2191 0.7 56.3 0.73 18.5 2031 0.6 59.5 0.67 20.5 1913 0.6 61.8 0.62 26.0 0.6 29.0 968 2.2 80.7 0.58 32.0 772 4.0 84.6 0.55 34.0 830 4.2 83.4 0.51 6302 37.0 584 4.3 88.3 0.49 42.0 541 5.6 89.2 0.44 Average 5810

B-8 Table B-21 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed wastewater at 45 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0.0 5010 5915 2.5 0.3 5.5 4030 0.2 19.6 0.67 7.5 3830 0.4 23.6 0.59 10.5 3690 0.4 26.3 0.47 16.0 3230 0.5 35.5 0.42 18.5 2700 0.4 46.1 0.47 20.5 2590 0.5 48.3 0.44 26.0 0.5 29.0 1500 0.8 70.1 0.45 32.0 1330 1.1 73.5 0.43 34.0 1240 2.5 75.2 0.41 6946 37.0 950 3.9 81.0 0.41 42.0 920 4.1 81.6 0.36 Average 6430

Table B-22 COD and DO profile data of the mixed bacterial batch culture with fish-protein- feed wastewater at 20 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0 1020 0.25 805 1.9 21 4.80 3730 1.25 619 2.1 39 1.79 2.25 416 4.2 59 1.50 3.5 300 4.5 71 1.15 6.5 143 5.9 86 0.75 9.0 40 6.5 96 0.61 4870 11.5 25 6.4 98 0.48 14.5 35 6.5 97 0.38 Average 4300

B-9 Table B-23 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed wastewater at 32 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0 976 3270 0.25 870 1.6 11 2.80 1.25 645 1.8 34 1.75 2.25 568 2.5 42 1.20 3.5 509 4.0 48 0.88 6.5 383 5.7 61 0.60 9 320 6.0 67 0.48 11.5 200 6.2 80 0.45 14.5 110 6.3 89 0.39 18.5 65 6.2 93 0.33 21.0 50 6.3 95 0.29 4172 26.0 45 6.2 95 0.24 28.0 20 6.2 98 0.23 Average 3721

Table B-24 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed wastewater at 45 g/L salt

Time COD DO COD% U MLSS h mg/L mg/L g/g.d mg/L 0 985 3650 0.25 935 0.7 5 1.17 1.25 941 0.9 4 0.21 2.25 888 1.5 10 0.25 3.5 760 1.5 23 0.37 6.5 691 1.6 30 0.26 9.0 534 2.4 46 0.29 11.5 473 3.8 52 0.26 14.5 337 5.5 66 0.26 18.5 298 5.7 70 0.22 21.0 206 6 79 0.22 26.0 127 5.7 87 0.19 28.0 90 6.2 91 0.19 4589 30.0 75 6.3 92 0.21 4120

B-10 Salt 20 Yeast Time CODin CODeff COD%y MLSSy F/M 0 4930 5450 2 5120 1587 69 5400 0.63 4 5210 1667 68 5870 0.59 6 5020 1456 71 6780 0.49 8 4970 1243 75 7920 0.42 10 4760 809 83 9250 0.34 12 5010 752 85 9350 0.36 14 5150 567 89 10850 0.32 16 5090 458 91 10750 0.32

Bacteria Time CODin CODeff COD% MLSS F/M 0 3120 2 1020 92 91 3650 0.28 4 990 79 92 4270 0.23 6 1070 86 92 4750 0.23 8 1120 56 95 5120 0.22 10 1010 51 95 5250 0.19 12 980 29 97 5430 0.18 14 990 50 95 5910 0.17 16 1130 45 96 5670 0.20 Salt 32 Yeast Time CODin CODeff COD%y MLSSy F/M 0 5010 5700 2 4850 1795 63 5400 0.60 4 5120 1640 68 5990 0.57 6 4950 1730 65 5930 0.56 8 4760 1190 75 6950 0.46 10 5230 1200 77 7050 0.49 12 5100 870 83 8345 0.41 14 5010 701 86 9050 0.37 16 5120 770 85 9710 0.35

Bacteria Time CODin CODeff COD% MLSS F/M 0 3560 2 1100 165 85 3750 0.29 4 980 78 92 4340 0.23 6 995 90 91 4950 0.20 8 1020 51 95 5120 0.20 10 1050 73 93 5010 0.21 12 950 38 96 5700 0.17 14 970 78 92 5600 0.17 16 1105 55 95 6310 0.18 Salt 45 Yeast Time CODin CODeff COD%y MLSSy F/M 0 4790 5500 2 5100 2091 59 5200 0.65 4 5020 1640 61 5350 0.63 6 4970 1730 61 5320 0.62 8 5120 1587 69 6240 0.55 10 5070 1200 72 8705 0.39 12 5025 870 83 8956 0.37 14 4790 701 84 9310 0.34 16 4990 770 83 9420 0.35

Bacteria Time CODin CODeff COD% MLSS F/M 0 3950 2 1010 222 78 3650 0.28 4 1200 228 81 3750 0.32 6 1105 221 80 4210 0.26 8 1030 175 83 4760 0.22 10 970 175 82 4790 0.20 12 990 109 89 5700 0.17 14 1025 113 89 5970 0.17 16 990 89 91 6105 0.16 CODBf

1200

1000

800

600 CODBf

400

200

0 0 5 10 15 20 25 30 35 S20 Time5000 CODy-pr DOy-pr %COD U MLSS 0.0 5010 5432 2.5 0.6 5.5 3180 0.7 36.5 1.32 7.5 2800 0.7 44.1 1.17 9.0 10.5 2490 0.6 50.3 0.95 12.5 16.0 2191 0.7 56.3 0.70 18.5 1650 0.6 67.1 0.72 20.5 1500 0.6 70.1 0.68 26.0 0.6 29.0 1010 2.2 79.8 0.55 31.0 790 4.5 84.2 0.54 34.0 680 4.9 86.4 0.51 6670 37.0 490 5.1 90.2 0.48 42.0 540 5.6 89.2 0.42 6050

S32 Time5000 CODy-pr DOy-pr COD% U MLSS 0.0 5010 5325 2.5 0.6 5.5 3234 0.7 35.4 1.33 7.5 2707 0.7 46.0 1.27 9.0 10.5 2392 0.6 52.3 1.03 12.5 16.0 2191 0.7 56.3 0.73 18.5 2031 0.6 59.5 0.67 20.5 1913 0.6 61.8 0.62 26.0 0.6 29.0 968 2.2 80.7 0.58 32.0 772 4.0 84.6 0.55 34.0 830 4.2 83.4 0.51 6302 37.0 584 4.3 88.3 0.49 42.0 541 5.6 89.2 0.44 5810 S45 Time5000 CODy-pr DOy-pr COD% U MLSS 0.0 5010 5915 2.5 0.3 5.5 4030 0.2 19.6 0.67 7.5 3830 0.4 23.6 0.59 9.0 10.5 3690 0.4 26.3 0.47 12.5 16.0 3230 0.5 35.5 0.42 18.5 2700 0.4 46.1 0.47 20.5 2590 0.5 48.3 0.44 26.0 0.5 29.0 1500 0.8 70.1 0.45 32.0 1330 1.1 73.5 0.43 34.0 1240 2.5 75.2 0.41 6946 37.0 950 3.9 81.0 0.41 42.0 920 4.1 81.6 0.36 6430 S20 Time5000 CODy-pr DOy-pr %COD U MLSS 0.0 5010 5432 2.5 0.6 5.5 3180 0.7 36.5 1.32 7.5 2800 0.7 44.1 1.17 9.0 10.5 2490 0.6 50.3 0.95 12.5 16.0 2191 0.7 56.3 0.70 18.5 1650 0.6 67.1 0.72 20.5 1500 0.6 70.1 0.68 26.0 0.6 29.0 1010 2.2 79.8 0.55 31.0 790 4.5 84.2 0.54 34.0 680 4.9 86.4 0.51 6670 37.0 490 5.1 90.2 0.48 42.0 540 5.6 89.2 0.42 6050

S32 Time5000 CODy-pr DOy-pr COD% U MLSS 0.0 5010 5325 2.5 0.6 5.5 3234 0.7 35.4 1.33 7.5 2707 0.7 46.0 1.27 9.0 10.5 2392 0.6 52.3 1.03 12.5 16.0 2191 0.7 56.3 0.73 18.5 2031 0.6 59.5 0.67 20.5 1913 0.6 61.8 0.62 26.0 0.6 29.0 968 2.2 80.7 0.58 32.0 772 4.0 84.6 0.55 34.0 830 4.2 83.4 0.51 6302 37.0 584 4.3 88.3 0.49 42.0 541 5.6 89.2 0.44 5810 S45 Time5000 CODy-pr DOy-pr COD% U MLSS 0.0 5010 5915 2.5 0.3 5.5 4030 0.2 19.6 0.67 7.5 3830 0.4 23.6 0.59 9.0 10.5 3690 0.4 26.3 0.47 12.5 16.0 3230 0.5 35.5 0.42 18.5 2700 0.4 46.1 0.47 20.5 2590 0.5 48.3 0.44 26.0 0.5 29.0 1500 0.8 70.1 0.45 32.0 1330 1.1 73.5 0.43 34.0 1240 2.5 75.2 0.41 6946 37.0 950 3.9 81.0 0.41 42.0 920 4.1 81.6 0.36 6430 S20 S45 Time CODBf DOBf COD% U MLSS Time CODBf DOBf COD% U MLSS 0 1020 0 985 3650 0.25 805 1.9 21 4.80 3730 0.25 935 0.7 5 1.17 1.25 619 2.1 39 1.79 1.25 941 0.9 4 0.21 2.25 416 4.2 59 1.50 2.25 888 1.5 10 0.25 3.5 300 4.5 71 1.15 3.5 760 1.5 23 0.37 6.5 143 5.9 86 0.75 6.5 691 1.6 30 0.26 9 40 6.5 96 0.61 4870 9 534 2.4 46 0.29 11.5 25 6.4 98 0.48 11.5 473 3.8 52 0.26 14.5 35 6.5 97 0.38 14.5 337 5.5 66 0.26 4300 4300 18.5 298 5.7 70 0.22 21.0 206 6 79 0.22 26.0 127 5.7 87 0.19 28.0 90 6.2 91 0.19 4589 30.0 75 6.3 92 0.21 Time CODBf DOBf COD% U MLSS 4120 0 976 3270 0.25 870 1.6 11 2.80 1.25 645 1.8 34 1.75 2.25 568 2.5 42 1.20 3.5 509 4.0 48 0.88 6.5 383 5.7 61 0.60 9 320 6.0 67 0.48 11.5 200 6.2 80 0.45 14.5 110 6.3 89 0.39 18.5 65 6.2 93 0.33 21.0 50 6.3 95 0.29 4172 26.0 45 6.2 95 0.24 28.0 20 6.2 98 0.23 3721 20 g/L Time CODB2 DOB2 COD% U MLSS 20 g/L 32 g/L 45 g/L 0 1036 2752 3050 3650 3200 0.25 821 1.97 18 5.84 2.5 8 17 1.25 146 6.3 85 5.57 3.27 0.84 0.44 2.5 20 6.3 98 3.20 20 30 70 3.5 26 6.5 97 2.27 98 97 93 6.5 45 6.4 96 1.20 3.2 0.81 0.41 8.5 15 6.4 99 0.94 3351 3050

32 g/L Time CODB2 DOB2 COD% U MLSS 0 1020 3245 0.25 760 2.1 24 6.44 1.25 320 4.95 68 3.65 2.25 235 5.7 77 2.28 3.5 160 6.2 84 1.61 6.5 45 6.3 96 0.99 8 30 6.4 97 0.81 4053 11.5 35 6.3 97 0.56 14.5 25 6.4 98 0.45 3650

45 g/L Time CODB2 DOB2 COD% U MLSS 0 1000 2950 0.25 945 0.9 6 1.65 1.25 836 1.2 16 0.98 2.25 769 2.3 23 0.77 3.5 578 3.4 42 0.90 6.5 405 4.7 60 0.69 8.5 259 5.4 74 0.65 3450 11.5 218 5.9 78 0.51 14.5 113 6.1 89 0.46 17 70 6.2 93 0.41 3200 Table C-1 Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 20 g/L salt OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 10 10.4 2.74 3.33 7.61 0.336 21.8 0.664 0.468 0.24 15 16.1 3.09 5.27 13 0.355 37.3 0.645 0.454 0.41 20 24.3 2.73 7.17 22 0.362 61.8 0.638 0.449 0.68 30 38.5 3.88 10.2 35 0.342 99.1 0.658 0.463 1.09 50 57.0 3.99 16.9 53 0.341 152 0.659 0.464 1.67 100 60.7 5.16 35.6 56 0.360 159 0.640 0.451 1.75 200 98.0 4.47 94 268 2.95 300 131.4 3.55 128 366 4.03 500 136.1 4.43 132 377 4.15 Average Y 0.457

Table C-2 Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 32 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 10 8.1 2.81 3.10 5.2 0.313 16 0.687 0.484 0.19 15 12.8 2.89 4.83 9.9 0.325 31 0.675 0.475 0.36 20 23.1 2.72 6.12 20 0.309 64 0.691 0.487 0.74 30 25.1 2.79 9.50 22 0.320 70 0.680 0.479 0.81 50 42.9 5.12 15.69 38 0.317 119 0.683 0.481 1.37 200 95 5.43 63.56 90 0.321 282 0.679 0.478 3.25 300 103 5.29 98 308 3.55 500 102 3.60 98 309 3.56 Average Y 0.480

C-1 Table C-3 Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 45 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 10 9.2 2.82 4.23 6.4 0.427 15.2 0.573 0.404 0.15 30 28.1 2.63 12.62 25 0.425 61.0 0.575 0.405 0.60 50 28.4 2.05 20.00 26 0.404 63.0 0.596 0.420 0.62 100 57.5 2.73 41.48 55 0.419 131.1 0.581 0.409 1.29 200 64.2 2.62 82.17 62 0.415 147.4 0.585 0.412 1.45 300 90.1 2.57 88 209.4 2.06 500 91.8 2.11 90 214.5 2.11 Average Y 0.411

5.0 20 g NaCl/L 20 g/L NaCl:

32 g NaCl/L 4.0 S 45 g NaCl/L P 5.60 -1 158  S day 2 P 3.0 R = 0.971 32 g/L NaCl:

2.0 S P 4.74 118  S

Specific Growth Rate Rate Growth Specific 2 1.0 R = 0.982 45 g/L NaCl: 0.0 S 0 100 200 300 400 50 0 P 2.70 129 S COD concentration S ( mg/ L COD)  R2 = 0.967 Fig. C-1 Variation of specific growth rate of yeast sludge versus COD concentration at different salt contents for glucose-feed wastewater

C-2 Table C-4 Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 20 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 5 17 2.64 1.04 14 0.210 75 0.790 0.556 1.03 7 22 2.49 1.42 19 0.205 101 0.795 0.560 1.38 10 24 2.48 1.79 21 0.181 112 0.819 0.577 1.54 30 48 2.88 5.05 46 0.170 240 0.830 0.585 3.28 50 84 3.15 8.91 81 0.180 427 0.820 0.577 5.85 100 109 3.79 18.91 105 0.191 555 0.809 0.570 7.60 200 109 3.69 105 554 7.58 Average Y 0.570

Table C-5 Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 32 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 5 7.2 4.79 0.92 2.42 0.185 13.7 0.815 0.574 0.19 10 10.9 4.14 1.79 6.74 0.181 38.1 0.819 0.577 0.53 15 11.6 3.82 2.61 7.76 0.176 43.9 0.824 0.580 0.61 20 12.9 3.72 3.50 9.16 0.177 51.8 0.823 0.580 0.72 30 14.7 3.64 5.11 11.07 0.172 62.5 0.828 0.583 0.87 50 22.0 3.90 8.37 18.07 0.169 102.1 0.831 0.585 1.42 100 30.5 4.20 0.00 26.34 148.8 1.000 0.704 2.07 200 31.1 4.62 26.47 149.5 2.08 Average Y 0.583

C-3 Table C-6 Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 45 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 10 6.8 3.85 2.58 2.91 0.261 11.8 0.739 0.520 0.15 20 10.9 3.75 5.09 7.18 0.257 29.1 0.743 0.523 0.37 30 12.2 4.61 6.89 7.57 0.232 30.6 0.768 0.541 0.39 50 13.6 4.05 11.93 9.51 0.241 38.5 0.759 0.535 0.49 100 20.5 4.00 24.26 16.50 0.245 66.8 0.755 0.532 0.85 200 20.8 4.10 16.69 67.6 0.86 Average Y 0.531

20 g/L NaCl: 20 g NaCl/L 8.0 S 32 g NaCl/L P 9.95 45 g NaCl/L 44  S -1 2 day 6.0 R = 0.965 P 32 g/L NaCl: S 4.0 P 2.80 52  S R2 = 0.969 Specific G rowthRate 2.0 45 g/L NaCl: S 0.0 P 1.14 0 40 80 120 160 200 53  S 2 COD concentration ( mg/ L COD ) R = 0.947 Fig. C-2 Variation of specific growth rate of mixed bacterial sludge versus COD concentration at different salt contents for glucose-feed wastewater

C-4 Table C-7 Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 20 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 20 18 1.30 6.8 16 0.435 41.8 0.565 0.398 0.43 50 44 1.26 16.0 43 0.410 110.4 0.590 0.415 1.14 100 49 1.46 28.9 47 0.371 121.1 0.629 0.443 1.25 300 118 1.30 80.7 116 0.345 298.3 0.655 0.461 3.08 500 123 1.46 121 311.4 3.21

Table C-8 Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 32 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 45 14 3.81 14.9 10 0.425 26.3 0.575 0.405 0.28 100 37 4.21 30.5 32 0.391 87.1 0.609 0.429 0.92 200 47 3.10 54.8 44 0.351 117.7 0.649 0.457 1.25 300 75 4.20 74.6 71 0.319 189.6 0.681 0.480 2.01 500 77 4.10 73 196.9 2.09

C-5 Table C-9 Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 45 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 20 11 3.51 7.4 7 0.472 16.6 0.528 0.372 0.16 50 13 3.51 17.6 9 0.452 21.8 0.548 0.386 0.21 100 41 3.29 31.6 38 0.405 88.2 0.595 0.419 0.85 300 73 3.29 91.5 70 0.391 163.0 0.609 0.429 1.57 500 70 70 163.0 1.57 Average Y 0.403

4.0 20 g NaCl/L 15 g/L NaCl: 32 g NaCl/L S 45 g NaCl/L P 4.69 -1 3.0 201  S day 2 P R = 0.964

2.0 32 g/L NaCl: S P 3.62 322  S 1.0 Specific G rowthRate R2 = 0.942 45 g/L NaCl: 0.0 S 0 100 200 300 400 500 P 2.46 228 S COD concentration S ( mg/ L COD)  R2 = 0.943 Fig. C-3 Variation of specific growth rate of yeast sludge versus COD concentration at different salt contents for fish-protein-feed wastewater

C-6 Table C-10 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 20 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 20 100 3.73 7.2 96 0.461 223.2 0.539 0.380 2.15 50 148 3.43 17.4 145 0.445 337.4 0.555 0.391 3.25 100 196 4.16 32.1 192 0.411 446.3 0.589 0.415 4.30 150 221 4.26 47.0 216 0.402 503.4 0.598 0.421 4.85 300 220 220 510.7 4.92 Average Y 0.402

Table C-11 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 32 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 20 14.1 3.81 5.5 10 0.352 31.0 0.648 0.456 0.35 36 18.9 2.82 9.7 16 0.345 48.6 0.655 0.461 0.55 100 34.7 3.66 25.0 31 0.321 93.7 0.679 0.478 1.06 200 37.8 2.70 47.0 35 0.301 106.1 0.699 0.492 1.20 300 50.2 4.3 46 138.9 1.57 Average Y 0.471

C-7 Table C-12 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 45 g/L salt

OC/S S Rx,t Rx,e OC Rx,ox Rx YCOD Yvss P -1 mg/L COD mg O2/g VSS.h mg O2/gVSS. h mg/L mg O2/g VSS.d mg COD/g VSS.d g COD/g COD g VS/g COD day 20 14.8 4.06 7.0 11 0.449 24.9 0.551 0.388 0.24 50 23.7 3.65 17.1 20 0.438 46.7 0.562 0.396 0.45 100 33.3 2.94 32.4 30 0.415 70.6 0.585 0.412 0.68 150 43.2 3.93 48.9 39 0.418 91.3 0.582 0.410 0.88 300 41.8 3.9 38 88.2 0.85 Average Y 0.401

15 g/L NaCl: S 5.0 P 5.65 33  S 2

-1 R = 0.982 4.0 day

P 20 g NaCl/L 32 g/L NaCl: 3.0 32 g NaCl/L S P 1.95 45 g NaCl/L 93  S 2.0 R2 = 0.973

Specific G rowthRate 45 g/L NaCl: 1.0 S P 1.11 64  S 0.0 2 0100200300 R = 0.944 COD concentration S ( mg/ L COD) Fig. C-4 Variation of specific growth rate of mixed bacterial sludge versus COD concentration at different salt contents for fish-protein-feed wastewater

C-8 Table D-1 Profile data of pH and nitrogen components of mixed yeast culture with glucose- feed wastewater at 32 g salt/L (mean MLSS = 9500 mg/L)

Time pH COD HN 3-N ON 3ON+ 2- N Total-N N removal h mg/L mg/L mg/L mg/L % 0 4950 365 365 0.25 3.50 1.25 2.88 1200 317 N/A 317 13.2 2.50 2.72 2750 267 2.13 269 26.3 3.50 2.64 N/A 237 2.00 239 34.5 6.50 2.60 950 172 2.01 174 52.3 9.00 2.65 255 139 2.05 141 61.4 11.50 2.71 210 127 2.00 129 64.7 14.50 2.80 245 117 2.20 119 67.3 17.00 2.85 N/A 112 N/A 112 69.3

Table D-2 Profile data of pH and nitrogen components of mixed bacterial culture with glucose-feed wastewater at 32 g salt/L (mean MLSS = 3650 mg/L)

Time pH COD NH 3-N ON 3ON+ 2- N Total-N N removal h mg/L mg/L mg/L mg/L % 0 1020 53 0 53 0.25 7.50 760 1.25 7.76 320 2.25 8.10 235 43 0.70 44 17.5 3.50 8.26 160 32 1.74 34 36.3 6.50 8.28 45 20 1.72 21 59.8 8.00 8.31 30 17 1.85 19 64.8 11.50 8.28 35 14 1.90 16 70.0 14.50 8.25 25 14 2.50 17 68.9

D-1 Table D-3 Profile data of pH and nitrogen components of mixed yeast culture with fish- protein-feed wastewater at 32 g salt/L (mean MLSS = 5810 mg/L)

Adjusted ON NO+ - Time COD pH Organic-NH N -N 3 2 Total N N removal pH 3 N h mg/L mg/L mg/L mg/L mg/L % 0.0 5010 745 45 ND 790 2.3 3.57 3.50 560 5.5 3234 4.26 3.47 63 ND 7.5 2707 3.78 3.45 458 97 554 37.8 10.5 2392 4.06 3.52 127 ND 16.0 2191 4.79 3.51 393 161 554 37.8 18.5 2031 4.00 3.46 209 ND 20.5 1913 4.14 3.50 290 234 524 41.3 26.0 4.37 3.56 29.0 968 4.09 3.50 109 329 1.20 438 50.9 32.0 772 4.21 3.52 31 396 1.50 427 52.1 34.0 830 4.63 3.50 38.0 584 4.47 3.41 424 2.10 42.0 541 3.99 3.46 16 430 2.30 446 50.0 D-N oneN detected

Table D-4 Profile data of pH and nitrogen components of mixed bacterial culture with fish- protein-feed wastewater at 32 g salt/L (mean MLSS = 3720 mg/L)

ON ON+ - Time COD pH Organic-N HN-N 3 2 Total N N removal 3 N h mg/L mg/L mg/L mg/L mg/L % 0 976 150 49 0 199 0.25 870 7.50 1.25 645 7.93 120 52 0.5 172 13.6 2.25 568 8.20 67 1.09 3.5 509 8.40 75 1.51 6.5 383 8.55 63 97 1.5 161 19.1 8.5 320 8.54 107 2.5 11.5 200 8.53 110 2.40 14.5 110 8.54 126 3.10 18.5 65 143 2.6 21.0 30 8.52 2.1 142 4.9 149 25.1 26.0 45 8.53 137 4.3 28.7 20 5.1 142 4.9 152 23.6

D-2 Table D-5 H2SO4 amount consumed to maintain pH 3.5 of the mixed yeast culture with fish-protein-wastewater at 32 g salt/L

1N H SO volume Accumulated acid Time pH Adjusted pH 2 4 consumed olumev h mL mL 0.0 0 6.6 2.3 3.57 3.50 5.5 4.26 3.52 0.9 7.5 7.5 3.78 3.47 0.7 8.2 10.5 4.06 3.54 0.9 9.2 16.0 4.79 3.50 1.7 10.8 18.5 4.00 3.46 0.7 11.5 20.5 4.14 3.47 0.7 12.3 26.0 4.37 3.49 0.9 13.2 28.7 4.09 3.55 0.5 13.7 31.7 4.21 3.51 0.6 14.3 34.8 4.63 3.50 0.8 15.2 38.3 4.47 3.41 0.9 16.1 41.8 3.99 3.46 0.5 16.6

Table D-6 Results of optimum pH experiment for mixed yeast sludge cultured with fish- protein-wastewater at 32 g/L salt (COD dose = 50 mg/L; MLVSS = 1190 mg/L)

pH OUR total OUR endo OUR ox mgO2/gVSS.h mgO 2/gVSS.h mgO 2/gVSS.h 2.5 4.2 2.3 2.0 3.0 13.9 4.2 9.7 4.0 14.9 4.2 10.7 5.1 16.1 3.8 12.4 5.5 16.3 3.8 12.5 6.6 14.7 2.9 11.8 7.5 14.9 2.7 12.1 7.9 13.4 3.0 10.4 8.7 11.7 2.5 9.2 9.1 8.1 2.2 5.8

D-3 Table D-7 Optimum pH experiment for mixed bacterial sludge cultured with fish-protein- wastewater at 32 g/L salt (COD dose = 50 mg/L; MLVSS = 1380 mg/L)

pH OUR total OUR endo OUR ox

mgO2/gVSS.h mgO 2/gVSS.h mgO 2/gVSS.h 4.5 2.8 1.10 1.7 5.3 7.8 2.31 5.5 6.3 14.8 4.14 10.7 7.6 21.3 5.45 15.8 8.1 21.0 5.23 15.3 8.9 21.0 5.88 15.6 9.7 17.7 4.8 12.9 10.1 12.0 3.4 8.6 10.8 7.1 2.2 4.9 10.9 7.5 2.3 5.2

Table D-8 Variation of MLSS during SRT experiments (Unit: mg/L) Time SRT day 5d 7d 10d 20d 45d 0 9000 9000 9000 9000 9000 3 6200 7950 8400 9100 9150 5 4140 7570 8300 8800 10130 7 3640 6800 8000 9300 9600 9 3920 6500 8500 8910 9860 11 2490 6200 8700 9140 9550 13 2770 6550 7640 9400 10270 15 2820 5770 8330 8900 10710 17 2950 5820 7770 9440 10230 19 3230 5110 8150 9740 10320 21 3450 5360 7950 9300 10510 25 3380 5210 8250 9520 10130

D-4 Table D-9 Experimental data of SRT ariationv runs for mixed yeast batch with fish-protein- feed wastewater at 32 g/L salt (Inititial COD = 5000 mg/L, HRT = 24 h)

SRT = 5 d SRT = 7 d SRT = 10 d Mean Mean Mean Parameter COD TKN COD TKN COD TKN eff eff MLSS eff eff MLSS eff eff MLSS mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L Batch 1 3120 467 3230 2075 532 5115 1054 495 8150 Batch 2 2655 534 3450 1811 502 5355 1145 497 7950 Batch 3 2776 572 3380 1964 512 5210 952 534 8250 Average 2850 524 3340 1950 515 5330 1050 509 8117 (continuous) SRT = 20 d SRT = 45 d Mean Mean Parameter COD TKN COD TKN eff eff MLSS eff eff MLSS mg/L mg/L mg/L mg/L mg/L mg/L Bach 1 854 517 9740 937 521 10320 Bach 2 1023 572 9295 925 576 10520 Bach 3 845 515 9525 988 553 10130 Average 907 535 9520 950 550 10320

Table D-10 Result of SRT experiment

SRT 5 7 10 20 45 Volume of wasted sludge, mL/d 400 286 200 100 44 F/M, g COD/g MLSS.d 1.54 0.93 0.62 0.53 0.49 COD removal, % 43 61 77 82 85 N removal, % 7.24 8.90 9.83 5.22 2.67 Amount of SS produced, mg/d 649 765 809 475 228 Amount COD removed, mg/d 2150 3050 3850 4100 4250 Observed yield coefficient 0.302 0.251 0.210 0.116 0.054

D-5 Table E-1 Experimental data for determination of initial membrane resistance of two membrane modules (A = 0.42 m2; pore size = 0.1 Pm, temperature = 31.7oC ) a. Module 1 b. Module 2 Pressure Pressure Flowrate Flux Flowrate Flux (L/h) (L/m2.h) (L/h) (L/m2.h) (mmHg) (kPa) (mmHg) (kPa) 12.8 30.5 37 4.93 2.6 6.1 6 0.80 10.4 24.6 31 4.13 4.2 10.1 12 1.60 8.9 21.1 27 3.60 7.1 17.0 22 2.93 7.3 17.4 22 2.93 10.0 23.9 31 4.13 5.9 14.1 18 2.40 12.7 30.2 37 4.93 4.3 10.2 12 1.60 2.7 6.3 6 0.80 1.2 2.8 2.5 0.33

Table E-2 Experimental data of YMBR in high COD loading

Time HRT F/M L MLSS CODinf CODeff COD% Time Pressure (day) (h) g/g.d (kg/m3.d) (mg/L) (mg/L) (mg/L) (%) (day) (kPa) 0 23.7 3650 4830 1691 0 0.3 5 24.2 0.60 4.8 8100 4780 1162 80 4 0.5 11 24.0 0.32 4.8 14200 4870 998 81 8 0.9 13 14100 4950 1105 16 12900 4750 1050 17 23.8 0.39 5.3 13500 5200 1456 83 13 0.0 19 24.4 0.34 5.0 12000 5100 969 85 19 0.8 22 23.5 0.23 3.4 13600 5120 870 86 25 0.9 25 32.3 0.23 3.4 11700 4780 884 86 30 0.8 29 31.7 0.25 3.3 11000 4900 784 84 33 1.0 31 32.2 0.53 6.3 10500 4780 1147 87 37 0.9 35 18.3 0.66 6.5 9890 4850 1261 76 39 0.9 38 17.9 0.69 7.0 10120 5290 1323 74 43 1.2 40 18.2 0.68 6.4 9530 4940 1136 75 46 1.2 43 18.4 0.93 9.7 10440 4882 1528 77 48 1.6 45 12.0 0.95 10.1 10610 5128 1436 71 51 2.0 47 12.2 0.88 9.9 11250 5100 1683 69 53 2.0 51 12.4 0.87 9.8 11200 4970 1541 68 54 2.3 54 12.2 1.43 14.7 10260 4872 1705 69 59 3.3 56 8.0 1.49 16.9 11290 5000 1909 62 62 4.3 59 7.1 1.59 17.1 10710 5120 2340 54 64 4.3 62 7.2 1.47 16.8 11450 4972 1756 65 67 15.0 65 7.1 2.22 23.3 10530 4870 3120 39 70 45.0 73 5.0 2.04 21.8 10730 4670 2870 37 73 54.0 75 5.1 2.15 23.7 11050 5120 3215 41 75 62.0 78 5.2 2.15 10050 5230 3430 76 65.0 79 65.0

E-1 Table E-3 Experimental data of BMBR in high COD loading

Time HRT F/M L MLSS CODinf CODeff COD% Time Pressure (day) (h) g/g.d (kg/m3.d) (mg/L) (mg/L) (mg/L) (%) (day) (kPa) 0 12.4 4005 1115 321 71.2 1 3 2 12.7 5200 1097 307 72.0 3 5 4 12.7 6200 1176 312 73.5 5 6 5 13.8 2.11 0.35 5970 1216 216 82.2 6 8 6 13.9 2.11 0.32 6700 1224 160 86.9 7 10 9 15.1 2.00 0.23 8600 1258 198 84.3 8 14 11 15.3 1.92 0.18 10950 1224 132 89.2 9 32 11 9.7 2.97 0.24 12500 1200 228 81.0 10 43 12 9.2 3.17 0.27 11540 1215 172 85.8 11 61 14 8.3 3.64 0.22 16500 1260 142 88.7 12 1 16 8.2 3.78 0.22 17450 1290 232 82.0 16 4 19 10.0 3.44 0.20 17400 1435 202 85.9 17 5 21 4.8 6.98 0.35 19800 1395 272 80.5 18 8 21 4.0 8.52 0.42 20100 1420 450 68.3 19 60 24 5.0 6.98 0.35 19850 1455 520 64.3 21 13 26 4.0 9.24 0.50 18500 1540 550 64.3 22 18 31 5.0 4.90 0.25 19400 1020 126 87.6 23 23 31 4.2 5.80 0.32 18200 1015 158 84.4 23 47 32 4.5 5.46 0.27 20050 1023 99 90.3 24 73 34 5.8 5.21 0.23 22400 1260 158 87.5 23 1 39 15.5 1.87 0.09 20150 1210 120 90.1 25 9 40 16.1 1.52 0.07 22500 1020 95 90.7 26 18 41 16.5 1.92 0.10 18950 1320 158 88.0 28 39 41 16.3 1.65 0.08 21750 1120 97 91.3 29 57 44 12.3 2.61 0.13 19970 1340 154 88.5 30 65 46 12.5 1.99 0.10 19750 1035 123 88.1 32 17 49 12.1 2.01 0.11 18769 1012 117 88.4 33 36 51 13.2 2.45 0.12 19700 1347 145 89.2 35 74 51 10.5 2.81 0.13 21720 1230 110 91.1 37 17 54 10.3 2.37 0.11 21434 1015 134 86.8 38 27 60 10.7 2.20 0.11 20352 980 85 91.3 41 2.0 63 3.2 9.38 0.48 19500 1250 320 74.4 43 2.7 66 3.7 9.41 0.45 21000 1450 420 71.0 46 5.0 70 4.1 8.78 0.43 20500 1500 570 62.0 48 7.0 72 3.2 10.50 0.53 19700 1400 630 55.0 49 15 75 3.6 9.00 0.42 21200 1350 790 41.5 50 35 51 65.0

E-2 Table E-4 Experimental data of YMBR in low COD loading

Time HRT F/M L MLSS CODinf CODeff COD% Time Pressure (day) (h) g/g.d (kg/m3.d) (mg/L) (mg/L) (mg/L) (%) (day) (kPa) 1 9.7 2.60 0.51 5120 1050 263 75 1 0.67 3 9.0 2.59 0.43 6040 970 233 76 3 0.93 5 8.3 2.66 0.44 6100 920 288 69 5 1.20 7 8.1 2.98 0.49 6100 1005 185 82 7 0.93 9 8.2 3.22 0.56 5700 1100 150 86 9 0.80 11 9.1 2.24 0.42 5400 850 45 94.7 9 0.80 12 9.2 2.48 0.52 4750 950 24 97.5 12 0.93 13 8.7 2.39 0.52 4600 866 25 97.1 13 0.93 15 8.7 2.61 0.47 5500 945 80 91.5 15 0.93 17 9.0 2.69 0.54 4970 1009 62 93.9 17 2.00 18 7.5 3.17 0.57 5520 990 40 96.0 17 2.00 19 7.8 2.99 0.58 5200 972 35 96.4 19 2.00 21 7.8 2.74 0.55 4950 892 45 95.0 21 1.86 22 7.5 3.23 0.67 4790 1010 24 97.6 22 2.13 23 7.5 2.86 0.58 4950 893 95 89.4 23 2.13 24 8.1 2.71 0.56 4800 913 80 91.2 24 6.00 25 6.1 3.44 0.72 4750 874 110 87.4 24 6.00 27 6.5 3.58 0.87 4133 970 170 82.5 27 6.00 29 5.9 3.46 0.80 4300 850 140 83.5 29 6.00 31 5.8 4.18 0.92 4560 1010 170 83.2 31 9.0 33 4.9 4.46 1.00 4450 910 240 73.6 31 9.0 35 5.1 4.00 0.86 4670 850 180 78.8 35 25.0 37 5.2 4.57 0.98 4670 990 270 73 36 45.0 38 5.1 4.64 1.03 4520 985 240 75.6 38 62.0 41 4.8 5.28 1.21 4350 1056 240 77.3 45 65.0 46 7.5 3.42 0.70 4900 1070 51 95.2 45 0.52 50 7.0 3.60 0.74 4850 1050 20 98.1 49 0.67 52 7.0 4.01 0.78 5120 1170 45 96.2 50 0.67 54 6.9 3.51 0.63 5600 1009 65 93.6 56 0.67 56 6.8 3.35 0.54 6200 950 45 95.3 56 0.67 59 6.3 4.03 0.39 10400 1059 70 93.4 60 1.20 62 6.2 4.53 0.42 10900 1170 45 96.2 61 2.30 64 6.2 3.91 0.34 11500 1009 20 98.0 62 2.30 66 5.8 4.63 0.35 13200 1120 65 94.2 62 2.30 67 5.3 5.00 0.38 13500 1120 75 93.3 66 5.00 70 5.4 5.00 0.35 14200 1125 65 94.2 66 5.00 71 5.0 5.09 0.36 14300 1060 120 88.7 70 12.0 72 5.3 4.51 0.32 14000 995 110 88.9 71 25.0 73 5.3 5.07 0.35 14700 1120 95 91.5 72 32 75 5.3 4.94 0.33 15050 1090 95 91.3 73 63 77 4.0 6.75 0.47 14500 1125 148 86.8 75 65 80 4.2 6.05 0.40 15150 1059 190 82.1 82 4.0 6.36 0.43 14750 1060 135 87.3 76 1.5 84 4.0 7.00 0.46 15200 1170 190 83.8 76 1.5 86 4.0 6.27 0.40 15500 1050 150 85.7 79 1.7 89 3.9 6.89 0.46 15100 1120 101 91.0 80 2.3 81 2.5

E-3 Table E-5 Experimental data of BMBR in low COD loading

Time HRT F/M L MLSS CODinf CODeff COD% Time Pressure (day) (h) g/g.d (kg/m3.d) (mg/L) (mg/L) (mg/L) (%) (day) (kPa) 1 7.8 3.23 0.29 5500 1050 50 95.2 1 20.0 3 8.1 2.87 0.28 4750 970 20 97.9 2 33.3 6 8.4 2.63 0.28 4500 920 45 95.1 3 79.8 9 7.9 3.34 0.46 5200 1100 25 97.0 4 82.5 12 8.2 2.78 0.51 4650 950 15 98.0 5 85.1 15 5.9 3.84 0.82 4700 945 45 95.2 7 20.0 17 6.5 3.73 0.76 4920 1009 45 95.5 8 33.3 19 6.0 3.89 0.86 4530 972 25 97.4 9 59.9 21 6.5 3.29 0.77 4300 892 20 97.8 10 79.8 25 6.8 3.08 0.68 4550 874 20 97.7 12 86.5 27 4.9 4.75 0.96 4950 970 110 88.7 13 86.5 29 5.1 4.00 0.78 5120 850 45 94.7 15 27.9 31 5.5 4.41 0.82 5400 1010 90 91.1 16 46.6 33 4.9 4.46 0.69 6500 910 75 91.8 17 85.0 35 5.2 3.92 0.62 6300 850 45 94.7 19 23.9 37 5.6 4.24 0.70 6100 990 70 92.9 20 59.9 41 6.8 3.73 0.61 6100 1056 20 98.1 21 86.0 42 6.8 4.13 0.70 5900 1170 45 96.2 22 86.5 44 7.1 3.21 0.59 5450 950 15 98.4 23 27.9 46 7.3 3.52 0.53 6700 1070 20 98.1 24 53.2 48 7.4 3.58 0.44 8100 1103 45 95.9 25 86.5 50 5.7 4.42 0.47 9500 1050 110 89.5 27 33.3 52 5.9 4.76 0.45 10600 1170 65 94.4 29 85.0 54 6.3 3.84 0.37 10500 1009 25 97.5 30 85.1 56 5.9 3.29 0.28 11700 810 20 97.5 31 86.5 59 4.4 5.78 0.48 12050 1059 45 95.8 34 58.5 62 4.7 5.97 0.46 12950 1170 95 91.9 35 82.0 64 5.2 4.66 0.35 13200 1009 65 93.6 36 85.1 66 4.6 5.84 0.41 14300 1120 76 93.2 37 86.5 67 3.9 6.89 0.46 15070 1120 155 86.2 42 20.0 70 4.1 6.59 0.45 14700 1125 120 89.3 43 32.0 71 4.3 5.92 0.38 15700 1060 45 95.8 44 78.0 72 3.8 6.28 0.38 16500 995 120 87.9 45 81.0 73 4.1 6.56 0.40 16300 1120 105 90.6 46 86.0 75 4.3 6.08 0.35 17400 1090 110 89.9 47 86.5 77 4.5 6.13 0.35 17100 1150 76 93.4 48 86.0 80 4.0 6.35 0.38 16900 1059 76 92.8 49 25.0 82 4.2 6.06 0.35 17200 1060 110 89.6 50 42.0 51 65.0 52 86.5 53 86

E-4 100

YMBR 90 BMBR 80

70

60

50 COD removal( %)

40

30

20 0 2 4 6 8 10 12 14 16 18 20 22 24 Volumetric Loading (kg COD/m3.d)

Table F-1 Regression analysis of Fig. YMBR BMBR Equation Y = 88.00264 - 1.04453 * X - 0.04398 * X2 Equation Y = 82.7394 + 3.5684* X - 0.5722 * X2

Degree = 2 Degree = 2 Number of data points used = 22 Number of data points used = 34 Average X = 10.45 Average X = 4.71309 Average Y = 70.3889 Average Y = 81.5303

Degree: 0 Coefficients: Residual sum of squares = 4949.26 Degree 0 = 82.7394 Coef of determination, R-squared = 0 Degree 1 = 3.5683 Degree 2 = -0.57221 Degree: 1 Residual sum of squares = 403.182 Orthogonal Polynomial Factors: Coef of determination, R-squared = 0.918537 X Shift = 6.010248447204969 X Scale = 0.4454589472227987 Degree: 2 Residual sum of squares = 350.384 Degree: 1 Coef of determination, R-squared = 0.929205 Residual sum of squares = 1091.09 Coef of determination, R-squared = 0.732687

Degree: 2 Residual sum of squares = 668.19 Coef of determination, R-squared = 0.836296

F-4 1.0

0.8

0.6

0.4

0.2 BMBR CO D removal rate( gCO D / gMLSS.d) YMBR

0.0 0.00.51.01.52.02.5 F/M ratio ( d-1)

Table F-2 Regression analysis of Fig. YMBR BMBR Equation Y = -0.10406 + 1.1556 * X - 0.33699 * X2 Equation Y = -0.03234 + 1.3306 * X - 1.2847 * X2

Degree = 2 Degree = 2 Number of data points used = 22 Number of data points used = 34 Average X = 0.951382 Average X = 0.264056 Average Y = 0.559181 Average Y = 0.202548

Coefficients: Coefficients: Degree 0 = -0.104058111 Degree 0 = -0.03233949848 Degree 1 = 1.155606227 Degree 1 = 1.330610919 Degree 2 = -0.3369879819 Degree 2 = -1.284738775

Degree: 1 Degree: 0 Residual sum of squares = 0.345839 Residual sum of squares = 0.273731 Coef of determination, R-squared = 0.76943 Coef of determination, R-squared = 0

Degree: 2 Degree: 1 Residual sum of squares = 0.0467869 Residual sum of squares = 0.0350683 Coef of determination, R-squared = 0.968807 Coef of determination, R-squared = 0.871888

Degree: 2 Residual sum of squares = 0.0169625 Coef of determination, R-squared = 0.9380

F-4 100

90

80

70 COD removal (%) removal COD 60 SRT of 50 d YMBR BMBR 100

90

80

70 COD removal( %) YMBR 60 SRT of 10 d BMBR

50 4.0 5.0 6.0 7.0 8.0 9.0 10.0

HRT (h)

Table F-3 Regression analysis of Fig. YMBR BMBR SRT 10 days Equation Y = -18.2938 + 26.02184 * X - 1.4905 * X2 Y = 91.219 - 9.232 * X + 2.896 * X2 - 0.2059 * X3

Degree = 2 Degree = 3 Number of data points used = 19 Number of data points used = 14 Average X = 7.13158 Average X = 6.24286 Average Y = 88.2956 Average Y = 94.8309

Coefficients: Coefficients: Degree 0 = -18.29383197 Degree 0 = 91.21936071 Degree 1 = 26.02183739 Degree 1 = -9.231993703 Degree 2 = -1.490548568 Degree 2 = 2.895593329 Degree 3 = -0.2059391806 Degree: 1 Residual sum of squares = 260.884 Degree: 2 Coef of determination, R-squared = 0.799519 Residual sum of squares = 36.227 Coef of determination, R-squared = 0.652589 Degree: 2 Residual sum of squares = 149.022 Degree: 3 Coef of determination, R-squared = 0.885482 Residual sum of squares = 35.8103 Coef of determination, R-squared = 0.656585

F-4 (Continuous) YMBR BMBR SRT 50 days Equation Y = 53.6689 + 10.8237 * X - 0.6825 * X2 Equation Y = 67.0097 + 7.8151 * X - 0.50207 * X2

Degree = 2 Degree = 2 Number of data points used = 21 Number of data points used = 22 Average X = 5.49667 Average X = 5.24091 Average Y = 91.6494 Average Y = 93.4416

Coefficients: Coefficients: Degree 0 = 53.66891328 Degree 0 = 67.00973409 Degree 1 = 10.82366557 Degree 1 = 7.815103512 Degree 2 = -0.6825361179 Degree 2 = -0.5020654975

Degree: 0 Degree: 1 Residual sum of squares = 419.6 Residual sum of squares = 118.846 Coef of determination, R-squared = -2.22045E-016 Coef of determination, R-squared = 0.576309

Degree: 1 Degree: 2 Residual sum of squares = 123.575 Residual sum of squares = 113.208 Coef of determination, R-squared = 0.705495 Coef of determination, R-squared = 0.59641

Degree: 2 Residual sum of squares = 110.933 Coef of determination, R-squared = 0.735623

F-4