Treatment of Ammonia and Phosphate from Water ...

4 downloads 0 Views 4MB Size Report
and diluted to 1L, stored in a rubber Stoppard boro silicate glassware, out of sunlight to be ...... D. (Ed.), Encyclopedia of Soils in the Environment, vol. 1. Elsevier ...

Treatment of Ammonia and Phosphate from Water Resources Through the Use of Free and Immobilized Microalgae A Thesis Submitted for the Degree of Master of Science as a Partial Fulfillment for the Requirements of the Master of Science Chemistry (Inorganic Chemistry)

By Ahmed Mohamed Bayomi Hassan (B.Sc., Applied Chemistry, Ain Shams University)

To Chemistry Department Faculty of Science Ain Shams University

Supervised by: Prof. Dr. Mohamed Sabry Abdel-Mottaleb Prof. Dr. Hosam Ahmed Shawky Prof. Dr. Amr Mahmoud Abdel Gwad

APPROVAL SHEET “Treatment of Ammonia and Phosphate from Water Resources Through the Use of Free and Immobilized Microalgae”

Submitted by Ahmed Mohamed Bayomi Hassan (B.Sc., Applied chemistry, Ain Shams University) For the Master Degree in Inorganic Chemistry

This thesis has been approved by supervisors committee SUPERVISORS Prof. Dr. Mohamed Sabry Abdel-Mottaleb Professor of Inorganic and Photochemistry, Faculty of science, Ain Shams University Prof. Dr. Hosam Ahmed Shawky Professor of water Chemistry, Director of Egyptian Desalination Research Center of Excellence, Desert Research Center Prof. Dr. Amr Mahmoud Abdel Gwad Professor of Soil Microbiology, Head of Soil Fertility and Microbiology Department, Desert Research Center Head of chemistry Department

Prof. Dr. Hamed Ahmed Younes Derbala

ACKNOWLEDGEMENT

ACKNOWLEDGMENT Acknowledgment First of all, all thanks & praise are to Allah for giving me prosperity & strength to fulfill this work.

Special thanks & deep gratitude are devoted to Prof. Dr. Mohamed Sabry Abdel Mottaleb (Chemistry Department, Faculty of Science, Ain Shams University) for supervision, generous guidance, valuable advice & sincere help throughout this work.

All appreciation & debt are to Prof. Dr. Hosam A. Shawky (Desert Research Center (DRC)) for planning, supervising, offering facilities & fruitful discussions in all steps of this work.

All gratitude & thanks are to Prof. Dr. Amr M. Abd El-Gawad (DRC) for supervising, continuous encouragement & sincere help in field work.

And last, but by certainly no means least, I wish to thank my family for the support they have given. I know without the support and confidence of my family would never have been able to achieve what I have.

I

ABSTRACT

ABSTRACT ABSTRACT Name: Ahmed Mohamed Bayomi Hassan Title of thesis: Treatment of Ammonia and Phosphate from Water Resources Through the use of Free and Immobilized Microalgae. Degree: (M.Sc.) Master of Science thesis, faculty of science, Ain Shams, 2015. Laboratory experiments were performed to study nitrogen and phosphorus uptake by the green microalga Scenedesmus quadricauda. The treatment process was studied using different forms of microalga i.e. free, immobilized and co-immobilized microalga in sodium alginate beads. The study revealed that the maximum removal percentage of 1 mg/l ammonia and phosphate solutions reached up to 100% and 86%, respectively, after 4 days using free microalga; meanwhile, at 5mg/l ammonia and phosphate concentrations, the maximum removal percentage reached 91.8% after 4 days and 61% after 6 days, respectively. To solve the harvesting problem, immobilization process was carried out for the microalga and the removal percentage of immobilized microalga was nearly close to the removal percentage of free microalga. The removal percentage was enhanced by the addition of microalga growth promoting bacteria MGPB Azotobacter chroococcum and Bacillus megatherium, where the removal capacity reached up to 100% for ammonia, and 80.4% for phosphate after 2 and 6 days, respectively, as compared to 100% ammonia, and 61% phosphate after 4 and 6 days, respectively, by using the immobilized microalga. Keywords: ammonia, phosphate, Scenedesmus, immobilized microalga, microalga growth-promoting bacteria.

II

LIST OF ABBREVIATION

LIST OF ABBREVIATION LIST OF ABBREVIATION No. 1

Abbreviations DEFRA

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

WHO EEA MGPB DO RO WEF PAO PVA EPA THM AOX DBPs EBPR HRT AWW GPM SWW PGPB BBM APHA ATP ADP RNA DNA BW

III

Meaning Department for Environment, Food, and Rural Affairs World Health Organization European Environment Agency Microalgae growth promoting bacteria Dissolved oxygen Reverse osmosis Water Environment Federation Polyphosphate accumulating organism Polyvinyl alcohol Environmental Protection Agency Trihalomethanes Absorbable organic halides Disinfectant By-Products Enhanced biological phosphate removal Hydraulic retention time aquarium wastewater sterile enriched natural aquarium wastewater synthetic wastewater media Plant growth-promoting bacteria Bold’s Basel medium American Public Health Association Adenosine Tri-Phosphate Adenosine Diphosphate Ribonucleic acid Deoxyribonucleic acid Brackish water

LIST OF FIGURES

LIST OF FIGURES LIST OF FIGURES Figure No.

Title

Page No.

Figure (1)

Scenedesmus diagram and image

39

Figure (2)

Counting microalga using Sedgwick—Rafter slide.

40

Figure (3)

Neat polymer (a) and immobilized microalga beads (b).

45

Figure (4)

A. chroococcum (a) and B. megatherium (b).

47

Scenedesmus quadricauda at magnification power 200X (a) and Figure (5)

Figure (6) Figure (7) Figure (8)

Figure (9) Figure (10) Figure (11) Figure (12) Figure (13) Figure (14) Figure (15)

IV

400X (b) Time course of ammonia removal and development of chlorophyll (a) at algal concentration ratio (95%). ([NH3] = 1 mg/l & [NH3] = 5 mg/l) Effect of algal concentration ratio on the removal percent of ammonia. ([NH3] = 1 mg/l & [NH3] = 5mg/l) Effect of algal concentration ratio on the developed concentration of chlorophyll (a). ([NH3] = 1 mg/l & [NH3] = 5 mg/l) Time course of phosphate removal and development of chlorophyll (a) at algal concentration ratio (95%). ([PO4-3] =1 mg/l & [PO4-3] =5 mg/l) Effect of algal concentration ratio on the removal percent of phosphate. ([PO4-3] = 1 mg/l & [PO4-3] = 5 mg/l) Effect of algal concentration ratio on the developed concentration of chlorophyll (a). ([PO4-3] = 1 mg/l & [PO4-3] = 5 mg/l) Effect of time on the removal of ammonia, using low, medium and high stocking beads. ([NH3] = 5 mg/l) Effect of time on the removal of phosphate, using low, medium and high stocking beads. ([PO4-3] = 5 mg/l) Effect of time on the removal of ammonia, using co-immobilized low, medium and high stocking beads. ([NH3] =5 mg/l) Effect of time on the removal of phosphate, using coimmobilized low, medium and high stocking beads. ([PO4-3] = 5 mg/l)

51

53 55 56

57 61 62 64 66 68 70

LIST OF TABLES

LIST OF TABLES LIST OF TABLES Table No. Table name Bold’s Basal Medium (BBM). Table(1) Trace metal solution. Table(2)

V

Page No. 35 35

SUMMARY

SUMMARY SUMMARY 1) Chapter (І) Introduction about water importance, water supply approach in Egypt, sources of ammonia and phosphate in water resources, impact and their ways of treatment. 2) Chapter (II) Literature review declared recent general tools of treatment of ammonia and phosphate from contaminated water with a focus on removal of ammonia and phosphate using microalgae. 3) Chapter (III) Includes the experimental part, materials, solutions, their preparation techniques, instruments, analysis used in this work, and the practical procedures that have been carried out in this study. 4) Chapter (IV) The main topics of this work can be classified into the following: A. Isolation and cultivation of Scenedesmus quadricauda microalga S. quadricauda was isolated and purified and applied to remove ammonia and phosphate form artificial samples by utilizing them with different techniques. B. Removal of ammonia and phosphate using free microalga S. quadricauda The maximum removal percentage of 1 mg/l ammonia and phosphate reaches up to 100% and 86% at 4th day, respectively. Meanwhile for 5 mg/l ammonia and phosphate, the maximum removal percentage reaches 91.8% at 4th day for ammonia and 61% at 6th day for phosphate. Ammonia and phosphate were efficiently removed by using free microalga S. quadricauda but the main obstacle was the harvesting problem. C. Application of Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol.

VI

SUMMARY S. quadricauda entrapped in calcium alginate as algal beads was employed to remove inorganic ammonia and phosphate in order to overcome the harvesting problem. The removal percentage for 5 ppm ammonia and phosphate was 100% at 4th day for ammonia and 61% at 6th day for phosphate utilizing the immobilized microalga. The practical work concluded that removal percentage of immobilized microalga was nearly close to the removal percentage of free microalga. D. Application of co-Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol. A combination of microalgae S. quadricauda and a microalgae growth promoting bacterium (Azotobacter chroococcum, and Bacillus megaterium), coimmobilized in small alginate beads, was developed to remove ammonia and phosphate form artificial samples. The removal percentage enhanced by the addition of microalga growth promoting bacteria MGPB A. chroococcum

and

B. megatherium reaching

removal capacity up to 100% ammonia, and 80.4% phosphate at 2nd and 6th days respectively.

VI

CONTENT

CONTENT CONTENT Subject

Page No. I II III IV V VI VII

ACKNOWLEDGMENT ABSTRACT LIST OF ABBREVIATIONS LIST OF FIGURES LIST OF TABLES SUMMARY CONTENT CHAPTER I : INTRODUCTION I.1. Water supply approaches in Egypt I.2. Source of ammonia and phosphate (nutrient) in water resources I.3. Problems of ammonia and phosphate I.4. Different methods for ammonia and phosphate removal I.4.1. Ammonia removal methods A. Biological treatment (controlled nitrification) B. Breakpoint chlorination C. Ion exchange D. Membrane filtration E. Combination of reverse osmosis and biological treatment F. Air stripping G. Emerging technologies H. Nitrification in the distribution system I.4.2. Phosphate removal methods A. Chemical precipitation B. Enhanced biological phosphorus removal I.5. Microalgae for wastewater treatment I.6. Aim of the present work CHAPTER II : REVIEW OF LITERATURE II.1. Review of literatures in the recent general tools for the removal of ammonia and phosphate II.2. Review of literatures concerning the removal of ammonia and phosphate using microalgae II.2.1.Wastewater treatment with suspended microalgae II.2.2.Wastewater treatment with immobilized microalgae II.2.3. Enhanced wastewater treatment with co-immobilized microalgae CHAPTER III : EXPERIMENTAL III.1. Materials III.2. Instruments and methods

VII

2 2 3 5 5 5 5 6 6 6 6 7 8 8 8 8 9 11 12 19 19 23 25

33 33

CONTENT III.3. Solutions III.3.1. Aqueous acetone solution: III.3.2. Magnesium carbonate suspension (1%): III.3.4. Bold’s Basal Medium (BBM) III.3.5. Trace Metal Solution (Micronutrients): III.3.6. Stabilizer reagent (Rochelle salt solution) III.3.7. Nessler reagent III.3.8. Stock ammonium solution 3.8.1. (1 ppm) Ammonia solution: 3.8.2. (5 ppm) Ammonia solution: III.3.9. Vanadate-molybdate reagent: III.3.9.1. Solution A III.3.9.2. Solution B III.3.10. Standard phosphate solution III.3.10.1. (1 ppm) phosphates Solution III.3.10.2. (5 ppm) phosphates Solution III.4. Procedures III.4.1. Isolation and cultivation of Scenedesmus quadricauda microalga III.4.2. Counting phytoplankton organisms using Sedgwick— Rafter slide: III.4.3. Chlorophyll (a) measurement III.4.4. Ammonia measurement III.4.5. Phosphate measurement: III.4.6. Application of S. quadricauda on the removal of ammonia and phosphate from artificial water samples III.4.6.1. Studying the effect of time on two different concentrations of ammonia using fixed concentration of algal suspension: III.4.6.2. Studying the effect of different ratios of microalga against residual ammonia concentration: III.4.6.3. Studying the effect of different concentrations of phosphate with fixed count algal suspension against time: III.4.6.4. Studying the effect of different ratios of microalga against phosphate concentration: III.4.6.5. Time course of development of chlorophyll (a) III.4.7. Preparation of Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removal III.4.7.1. Preparation of composite of sodium alginate and poly vinyl alcohol A. Preparation of 5% sodium alginate

VII

34 34 34 34 35 35 36 36 36 36 36 36 36 36 37 37 37 37 37 38 41 41 42 42

42 42 42 43 43

43 43

CONTENT B. Preparation of 5% poly vinyl alcohol C. Preparation of 2% CaCl2 D. Preparation of Neat polymer III.4.7.2. Preparation of three different concentrations of Immobilized microalga: A. High stocking beads B. Medium stocking beads C. Low stocking beads III.4.8. Application of Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removal III.4.8.1. Time course of ammonia removal using high, medium and low stocking bead III.4.8.2. Time course of phosphate removal using high, medium and low stocking bead: III.4.9. Preparation of three different concentrations of Coimmobilization microalga III.4.9.1. Co-immobilization High stocking beads III.4.9.2. Co-immobilization Medium stocking beads III.4.9.3. Co-immobilization Low stocking beads III.4.10. Application of co-Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removal III.4.10.1. Time course of ammonia removal using coimmobilizing high, medium and low stocking bead III.4.10.2. Time course of phosphate removal using coimmobilizing high, medium and low stocking bead CHAPTER IV : RESULTS AND DISCUSSION IV.1. Identification and classification of the microalgal taxa under investigation: IV.2. Removal of ammonia and phosphate using free microalga S. quadricauda IV.2.1. Removal of ammonia using free microalga S. quadricauda IV.2.1.A. Effect of time and initial concentration of ammonia IV.2.1.B. Effect of algal concentration ratio on ammonia removal IV.2.2. Removal of phosphate using free microalga S. quadricauda IV.2.2.A. Effect of time and initial concentration of phosphate IV.2.2.B. Effect of algal concentration ratio on phosphate removal

VII

43 43 43 44 44 44 44 44

44 46 46 46 47 47 47

47 48

50 52 52 52 54 55 56 59

CONTENT IV.3. Immobilization of microalga S. quadricauda in alginate beads IV.3.1. Removal of ammonia using immobilized microalga S. quadricauda in alginate beads IV.3.2. Removal of phosphate immobilized microalga S. quadricauda in alginate beads

60

IV.4. Co-immobilization of microalga and bacteria in alginate beads IV.4.1. Removal of ammonia using co-immobilized microalga and bacteria in alginate beads. IV.4.2. Removal of phosphate using co-immobilized microalga and bacteria in alginate beads. CHAPTER V : CONCLUSION REFERENCES ARABIC SUMMARY

67

VII

63 65

68 69 71 73

CHAPTER I INTRODUCTION

CHAPTER I INTRODUCTION CHAPTER I INTRODUCTION General outlines: Water is an essential commodity to mankind. With the advent of rapid industries development, cultivation and population growing, this common and abundant commodity is becoming insufficient. As a result, many attempts are done to search for non-conventional water resources of good quality. Unfortunately, the contamination of many water resources is one of the most important problems that restrict the different uses of such water. The quick development of human activities has greatly increased the input of ammonia and phosphate into water bodies. This input induces eutrophication and causes deterioration in natural water quality. As such, the removal of nitrogen and phosphorus from wastewater is a fundamental way to prevent eutrophication and water bloom. Effluents from secondary domestic and agricultural wastewater treatment plants contain high concentrations of inorganic nitrogen and phosphorus that may lead to eutrophication of the water bodies that they discharge (Martínez et al., 2000; Mallick, 2002; De-Bashan et al., 2004). Microalgae offer a low cost and effective approach to remove the excess nutrients and other contaminants because of a high capacity for inorganic nutrient uptake for tertiary wastewater treatment, while producing potentially valuable biomass (Martínez et al., 2000; Muňoz and Guieysse, 2006; Bolan et al., 2004; García et al., 2006; Chevalier et al., 2000). However, one of the major drawbacks of using microalgae in wastewater purification is the harvesting of biomass from the treated effluent (Mallick, 2002; Aslan and Kapdan, 2006).

1

CHAPTER I INTRODUCTION Water treatment is not a recent development, there are many treatment methods are well known and already applied. These methods are sometimes restricted because of technical or economic constraints. The following items will be discussed in this chapter: 1. Water supply approaches in Egypt 2. Source of ammonia and phosphate (nutrient) in water resources 3. Problems of ammonia and phosphate 4. Different treatment methods for contaminated water with ammonia and phosphate. 5. Microalgae for wastewater treatment I.1. Water supply approaches in Egypt Egypt is located in a dry climate zone where rainfall is scarce and the desert covers most of the land. Water resources in Egypt are confined to the withdrawal quota from the Nile water; the limited amount of rainfall; the shallow and renewable groundwater reservoirs in the Nile Valley, the Nile Delta and the coastal strip; and the deep groundwater in the eastern desert, the western desert and Sinai, which are almost nonrenewable. The non-traditional water resources include reuse of agricultural drainage water and treated wastewater, as well as the desalination of seawater and brackish groundwater (Allam, 2007). Egypt is now capable of satisfying its water needs, which are 25% more than the available water resources, through recycling of agriculture wastewater and trapping water losses. The water shortage is the main constraint and a major limiting factor facing the implementation of the country’s future economic (Allam, 2007). I.2. Source of ammonia and phosphate (nutrient) in water resources Excessive nitrogen and phosphorus that washes into water bodies and is released into the air are often the direct result of human activities.

2

CHAPTER I INTRODUCTION The primary sources of nutrient pollution are: 

Agriculture: The use of fertilizers (often composed of phosphorus, nitrogen and potassium, manure, sludge, irrigation water or crop residues) to enhance production can contribute negatively to water quality (Verona et al., 2000).



Storm water: When precipitation falls on our cities and towns, it runs across hard surfaces - like rooftops, sidewalks and roads - and carries pollutants, including nitrogen and phosphorus, into local waterways (Dillon et al, 2005).



Wastewater: Our sewer and septic systems are responsible for treating large quantities of waste, and these systems do not always operate properly discharging into waterways.



In and around the home: Fertilizers, yard and pet waste, and certain soaps and detergents contain nitrogen and phosphorus, and can contribute to nutrient pollution if not properly used or disposed of. The amount of hard surfaces and type of landscaping can also increase the runoff of nitrogen and phosphorus during wet weather (DEFRA, 2008). I.3. Problems of ammonia and phosphate Ammonia in drinking water is not of immediate health relevance, and therefore no health-based guideline value is proposed. However, ammonia can compromise disinfection efficiency, result in nitrite formation in distribution systems, cause the failure of filters for the removal of manganese and cause taste and odor problems (WHO guidelines, 2011). Ammonia in water is an indicator of possible bacterial, sewage and animal waste pollution. Ammonia is a major component of the metabolism of mammals. Exposure from environmental sources is insignificant in comparison with endogenous synthesis of ammonia.

3

CHAPTER I INTRODUCTION Toxicological effects are observed only at exposures above about 200mg/kg of body weight (WHO guidelines, 2011). Ammonia present in the raw water creates a high oxidant demand and decreases disinfection efficiency. The reaction between ammonia and chlorine is very rapid, and ammonia may negatively affect the removal of organic and inorganic compounds such as iron, manganese and arsenic by reducing chlorine’s availability for oxidation (Lytle et al., 2007; White et al., 2009). The presence of ammonia at higher levels is an important indicator of fecal pollution. The reasons for ammonium removal from ground water are as follows: a) Till ammonium ions are not removed from water, manganese cannot be removed. b) Chlorination of water with a larger quantity of ammonium ions will cause formation of unpleasant scented chloramines. c) When ammonium ions are in water, the growth of nitrifying bacteria is accelerated. Because of the metabolic processes of ammonium oxidizing bacteria, the quantities of nitrites and organic compounds increase in water. Therefore, the total growth of bacteria can increase in pipelines. Usually the contaminants mainly removed are: organic substances, nitrogen, phosphorous and solid particles. Discharging organic matter lead to oxygen consumption; heterotrophic bacteria present in the aquatic environment use the organic matter as substrate to grow and hence consuming oxygen. If the oxygen consumption rate is greater than the capability of the atmospheric oxygen to dissolve in water, critical conditions can be reached. In these conditions many living organism die Durand et al., 2011). Excessive nitrogen and phosphorus loading is a major ongoing threat to water quality and lead to increased rates of eutrophication. Nowadays

4

CHAPTER I INTRODUCTION large discharges of input from wastewater treatment plant may result in the permanent eutrophication of a water system. Eutrophication has been identified as a major environmental issue in both freshwater and marine waters in Europe’s environment (EEA, 2001; Smith, 2003; Smith and Schindler, 2009). Phosphorus has been identified as a key grow-limiting nutrient for algae in lakes and reservoirs (Schindler, 1977). Issues associated with freshwater eutrophication include increased algal biomass, decreased water transparency, low dissolved oxygen (DO) levels, increased fish mortality and more frequent incidence of toxic phytoplankton. Therefore, eutrophication related water quality impairment could have very substantial negative economic effects, for example, higher treatment costs and health hazards due to algal toxins for drinking water (Smith, 2003). I.4. Different methods for ammonia and phosphate removal I.4.1. Ammonia removal methods (A) Biological treatment (controlled nitrification) Biological treatment processes are based on the ability of microorganisms (non-pathogenic bacteria) to catalyze the biochemical oxidation or reduction of drinking water contaminants and produce biologically stable water (Rittmann and Snoeyink, 1984). The method involves biological denitrification of wastewater to convert ammonia into harmless ammonia gas with the use of bacteria. The main disadvantages of this process are that growth of denitrifying bacteria is slow and the required reaction time is long. (B) Breakpoint chlorination Breakpoint chlorination can eliminate ammonia from water through the formation of free chlorine residual. Breakpoint chlorination is described as a process in which chlorine demand is satisfied, combined chlorine compounds are destroyed, ammonia is oxidized to form nitrogen

5

CHAPTER I INTRODUCTION gas and free chlorine residual is achieved when additional chlorine is added (Pinter and Slawson, 2003) (C) Ion exchange Ion exchange is a physicochemical process that employs an exchange of ions (cations or anions) in the water to be treated with ions sorbed at the solid phase of the natural or synthetic resins. Cation exchange is capable of removing ammonia from drinking water. Studies have investigated natural zeolites, such as clinoptilolite, bentonite, sepiolite and mordenite (Hodi et al., 1995; Demir et al., 2002; Park et al., 2002; Weatherley and Miladinovic, 2004; Wang et al., 2007), and synthetic resins (Abd El-Hady et al., 2001) for the removal of ammonium ions from water. (D) Membrane filtration The available scientific information on the removal of ammonia from water supplies by membrane technologies is limited. These processes are based on forcing water across a membrane under pressure while the ionic species, such as ammonium, are retained in the waste stream (Cevaal et al., 1995). (E) Combination of reverse osmosis and biological treatment Nagy and Granlund (2008); Quail (2008) and McGovern and Nagy (2010) presented a combined process of an RO (75% water treated) system and biological treatment (25% water treated) to remove inorganic contaminants found in groundwater simultaneously and to address copper corrosion control. The maximum design capacity of the water treatment plant was 6.5 MGD (24 605 m3/day). (F) Air stripping This method involves the removal of ammonia from wastewater by gasifying it with air or steam. The key shortcoming of this method is that it is not cost-effective in achieving low concentration of ammonia in

6

CHAPTER I INTRODUCTION wastewater. Although air stripping is a common practice for removing ammonia from wastewater, its treatment efficiency for drinking water is expected to be marginal due to the low Henry’s Law constant (0.0006 at 20ºC) in combination with relatively low concentrations of ammonia encountered in source water (Crittenden et al., 2005). (G) Emerging technologies Several drinking water treatment technologies for ammonia are being developed but are still primarily in the experimental stage or do not have peer-reviewed information on the effectiveness of pilot-scale or largescale application. Some of the emerging technologies include the following:  Trickling filters: A pilot-scale study evaluated trickling filters for simultaneous biological removal of ammonia, iron and manganese from potable water. Influent ammonia concentrations in the range of 0.5–3.0 mg/L were reduced up to 82% in the finished water under a variety of operating conditions (Tekerlekopoulou and Vayenas, 2007, 2008).  Electrochemical removal: A pilot-scale charge barrier capacitive deionization process is reported as effective in removing total dissolved solids, nitrate and ammonia from water. The process employs an adsorption of ions on the surface of two oppositely charged electrodes. The process achieved ammonia removal up to 88.1% at 1000 mg/L as feed concentration (Broseus et al., 2009).  Submerged membrane bioreactors: Laboratory studies examined the effectiveness of hollow fibre membrane modules directly immersed inside the activated sludge reactors for ammonia removal. Removal efficiencies in the range of 89–98% were achieved by the submerged membrane bioreactors through biological nitrification (Li and Chu, 2003; Tian et al., 2009).

7

CHAPTER I INTRODUCTION (H) Nitrification in the distribution system One of the main concerns related to the presence of ammonia in drinking water is the potential for the formation of nitrite and nitrate, parameters with health risks and drinking water quality guidelines. Nitrite and nitrate are the products of nitrification, a two-step process that oxidizes ammonia either in natural water or in water that has been disinfected

by

chloramine.

The

occurrence

of

nitrification

in

chloraminated distribution systems has been well documented (Skadsen, 1993; Odell et al., 1996; Wilczak et al. 1996). Nitrification in the distribution systems can have adverse impacts on water quality. These impacts include increased nitrite and nitrate levels, reduced chloramine residuals, increased bacterial regrowth (i.e., increased heterotrophic plate count [HPC], with a possible detection of Escherichia coli), as well as a reduction of pH and dissolved oxygen (Lytle et al., 2007; Odell et al., 1996). I.4.2. Phosphate removal methods There are two fundamental ways to remove phosphorus from wastewater: A. Chemical precipitations B. Enhanced biological phosphorus removal. Combinations of these two methods and some other specific technologies are also used for phosphorus removal. (A) Chemical precipitation A common method of Phosphorus removal in wastewater treatment is the chemical precipitation of phosphorus. This is done with the addition of chemical coagulants to the wastewater. These chemical coagulants are made of multivalent cations including aluminum and iron (WEF, 2011). (B) Enhanced biological phosphorus removal Phosphorus removal is performed mainly by a group of microorganisms known as the polyphosphate accumulating organisms

8

CHAPTER I INTRODUCTION (PAO). These organisms can consume and store P in form of intracellular polyphosphate. This fact leads to the decreasing phosphorus content in the liquid phase and a concentration of P in the activated sludge (Mino et al., 1998). I.5. Microalgae for wastewater treatment The above-discussed nitrogen and phosphorus removal methods normally consume significant amounts of energy, chemicals and carbon source, and therefore are cost intensive. Furthermore, chemical based treatments often lead to the contamination of the sludge by-product (Hoffmann, 1998; Tchobanoglous et al., 2003). For those reasons, more researches and further methods have been studied toward development and application of a more economical nitrogen and phosphors removal process. As a novel ‘‘green technology,” microalgae have many advantages in the removal of nitrogen and phosphorus, including the following: (1) low cost due to sufficient solar energy, (2) simultaneous fixation of CO2, (3) lack of extraorganic carbon requirement (as compared to biological nitrification–denitrification), (4) discharge of oxygenated effluents into water bodies, (5) avoidance of sludge handling problems, and (6) high economic potential of harvested algal biomass (for feedstock, fertilizers, biogas, biofuels, and so on) (Aslan and Kapdan, 2006; VergaraFernandez et al., 2008) . De la Noüe (1992) summarized the advantages of using microalgae for wastewater treatment in comparison with conventional methods: firstly, nutrients can be removed more efficiently; secondly, secondary pollution in the by –product caused by chemical additives is avoided, so it does not generate additional pollution; thirdly, when the biomass is harvested, it generates recycling of nutrients; and fourthly, the system is less expensive

9

CHAPTER I INTRODUCTION Microalgae offer a low cost and effective approach to remove the excess nutrients and other contaminants because of a high capacity for inorganic nutrient uptake for tertiary wastewater treatment, while producing potentially valuable biomass (Bolan et al., 2004; García et al., 2006; Chevalier et al., 2000; Abdel-Raouf et al., 2012). However, one of the major drawbacks of using microalgae in wastewater purification is the harvesting of biomass from the treated effluent (James, 1998; Mallick, 2002; Aslan and Kapdan, 2006). Recently, research efforts have increasingly focused on the use of non-suspended algae; either attached or immobilized, as a valid method that avoids the harvesting problem (Vílchez et al., 2001; Jiménez-Pérez et al., 2004). Studies have consistently indicated efficient and rapid removal of nitrogen and phosphorus from wastewater by immobilized algae. Carrageenan, chitosan and alginate are the polymers often used in these algal systems (Hernandez et al., 2006; Shawky et al., 2012), with alginate beads being used most frequently (Thakur and Kumar, 1999; Tam and Wang, 2000; De-Bashan and Yoav, 2004; Susana et al., 2006). Calcium alginate is the most commonly employed system for its easiness in gel formation. Once liquid alginate solutions are contacted with polycation (Ca2+), they immediately transformed into gel by binding between guluronic acid blocks in alginate and Ca2+ (Sugiura et al., 2005; Shawky, 2011). The major advantage of alginate gel entrapment is that immobilized cells do not suffer extreme physical-chemical condition changes during the immobilization process. Permeability, null toxicity and transparency of formed matrix imply a very gentle environment for immobilized cells (Kadimpati et al., 2013). Polyvinyl alcohol (PVA) is a promising polymer due to its attractive characteristics especially for various biotechnological and biomedical

10

CHAPTER I INTRODUCTION applications (Shawky, 2009; Bayramoglu and Arica, 2011). PVA also offers assorted advantages over the conventional alginate matrix including lower production cost, higher robustness, higher degree of chemical stability, and nontoxicity to viable cells (Hassan and Peppas 2000; Shawky, 2009), because the flexibility of PVA beads provides them with stronger mechanical strength compared to the brittle alginate beads. Moreover, PVA beads also demonstrate high stability within the pH range of 1.0– 13.0, whereas alginate beads are relatively stable only in the pH range of 6.0–9.0 (Lozinsky and Plieva 1998). The removal percentage of immobilized microalga was nearly close to the removal percentage of free microalga. The removal percentage enhanced by the addition of microalga growth promoting bacteria MGPB Azotobacter chroococcum and Bacillus megaterium. These bacteria are capable of fixing atmospheric nitrogen and solubilizing phosphorus, it demonstrated that the observed growth promotion might improve the capabilities of microalgae to remove nutrients from natural wastewater (El-Komy, 2005; Ali et al, 2012). 1.6. Aim of work: The aim of this investigation is to evaluate the use of microalga Scenedesmus quadricauda in free, immobilized and co-immobilized form in the removal of ammonia and phosphate from polluted water resource.

11

CHAPTER II LITERATURE REVIEW

CHAPTER II REVIEW OF LITERATURE CHAPTER II REVIEW OF LITERATURE General outlines: This chapter is dealing with the review of literature concerning with the removal of ammonia and phosphate from contaminated water or aqueous solutions. According to the applicable tools in ammonia and phosphate removal process, this chapter will be discussed under the following items: 1- Review of literatures in the recent general tools for the removal of ammonia and phosphate. 2- Review of literatures concerning the removal of ammonia and phosphate using microalgae II.1. Review of literatures in the recent general tools for the removal of ammonia and phosphate: Cooney et al. (1999) undertook a laboratory study of a natural Australian zeolite, clinoptilolite, to remove ammonium from water. They investigated the equilibria and kinetic characteristics of ammonium exchange in the zeolite under binary and multi-component conditions. The results revealed that the highest ammonium removal efficiency was achieved when the zeolite’s exchange sites were converted to the sodium form. For a multi-component system there was a competitive effect between ammonium and other cations such as Ca2+, Mg2+, K+ and the zeolite’s selectivity for ammonium ions was over other cations typically present in sewage. Rozic et al. (2000) investigated the removal of ammonium ion from aqueous solutions using a Croatian clinoptilolite and bentonite. The highest removal efficiency for N–NH4+ (61.1 wt. %) was achieved with the natural zeolite at the lowest initial concentration, i.e. 100 mg N–

12

CHAPTER II REVIEW OF LITERATURE NH4+/L. With the increase of the initial concentration of ammoniacal nitrogen, the removal efficiency quickly decreased. For the clay, the ammoniacal nitrogen removal efficiency was higher in a liquid colloidal state and the acid modification of the clay decreased the efficiency of ammoniacal nitrogen removal. Campos et al. (2013) evaluated the air stripping technology for the removal of ammonia from landfill leachates. In this process, pH, temperature, airflow rate and operation time were investigated. Furthermore, the relationship between the leachate alkalinity and the ammonia removal efficiency during the process was studied. The leachate used in the tests was generated in the Gramacho Municipal Solid Waste Landfill (Rio de Janeiro State, Brazil). The best results were obtained with a temperature of 60oC, and they were independent of the pH value for 7 h of operation (the ammonia nitrogen removal was greater than 95%). A strong influence of the leachate alkalinity on the ammonia nitrogen removal was observed; as the alkalinity decreased, the ammonia concentration also decreased because of prior CO2 removal, which increased the pH and consequently favored the NH3 stripping. The air flow rate, in the values evaluated (73, 96 and 120 L air.h−1. L−1 of leachate), did not influence the results. Disadvantages of this process are Pumping requires higher maintenance, power requirements, Ammonia stripping cannot be performed in freezing conditions, Scale formation can be removed hydraulically in most cases but not all, resulting in a need to pilot test at most locations, Ammonia stripping does not remove nitrite and organic nitrogen, Air pollution problems may result from ammonia and sulfur dioxide reaction, Air stripping often requires the addition of lime to

13

CHAPTER II REVIEW OF LITERATURE control pH, which may create operation and maintenance concerns and Noise may be a problem, (USEPA, 2000). Electrochemical nitrite and ammonia removal from aqueous solution was investigated by (Lin and Wu, 1996). Operating variables of the electrochemical method including the current input, pH, conductivity, buffer solution and initial nitrite and ammonia concentrations were considered to determine their respective effect on the efficiency of nitrite and ammonia removal. The experimental results indicated that the time for complete nitrite removal and power consumption decrease with an increase in conductivity. For nitrite removal, the electrochemical process was found to consume less power when utilizing a current input larger than 2 A. The pH effect on the nitrite removal was observed to be significantly smaller than those of conductivity and current input. While the acidic environment favored the nitrite removal, the alkaline environment appeared to be beneficial to the ammonia removal. The ammonia removal was found to be a much slower process than the nitrite removal. Takó and Laky (2012) mentioned that the breakpoint chlorination is a commonly applied and well-known method to remove the ammonium. The method has been used for decades. The ammonium reacts with the chlorine based on the following reactions (Öllős, 1997).

The ammonium in the water converts into chloramines as a result of these three-step reactions, so called breakpoint chlorination. As a first step monochloro-amine (NH2Cl), then dichloro-amine (NHCl2) are formed followed by, trichloro-amine (NCl3) formation (Sathasivan et al, 2008).

14

CHAPTER II REVIEW OF LITERATURE Theoretically the required chlorine: ammonium-nitrogen ratio is 7.6 to achieve complete ammonium removal (Griffin, 1941). However, in practice higher chlorine dose is needed, since the hypochlorous acid reacts not only with ammonium but the reduced state of iron, manganese, organic materials, etc. Due to the reactions with organic substances, harmful THM (trihalomethanes) or AOX (absorbable organic halides) may be also formed. They can cause cancer and mutagenicity (Takó and Laky, 2012). Therefore, as a last step of this technology usually activated carbon adsorption is needed in order to remove these by-products, catalysing the degradation of trichloroamine to nitrogen gas and also to decrease the chlorine concentration in the finished water. Kumar (2013) studied chlorination of raw Yamuna River water. Samples were chlorinated with increasing doses of standard chlorine water and residual chlorine (Cl2) was measured by Starch- Iodide method. For each sample, the chlorination curve (chlorine residual versus chlorine dose) was obtained. Curves showed the typical irregularity attributed to the formation and destruction of chloramines and transformation of toxic cyanobacteria (blue-green algae) by chlorine. It was observed that, after reactions with strong reductants and chloramines forming compounds, the remaining organic matter exerted a certain demand of chlorine. The evolutions of chlorination curves were studied. Till date, study on breakpoint chlorination revealed only single breakpoint but, in present study single breakpoints during chlorination of raw waters were not established in many cases. Double breakpoints have been reported in this study. The evolutions of different breakpoint curves might be attributed to formation and destruction of numerous chemical disinfection byproducts (DBPs) and biological bacteria/alga, due to variant pollution conditions of raw water.

15

CHAPTER II REVIEW OF LITERATURE The study of (Koyuncu et al. 2001) revealed that the removal efficiency of ammonium ion from a solution containing ammonium ion which is not complexes is varying in the range of 5–60%. On the contrary, ammonium ion forms complexes with other ions in water and wastewater removal efficiency was as high as 99% for the synthetic solution which contained a complex of iron and ammonium ion. Elmali reservoir which is one of the water sources of the Istanbul city is suffering from high ammonia content due to discharges of untreated wastewater. Application of the brackish water membrane to Elmali Reservoir water resulted in ammonia removal efficiency of about 95%. The permeate ammonia concentration of 0.2 mg/l was achieved. High removal ratio of ammonia ions in complex form is due to increased molecular diameters of the ammonium ion complexes. Increased pH values were observed in parallel to increase the ammonium ion concentrations when ammonium ion was not in complex form. Ammonium ion removal efficiency decreased with increasing pH. On the other hand, when the concentration of ammonium ion complexes with iron was increased, pH value was decreased. In parallel to the decreasing pH, an increase of removal efficiency was observed. Membrane types did not significantly affect the flux. No fouling problems were observed during the test runs and thus fluxes were practically constant throughout the experimental run. The total estimated cost of treatment will be in the range of $0.95 to $1.06/m3 for the investigated drinking water source. Janda and Rudovský (1994) studied biological nitrification. The process is performed in fixed-bed open filters with pre-aerated water. Common silica sand covered with manganese oxides is used as a filtration material. Manganese oxides layer serves as a rough support for the biofilm of nitrifying bacteria that are not thus removed from the

16

CHAPTER II REVIEW OF LITERATURE packing during backwashing. Ammonia is under controlled conditions biologically oxidized to nitrate. The process was applied in several water treatment plants in central Bohemian region in full scale. Long term operational results showed that concentration of ammonia up to 3 mg/1 can be removed with higher than 90 % efficiency. For a concentration of ammonia higher than 3 mg/l in raw water two step technologies

has

to

be

applied

(1st

aeration, 1st

filtration, 2nd

aeration and 2nd filtration). Practicability of the two-step arrangement was also successfully verified in full scale. Tekerlekopoulou and Vayenas (2008) constructed and tested a pilot-scale trickling filter with dual layer support material for simultaneous biological removal of ammonia, iron and manganese from potable water. The performance of the trickling filter was tested at constant hydraulic loading of 226 m3/m2 .d while feed concentrations of iron, ammonia and manganese were varied between 0.5 and 4.0, 0.5 and 3.0, and 0.5 and 1.3 mg/l, respectively. The system was inoculated with a mixed culture and a series of experiments was performed to investigate the interactions among ammonia, iron and manganese removal when simultaneously present in the trickling filter. The oxidation reduction potential increased along the filter depth from about 150 to 600 mV, depending on the feed concentrations, thus enabling one-stage simultaneous removal of the three pollutants. Ammonia and iron drastically affected manganese oxidation and manganese was found to be the rate-limiting pollutant. The results are presented using an operating diagram of the system, that determines the range of operating conditions resulting in optimal operation, keeping iron, ammonia and manganese concentration under the maximum permitted limits in potable water

17

CHAPTER II REVIEW OF LITERATURE Mohammed and Shanshool (2009) studied a series of jar test was conducted to evaluate the optimum pH, dosage and performance parameters for coagulants alum and calcium chloride. Phosphorus removal by alum was found to be highly pH dependent with an optimum pH of 5.7-6. At this pH an alum dosage of 80 mg/l removed 83 % of the total phosphorus. Better removal was achieved when the solution was buffered at pH = 6. Phosphorus removal was not affected by varying the slow mixing period; this is due to the fact that the reaction is relatively fast. The dosage of calcium chloride and pH of solution play an important role in phosphorus removal. The removal efficiency increases with increasing pH, and the optimum dosage of CaCl2 was 60 mg/l. Alum demonstrated much better results in phosphorus removal than CaCl2. The tropical cyanobacterium Phormidium bohneri in domestic wastewater removed nitrogen and phosphate after growing 50 and 75 h, respectively. Adding monopotassium phosphate enhanced production of biomass by 56%, but did not significantly affect the time for completely removing these nutrients. The high biomass production by P. bohneri (23–57 mg d.w.L-1 day-1), along with high rates of ammonium and phosphate removal (up to 20 mg/L day-1), indicates that P. bohneri has promise for wastewater treatment (Laliberte et al., 1997). Mino et al. (1998) reviewed microbiological and biochemical aspects of the enhanced biological phosphate removal (EBPR) process. The discussion includes: microorganisms responsible for EBPR, isolation of polyphosphate accumulating organisms (PAOs), microbial diversity of the EBPR sludge, biochemical metabolisms of PAOs, energy budget in PAOs metabolism, denitrification by PAOs, glycogen accumulating nonpoly-P organisms (GAOs), etc. Since pure cultures which possess complete characteristics of PAOs have not been isolated yet, the

18

CHAPTER II REVIEW OF LITERATURE biochemical mechanism cannot be definitively described. The criteria to obtain a pure culture isolate are proposed. Based on the review, essential characteristics of PAOs are summarized in a table and directions for future research are identified. II.2. Review of literatures concerning the removal of ammonia and phosphate using microalgae The potential of microalgae as a source of renewable energy based on wastewater has received increasing interest worldwide in recent decades (Wang et al, 2013). The choice of microalgae to be used in wastewater treatment is determined by their robustness against wastewater and by their efficiency to grow in and to take up nutrients from wastewater (Olguín, 2003). II.2.1.Wastewater treatment with suspended microalgae Algae are composed of about 52% carbon, 9.2% nitrogen, and 1.3% phosphorus according to the algae biomass empirical formula below (Rittmann, 2001). 106 CO₂ + 65 H₂O + 16 NH₃ +H₃PO₄ = C₁₀₆H₁₈₁O₄₅N₁₆P +118 O₂ From this empirical formula, it can be deduced that as algae grow, they will consume carbon dioxide as well as smaller amounts of nitrogen and phosphorus nutrients. A similar empirical formula can be made with nitrate as a nitrogen source instead of ammonia. One of the more useful properties of algal nitrogen uptake is that it is able to uptake nitrogen in either ammonia or nitrate forms. This is important when considering algae as a means for tertiary treatment, because secondary effluent commonly contains high concentrations of nitrate nitrogen, which needs to be eliminated before discharge.

19

CHAPTER II REVIEW OF LITERATURE It is important to select and cultivate an algal culture which has a rapid growth rate, so that constant uptake is possible. Raw wastewater is not the most suitable environment for algal growth because of potentially fierce competition with faster growing bacteria in the presence of high concentrations of organic matter and nutrients, so it is important to select and cultivate a more resilient algae species for wastewater treatment, especially for tertiary treatment applications. Many studies incorporate Chlorella and Scenedesmus sp. for their fast growth rates and high tolerance to unsuitable conditions. These species will likely dominate a treatment system because of these characteristics (Muñoz et al., 2006). Some algae species have been known to store phosphorus in dry weight biomass percentages much higher than their stoichiometrically derived numbers. This can be from the following two mechanisms. The first mechanism features algae’s response to a period of phosphorus deficiency in its environment, followed by exposure to abundant phosphorus in its environment. The result is an overcompensation of sorts, with extra storage of phosphorus in polyphosphate form. The second mechanism is when algae exist in consistently favorable nutrient conditions, and the algae continue to store phosphorus in excess. In both instances, the name ‘luxury uptake’ is used (Eixler et al. 2006). The choice of microalgae to be used in wastewater treatment is determined by their robustness against wastewater and by their efficiency to grow in and to take up nutrients from wastewater (Olguín, 2003). Bio-treatment with microalgae is particularly attractive because of their photosynthetic capabilities, converting solar energy into useful biomasses and incorporating nutrients such as nitrogen and phosphorus causing eutrophication (Abdel-Raouf et al., 2012).

20

CHAPTER II REVIEW OF LITERATURE Martínez et al. (2000) studied the removal of phosphorus and nitrogen by the freshwater alga Scenedesmus obliquus, cultured in urban wastewater, previously submitted to secondary sewage treatment, was studied under different conditions of stirring and temperature. In all cases, the amount of NH3 lost, as well as biomass productivity and its biochemical composition, were evaluated. The specific growth rates proved greatest in the stirred cultures. The stirring increased biomass productivity in the linear growth phase after exponential growth, with the optimum appearing at 25°C. For the temperatures studied stirring was not necessary to provide the highest percentage of phosphorus removal, but did reduce the time needed to reach that percentage.

The highest

percentage of phosphorus removal was 98%, within the shortest time period at 94.33 h. Ammonium removal was determined by two factors, the consumption of ammonium for growth and elimination by desorption as ammonia.

The highest percentage of ammonium removal, 100%,

resulted at the final culture time of 188.33 h, in the stirred culture at 25°C. Wang et al. (2013) investigated the ability of fresh water microalga Chlarella sp. to remove both nitrogen and phosphorus from influent and effluent wastewaters which were diluted in four different proportions (namely, 100%, 75%, 50% and 25%). Chlarella sp. grew fastest under 50% influent and effluent wastewaters culture conditions, indicating the levels of nitrogen and phosphorus greatly influenced algal growth. High removal efficiency for total nitrogen (17.04-58.85%) and total phosphorus (62.43- 97.08%) was achieved. Further, more than 83% NH4-N in 75%, 50%, 25% influent wastewater, 88% NOX-N in effluent wastewater and 90% PO4-P in all treatments were eliminated after 24 days of incubation. Chlarella sp. grew well when PO4-P concentration

21

CHAPTER II REVIEW OF LITERATURE was very low, indicating that this might be not the limiting factor to algal growth. Our results suggest the potential importance of integrating nutrient removal from wastewater by microalgae cultivation as biofuel production feedstock. Han et al. (2014) studied four microalgae including Chlorella sp. SDEC-10, Chlorella ellipsoidea SDEC-11, Scenedesmus bijuga SDEC12 and Scenedesmus quadricauda SEDC-13 isolated from a local lake have been investigated for the properties of growth, nutrient removal and lipid accumulation in synthetic sewage. Their biomass ranged between 0.4 and 0.5 g/L. The total phosphorus removal efficiency of four strains was nearly 100%, but in the case of total nitrogen and ammonium the removal efficiency was relatively low. Their lipid content, ranging from 25.92% to 27.76% and corresponding to the lipid productivity 7.88–18.08 mg/L/d, was higher than that obtained in BG-11. Palmitic acid and oleic acid were the predominant compositions found through fatty acids analysis. S. quadricauda SDEC-13 performed best both in nutrient removal and in lipid production among the four strains. One of the main obstacles in establishing algal wastewater treatment as a dependable large scale method is the difficulty of biomass harvesting. Algae do not settle as well as bacteria in suspended growth processes. Thus, the traditional clarifier approach to biomass separation does not work well enough to keep biomass out of the effluent. If biomass remains in the effluent, not only are treatment plants missing a percentage of biomass that can be used for fuel or byproduct production, but also the nutrients and metals absorbed during biomass growth remain in the effluent, leading to water quality issues. Common industrial methods of harvesting

include

centrifugation,

filtration,

flocculation,

gravity

sedimentation, flotation, and recently studied electrophoresis techniques

22

CHAPTER II REVIEW OF LITERATURE (Chen and Yeh 2011). Thus far, none of the previously mentioned harvesting methods have proven themselves to be efficient or cost effective enough to be widely implemented (Muñoz et al., 2006). II.2.2.Wastewater treatment with immobilized microalgae A more synthetic approach to solving problems resulting from the difficulty of handling algal biomass is algae immobilization. Immobilized cells are prevented by natural or artificial means from moving independently to all parts of an aqueous system. The most common method of algae immobilization is flocculation in beads of alginate polymer. The polymer has pores smaller than algae diameter, which allow nutrients to enter the bead, and keeps biomass from exiting. The immobilization of algae allows algae to be more easily harvested, while the algae can still treat water by consuming nutrients. Immobilization often causes positive effects such as protection from other external aggressive organisms, reduction in competition with other organisms for nutrients, and improvements in metabolism and behavior of algae. Multiple tests indicate the superiority of immobilized algae over suspended cells in nutrient removal and metal removal rates (De-Bashan and Bashan, 2010). An immobilized cell is defined as a cell that by natural or artificial means is prevented from moving independent of its neighbours to all parts of the aqueous phase of the system. A major advantage of alginate gel entrapment is that immobilized cells do not suffer extreme physical chemical condition changes during the immobilization process. Permeability, null toxicity and transparency of formed matrix imply a very gentle environment for immobilized cells (Abdel Hameed and Hammouda, 2007)

23

CHAPTER II REVIEW OF LITERATURE Ruiz-Marin and Mendoza-Espinosa (2008) evaluated the ammonia removal efficiency, growth, biomass production, chlorophyll a, protein and lipids content of the microalgae Scenedesmus obliquus immobilized in alginate in semi-continuous bioreactors for the treatment of secondary effluent. Six 35 h experimental cycles were run. The growth of algal cells in the alginate matrix was not inhibited and the exponential growth rates were 0.273 d-1 and 0.374 d-1 for cycles II and III, respectively. Biomass harvest levels of 833.3 – 937.5 mg/l dry weight showed a high protein and lipids content. The low cell density in the first cycles allowed the cells to grow satisfactorily; in subsequent cycles, nitrogen removal and protein and lipids content decreased probably due to nitrogen and light limitation after 181 h of experimentation. The high pH reached within reactors appeared to have contributed to NH3 stripping from the medium of 37%-59% of the total initial ammonia. Part of the available nitrogen was incorporate into new protein (10.6 to 11.4 mg N/ l); but this was not only due to ammonia uptake, since one part of this is lost to the atmosphere. These results suggested that immobilized S. obliquus cells can be used as secondary treatment process for domestic wastewater and biomass with higher protein and lipid content than free cell cultures can be produced. Boonchai et al. (2012) determined the performance of microalgae photobireactor for advanced wastewater treatment and microalgal biomass production, Chlorella vulgaris was cultured with final effluent from sewage treatment plant in batch condition. The average specific growth rate was 0.103 d-1 because low P concentration inhibited algal growth. 60% of N and P concentrations were removed from the system in 2 days. When the system operated under a semi-continuous condition with hydraulic retention time (HRT) 2 days, the microalgae showed

24

CHAPTER II REVIEW OF LITERATURE growth rate of 0.452 d-1 and 0.277 d-1 in primary effluent and final effluent, respectively. 30% of N and 53% of P were removed from primary effluent and 44% of N and 84.2% of P were removed from final effluent. These results suggest that semi-continuous mode offers higher biomass production and appropriate HRT were needed for high N and P removals. II.2.3. Enhanced wastewater treatment with co-immobilized microalgae The most unusual combination of microalgae and bacteria suggested so far for wastewater treatment is to use plant growth-promoting bacteria (PGPB), used in agriculture (Bashan and de-Bashan, 2005), to enhance the growth and nutrient removal capacity of microalgae. The bacterial species of choice so far belongs to the genus Azospirillum and is widely used as an inoculant to promote the growth and yield of numerous crop plants, mainly by affecting hormonal metabolism and mineral absorption of the plants (Bashan et al., 2004). The underlying hypothesis assumes that the bacteria will enhance the performance of unicellular plants, that is, microalgae, and that the single-cell plant will respond to bacterial inoculation like higher plants (de-Bashan and Bashan, 2008). Immobilization of C. vulgaris and C. sorokiniana with Azospirillum brasilense in small alginate beads significantly enhanced all the growth parameters of the microalgae, including the general population, colony size, biomass, and in some strains, cell size (De-Bashan and Bashan, 2008; De-Bashan et al., 2002a; Gonzalez and Bashan, 2000). Furthermore, these artificial combinations, thus far not found in natural habitats, profoundly changed many cytological, physiological, and biochemical pathways and metabolites within the microalgal cells, such as photosynthetic pigments, lipid content, and the variety of fatty acids

25

CHAPTER II REVIEW OF LITERATURE (De-Bashan and Bashan, 2008; De-Bashan et al., 2002a, 2005; Gonzalez-Bashan

et

al.,

2000;

Lebsky

et

al.,

2001).

Duo

immobilization under semi-continuous synthetic wastewater cultivating conditions, significantly increased removal of ammonium and soluble phosphate ions, compared to microalgae immobilized alone (De-Bashan et al., 2002b; De-Bashan and Bashan, 2003; Yabur et al., 2007). Recently, these combinations were successful in reducing ammonium and phosphate levels of municipal wastewater (De-Bashan et al., 2004; Hernandez et al., 2006). Consequently, A. brasilense a PGPB has been called a ‘‘microalgae growth-promoting bacteria” or MGPB (De-Bashan et al., 2002a, b, 2004, 2005). Immobilization of the diazotrophic PGPB Bacillus pumilus from arid region soils with C. vulgaris did not enhance the capacity of the microalgae to remove nitrogen and phosphorus from the medium, as a culture. However, when the capacity of cells was evaluated, each cell could remove more nutrients (Hernandez et al., 2009). The mechanisms by which bacteria affect growth and nutrient metabolism of microalgae have only recently been investigated. General analysis of numerous experiments show that immobilization of microalgae with A. brasilense could result in two independent phenomena. These phenomena are directly affected by the nature of the nitrogen compound, the pH, and the presence of carbon in the substrate. First, growth of the microalgal population increased without an increase in the capacity of the single cells to take up nitrogen, or second, the capacity of cells to take up nitrogen increased without an increase of the total microalgal population. These phenomena were dependent on the population density of the microalgae, which was in turn affected by cultivation factors. This supports the conclusion that the size of the

26

CHAPTER II REVIEW OF LITERATURE microalgal population controls the uptake of nitrogen in C. vulgaris cells—the higher the population, the less nitrogen each cell takes up (DeBashan et al., 2005). The cellular mechanisms are probably related to (1) enhancement of enzymes involved in nitrogen metabolism of the microalgae, such as glutamate dehydrogenase and glutamine synthetase (De-Bashan et al., 2008b) and (2) the capacity of the PGPB to produce phytohormones that increase microalgae growth. Mutants of the PGPB that did not produce sufficient phytohormone indole- 3 acetic acid also failed to affect growth and metabolism of the microalgae (De-Bashan et al., 2008a). Starvation also plays a key role in this association by having a synergistic effect on absorption of phosphorus from wastewater and merits consideration in designing future biological treatments of wastewater (De-Bashan and Bashan, 2010). De-Bashan et al. (2004) studied the potential of co-immobilization of microorganisms in small beads to serve as a treatment for wastewater in tropical areas. A combination of microalgae (Chlorella vulgaris or C. sorokiniana) and a microalgae growth-promoting bacterium (MGPB, Azospirillum brasilense strain Cd), co-immobilized in small alginate beads, was developed to remove nutrients (P and N) from municipal wastewater. A. brasilense Cd significantly enhanced the growth of both Chlorella species when the co-immobilized microorganisms were grown in wastewater. A. brasilense is incapable of significant removal of nutrients from the wastewater, whereas both microalgae can. Coimmobilization of the two microorganisms was superior to removal by the microalgae alone, reaching removal of up to 100% ammonium, 15% nitrate, and 36% phosphorus within 6 days (varied with the source of the

27

CHAPTER II REVIEW OF LITERATURE wastewater), compared to 75% ammonium, 6% nitrate, and 19% phosphorus by the microalgae alone . Ali et al. (2012) mentioned that Chlorella vulgaris was an important

freshwater

microalgae

treatment, and increasing its achieved

with

of Chlorella

potential

existence

used

of

of N 2 -fixing

vulgaris with

brasilense or Azotobacter

which

the

in

wastewater

treatment c a n

be

bacteria. Co-culturing

diazotrophs, Azospirillum

chroococcum in

three

different

media;

aquarium wastewater (AWW), sterile enriched natural aquarium wastewater (GPM) and synthetic wastewater media (SWW). Biomass yield of the microalgae was estimated by determination of chlorophylls (a, b), total carotenoid and the dry weight of C. vulgaris. Also determination of ammonia, nitrite, phosphate and nitrate in the culture were done. The presence of diazotrophs significantly increased the biomass

of C.

vulgaris by

increasing

its

microalgae

pigments

(chlorophylls a and b, and total carotenoids). The highest pigments percentage was reported due to addition of A. brasilense to C. vulgaris (18.3-133.5%) compared to A. chroococcum (23.9-56.9%). As well as increased dry weight from 12 to 50%. There was also improved removal of nitrate, nitrite, ammonia and phosphate; where, the highest removal percentage was reported due to addition of A. chroococcum to C. vulgaris (0.0-52%) brasilense and A.

compared

to A.

chroococcum can

brasilense (0.6-16.4%). A.

support C.

vulgaris biomass

production and bioremediation activity in the aquarium to minimize the periodical water renewal. Bacillus sp. promoted plant growth by a number of mechanisms, including

the

solubilization

of

phosphorus

and

production

of

phytohormones such as Indole Acetic Acid (Choudhary and Johri,

28

CHAPTER II REVIEW OF LITERATURE 2009; Lal and Tabacchioni, 2009). Bacillus is abundantly found genus in the rhizosphere and play vital as phosphate solubilizer and growth promoting rhozobacteria (Probanza et al., 2002 and Gutiérrez et al., 2003). Co-inoculation of phosphate solubilizing bacteria with Rhizobium stimulated the plant growth more than their alone inoculation depending upon the soil conditions (Perveen et al., 2002; Zaidi et al., 2003; Akhtar et al, 2013). Azotobacter is used for studying nitrogen fixation and inoculation of plants due to its rapid growth and high level of nitrogen fixation. However, despite the considerable amount of experimental data concerning Azotobacteria stimulation of plant development, the exact mode of action by which Azotobacteria enhances plant growth is not yet fully understood. Three possible mechanisms have been proposed: N2 fixation, delivering combined nitrogen to the plant, the production of phytohormones-like substances that alter plant growth and morphology, and bacterial nitrate reduction, which increases nitrogen accumulation in inoculated plants (Mrkovacki and Milic, 2001). Nitrogen and phosphorus are essential nutrients required by both plants and microorganisms, their major physiological roles are the accumulation and release of energy during cellular metabolism (Marchner, 1995). Phosphorus is generally deficient in most natural soils, because it is fixed as water-insoluble iron and aluminum phosphates in acidic soils or calcium phosphate in alkaline soils (Singh and Kapoor, 1994). However, calcium phosphate, which is of low solubility, can be dissolved

and

made

available

to

plants

by

soil

rhizosphere

microorganisms through the production of organic acids and chelating oxo acids from sugars (Rodriguez and Fraga, 1999) Therefore, the

29

CHAPTER II REVIEW OF LITERATURE inoculation of soil with phosphate solubilizing microorganisms may alleviate this problem (Illmer, et al, 1995; Johri et al, 1999). Plant growth-promoting bacteria (PGPB) of the genus Azospirillum are widely distributed in the rhizosphere of tropical and subtropical grasses (Bashan and Holguin, 1997). The mechanisms by which Azospirillum spp. can exert a positive effect on plant growth is probably composed of multiple effects including synthesis of phytohormones, N2fixation, nitrate reductase activity and enhancing minerals uptake (Steenhoudt and Vanderleyden, 2000; El-Komy et al., 2003). However, very few reports have indicated the P-solubilizing activity by different Azospirillum spp. (El-Komy et al., 2003; Seshadri et al., 2000). Therefore, a promising trend for increasing nitrogen and phosphorus availability to plants has been increased using combined inoculation of nitrogen fixing and P-dissolving organisms. There have been many successful attempts to improve plant development by using mixtures of Azospirillum and VA mycorrhiza (Vazquez et al., 2000; Alarcon et al., 2002). Similarly, the combined inoculation of Azospirillum and Psolubilizing bacteria was successfully used for plant N and P nutrition and growth yield (Alagawadi and Gaur et al., 1992; Belimov et al., 1995). In the last few years, several new inocula formulations have been proposed including alginate and agar immobilization inoculants (Bashan et al., 2002; El-Komy, 2001). These carriers permit entrapment of living cells, protecting the organisms against stresses. In addition, microbial immobilization promotes slow release of bacteria into soil (Shaban and El-Komy, 2000). (Bashan and Gonzalez, 1999) reported that Azospirillum can survive in dry alginate inoculants for prolonged periods without losing effectiveness. Moreover, (El-Katatny et al, 2003)

30

CHAPTER II REVIEW OF LITERATURE demonstrated that microbial immobilization gives prolonged metabolic activity when microbial cells are reused. Organisms could be immobilized separately or coimmobilized together (De-Bashan et al 2002; 2004; EL-Komy, 2005) Muňoz and Guieysse (2006) reviewed the interactions between algae and bacteria in processes designed for the treatment of hazardous contaminants. Production of oxygen by algae improves degradation of substances that must be degraded aerobically. Both bacteria and algae could produce defending substances against the other co-immobilized organism. Increase of pH values due to photosynthesis and increase of oxygen in the media could also slow down bacterial growth when coimmobilized with algae. On the other hand, consumption of CO2 and extracellular matter production (such as exopolysaccharydes) by algae can enhance bacterial growth rate, as well as CO2 and growth promoter substances production by bacteria can enhance microalgal growth. Lananan et al. (2014) mentioned that Bioremediation of aquaculture wastewater utilizing naturally occurring bacteria and microalgae have been widely used since 1990s in open pond. However, the relationship between both bioremediators especially in term of nutrient reduction had not been studied thoroughly in enclosed treatment system. Bioremediation of either Effective Microorganisms or microalgae independently required additional supply of oxygen and carbon dioxide, respectively to sustain their growth and treatment efficiency. Conversely, symbiotic bioremediation could omit this requirement due to the associate relation

between

both

in

term

of

respiration.

Microorganism

bioremediation would produce CO2 and consume O2 whereas microalgae are vice versa. On top of that, both EM and microalgae simultaneously function as degradation of organic matter. With proper optimization of

31

CHAPTER II REVIEW OF LITERATURE inoculation volume and bioremediation mode, symbiotic relations of these two microorganisms would benefits in designing more robust, economical and least maintenance on the wastewater treatment system.

32

CHAPTER III EXPERIMENTAL

CHAPTER III EXPERIMENTAL CHAPTER III EXPERIMENTAL III.1. Materials All reagents, Chemical and solvents used in microalgae cultivation, physicochemical, organic and microbiological analysis were very pure chemicals and provided from international companies such as SigmaAldrich, Merck, Fisher Scientific, Sharlau, Honey Well and Panreac. III.2. Instruments and methods The analyses of ammonia, phosphate, chlorophyll (a), algal count were carried out in Embaba laboratories, Embaba water treatment plant, Giza  Algal count was counted using compound microscope (Zeiss, Model Axio-Imager.A1).  Ammonia, phosphate, chlorophyll (a) were determined using spectrophotometer (Jenway, Model 6715 UV/VIS).  Sedgwick—Rafter slide was used for counting microalga.  Azotobacter chroococcum and Bacillus megatherium bacteria were counted using colony counter (DCC 560, HUMAN).  pH value of water measured electrometrically using pH meter (HACH (U.S.A), ECO 10).  Plastic syringe without needle used in cultivation.  Fluorescent lamp.  Stainless steel sieve to recover polymer.  Some glassware and lab tools.  Microwave was used in the preparation of the PVA. 

Magnetic stirrer - hot plat (VELP SCIENTIFIC, Model: F20700080) was used in the preparation of sodium alginate.

 Light intensity was measured using Lux-meter (YEW, Model 03681

33

CHAPTER III EXPERIMENTAL U, Japan).  Vacuum cultivation manifold was used for harvesting microalgae.  Centrifuge (J.P SELECTA, Model CE 95) was used in chlorophyll extraction.  Electrical balance (ADAM, Model PW 214) was used in chemicals, reagents, polymer and algal media preparation.  Refrigerator (KW APPARECCHI SCIENTIFICI, KLAB) was used to in chlorophyll a extraction by acetone and preservation of culture medium. III.3. Solutions III.3.1. Aqueous acetone solution: Aqueous acetone solution was prepared by mixing 90 parts reagent grade acetone with 10 parts Milli-Q water (Millipore Reagent Grade Water System). III.3.2. Magnesium carbonate suspension (1%): Magnesium carbonate suspension (1%) was prepared by Add 1.0 g of magnesium carbonate powder to 100 mL of Milli-Q water. III.3.3 Saline solution (0.85%): It was prepared by dissolving 8.5 g NaCl in 1000 ml distilled water (De-Bashan et al., 2004). III.3.4. Bold’s Basal Medium (BBM) Bold’s Basal Medium (Table 1, 2) is a freshwater algae medium that has been used to grow a variety of green algal cultures (e.g. Trichosarcina, Chlorococcum, and Chlorella) without the need for soilextract or vitamins (Brown et al., 1964; Nichols and Bold, 1965). The predominantly inorganic nature of this medium facilitates itself as an axenic-culture maintenance medium (Nichols and Bold, 1965; Nichols, H. W. 1973).

34

CHAPTER III EXPERIMENTAL Table (1): Bold’s Basal Medium (BBM) STOCK

STOCK SOLUTION

ml/Litre

NaNO3

5 g/200 ml

10 ml

MgSO4.7H2O

1.5 g/200 ml

10 ml

NaCl

0.5 g/200 ml

10 ml

K2HPO4

1.5 g/200 ml

10 ml

KH2PO4

3.5 g/200 ml

10 ml

CaCl2.2H2O

0.5 g/200 ml

10 ml

Na2EDTA. 2H2O

5 g/100 ml

KOH

3.1 g/100 ml

FeSO4.7H2O

4.98 g/L

H2SO4 (concentrated)

1 ml/L

Trace Metal Solution

See below (Table 2)

1 ml

H3BO3

1.14 g/100 ml

1 ml

1 ml

1 ml

III.3.5. Trace Metal Solution (Micronutrients): Table (2): Trace Metal Solution Substance

g/Litre

ZnSO4.7H2O

8.82 g

MnCl2.4H2O

1.44 g

MoO3

0.71 g

CuSO4.5H2O

1.57 g

Co(NO3)2.6H2O

0.49 g

III.3.6. Stabilizer reagent (Rochelle salt solution) Rochelle salt was prepared by dissolving 50 g potassium sodium tartrate tetra hydrate KNaC4H4O6.4H2O in 100 ml water. Remove

35

CHAPTER III EXPERIMENTAL ammonia usually present in the salt by boiling off 30 ml of solution .After cooling. Dilute to 100 ml. III.3.7. Nessler reagent Nessler reagent was prepared by dissolving 100 g HgI2 and 70g KI in a small quantity of water and this mixture was added slowly, with stirring, to a cooled solution of 160 g NaOH and dissolved in500 ml water, and diluted to 1L, stored in a rubber Stoppard boro silicate glassware, out of sunlight to be stable up to a year. III.3.8. Stock ammonium solution Stock ammonium solution was prepared by dissolving 0.382 g anhydrous NH4Cl, dried at 100 ºc, in water, and diluted to 1000 ml, 1.00 ml = 0.1 mg NH3 -N. III.3.8.1 (1 ppm) Ammonia solution: 1 ml of stock ammonia solution was added to 100 ml distilled water then measured using Nesslerization method. III.3.8.2 (5 ppm) Ammonia solution: 5 ml of stock ammonia solution was added to 100 ml distilled water then measured using Nesslerization method. III.3.9. Vanadate-molybdate reagent: III.3.9.1. Solution A: was prepared by dissolving 25 g ammonium molybdate, (NH4)6Mo7O24.4H2O, in 300 mL distilled water. III.3.9.2. Solution B: was prepared by dissolving 1.25 g ammonium metavanadate, NH4VO3, by heating to boiling in 300 mL distilled water. Then the solution was cooled and 330 mL conc. HCl was added. Then the solution B was cooled to room temperature, the solution A was poured into solution B, mixed, and diluted to 1 L. III.3.10. Standard phosphate solution: was prepared by dissolving in distilled water 0.438 g anhydrous KH2PO4 and diluted to 1000 mL; 1.00 mL = 0.1 mg PO4 -P.

36

CHAPTER III EXPERIMENTAL III.3.10.1 (1 ppm) phosphates Solution: 1 ml of stock phosphates solution was added to 100 ml distilled water then measured using Vanadomolybdophosphoric acid colorimetric method. III.3.10.2 (5 ppm) phosphates Solution: 5 ml of stock phosphates solution was added to 100 ml distilled water then measured using Vanadomolybdophosphoric acid colorimetric method. III.4. Procedures III.4.1. Isolation and cultivation of Scenedesmus quadricauda microalga S. quadricauda was isolated and purified by National Research Centre NRC, Dokki, Giza, Egypt. S. quadricauda were cultivated in Bold’s Basel inorganic culture medium table (1), aerated by filtered air pumped at a rate of 4 L min-1 and maintained at temperature of 25 ± 2ºC, fluorescent lamp with light intensity 3100 lux and 16/8 h light/dark cycle was used as light source, (Fig. 1) The growth of algae was monitored by measuring the algal count daily. After two weeks of cultivation, algal cells were harvested by filtration through manifold membrane with pore size 0.45 µm, the algal cells washed several times with saline solution (0.85 % NaCl) afterwards re-suspended in saline solution which gave a solution with cell count =6.8*105 cell/ml. The free microalga became ready to be used in treating the inorganic pollutants; the algal suspension was used to remove ammonia and phosphate from artificial samples. III.4.2. Counting phytoplankton organisms using Sedgwick—Rafter slide:

37

CHAPTER III EXPERIMENTAL Sedgwick—Rafter cell was used for quantification of phytoplankton with a volume of 1 ml [50mm (length) *20mm (width)* 1 mm (height)]. The bottom plate of Sedgwick—Rafter cell is divided into 50 columns and 20 rows forming 1000 square altogether, This method is based on standard Methods 10200-F (palmer 1980; APHA, 2005), (Fig. 2) . Counting steps: 1. The processed sample is shaken well to be homogenous 2. Take 5 ml from sample to be dilute (Because the sample is very dense), the final volume after dilution is a definite volume. 3. Transfer the net volume to brown bottle and this volume is recorded on the bottle label. 4. Shake the sample well before investigation to keep the homogeneity of the sample 5. Take 1ml from Diluted sample and put it on the Sedgwick rafter cell the cover slip Will rotate slowly and cover the inner portion of S-R cell 6. Let the sample to settle on the rafter cell at least 15 minute. Calculations

III.4.3. Chlorophyll (a) measurement: Chlorophyll (a) was determined by spectrophotometer. This method is based on standard Methods 10200H (APHA, 2005). Procedure 1. Collect the sample of volume 10 ml. 2. Add 1ml /L of magnesium carbonate 1% to the sample for fixation. 3. The sample was Centrifuged at 2000 rpm for 15 min

38

CHAPTER III EXPERIMENTAL

Figure 1: Scenedesmus diagram and image

39

CHAPTER III EXPERIMENTAL

Figure 2: Counting microalga using Sedgwick—Rafter slide 4. Discard supernatant and add 10 ml acetone 90% to the residue and shake well to extract chlorophyll (a). 5. Collect the sample of volume 10 ml. 6. Add 1ml /L of magnesium carbonate 1% to the sample for fixation. 7. The sample was centrifuged at 2000 rpm for 15 min. 8. Discard supernatant and add 10 ml acetone 90% to the residue and shake well to extract chlorophyll (a). 9. close the cap to prevent evaporation of acetone. 10.Take the extract and put it in refrigerator at 4 ºC for 18-24 hr. 11.The extract was centrifuged for 20 minute at 2000 rpm. 12.Transfer the extract to 1-cm cuvette and measure the optical density (OD) at 750,664,647 And 630 nm. 13.Correct the turbidity by reading OD at 750 nm. Calculations: The concentration of chlorophyll (a) in the extract by inserting the corrected optical density in the following equations:

Where: Ca = Concentration of chlorophyll (a).

40

CHAPTER III EXPERIMENTAL OD630 = Corrected optical densities (with a 1-cm light path) at respective wave length after determination of concentration of chlorophyll (a) in extract, calculate the amount of chlorophyll a per unit volume as follow:

III.4.4. Ammonia measurement: Ammonia is measured spectrophotometry by Nesslerization Method according to SMWW Method No. 4500-N (APHA 1992). Procedure 1. 50 ml sample was used or a portion diluted to 50 ml with water. 2. 1 to 2 drops Rochelle salt solution was added to the sample. And mixed well. And 1 ml Nessler reagent was added. 3. Let reaction proceed for at least 10 min after adding Nessler reagent. 4. 1 ml was added Nessler reagent to the sample 5. Spectrophotometer at wave length 425 nm with alight bath 1 cm was used for measurement. Calculations:

III.4.5. Phosphate measurement: Phosphate was determined spectrophotometry according to SMWW Method No. 4500-P (APHA, 2005). Ammonium molybdate reacts under acid conditions to form a heteropoly acid, molybdophosphoric acid. In the presence of vanadium, yellow vanadomolybdophosphoric acid is formed. The intensity of the yellow color is proportional to phosphate concentration. Procedure 1. 35 mL of sample was placed in a 50-mL volumetric flask.

41

CHAPTER III EXPERIMENTAL 2. 10 mL vanadate-molybdate reagent was added and diluted up to the mark with distilled water. 3. A blank in which 35 mL distilled water is substituted for the sample was prepared to subtract the method background. 4. After 10 min or more, the absorbance of sample was measured versus a blank at a wavelength of 400 to 490 nm. Calculations:

III.4.6. Application of S. quadricauda on the removal of ammonia and phosphate from artificial water samples III.4.6.1. Studying the effect of time on two different concentrations of ammonia using fixed concentration of algal suspension: The artificial samples with the following concentrations of ammonia 1 and 5 mg/l were treated with constant concentration ratio of microalga 95%. III.4.6.2. Studying the effect of different ratios of microalga against residual ammonia concentration: The artificial samples with 1, 5 mg/l concentration of ammonia were treated with different concentration ratios of microalga (35%, 55%, 95% and 95%). III.4.6.3. Studying the effect of different concentrations of phosphate with fixed count algal suspension against time: The artificial samples with 1, 5 mg/l concentrations of phosphate were treated with constant concentration ratio of algae (95%). III.4.6.4. Studying the effect of different ratios of microalga against phosphate concentration:

42

CHAPTER III EXPERIMENTAL The artificial samples with 1, 5 mg/l concentrations of phosphate were treated with different concentrations ratios of microalga (35%, 55%, 75% and 95%). III.4.6.5. Time course of development of chlorophyll (a) In all previous cases chlorophyll (a) was measured daily against time which indication of algal growth rate. III.4.7. Preparation of Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removal III.4.7.1 Preparation of composite of sodium alginate and poly vinyl alcohol A. Preparation of 5% sodium alginate 12.5 g of sodium alginate was added to 250 mL of distilled water. Stirred and the mixture was warmed on a magnetic stirrer for about one hour. B. Preparation of 5% poly vinyl alcohol Weigh 5 g of polyvinyl alcohol into a 100 ml Pyrex® beaker. Fill the beaker to the 100 ml with hot distilled water; stir. Cover with microwaveable plastic wrap. Microwave on high for about 3 minutes; stir and heat for an additional 3 minutes. Stir and heat for another 2 to 3 minutes. Repeat this process one more time, if necessary. Using short increments of time between stirring will produce the best results. Time will vary widely depending on the power of your microwave. With a clean spatula, remove the polyvinyl alcohol film lying on top of the solution. Allow the solution to cool before use (Shakhashiri, 1989). C. Preparation of 2% CaCl2 By dissolving 10 g CaCl2 into 500 ml distilled water. D. Preparation of neat polymer

43

CHAPTER III EXPERIMENTAL By mixing 25 ml of 5% sodium alginate with 5 ml of 5% poly vinyl alcohol ratio (5:1) and completed to 50 ml, the final concentration of sodium alginate and poly vinyl alcohol was 2.5% and 0.5 % respectively. The mixture was titrated with 2% CaCl2 and the beads were allowed to harden in the CaCl2 solution for 1 hr. After that, the beads were washed several times with distilled water to remove any remains of CaCl 2, (Fig. 3a). III.4.7.2. Preparation of three different concentrations of Immobilized microalga: A. High stocking beads By mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 15 ml algal suspension has algal count (4.3*106 cell/ml), and the mixture was adjusted to 50 ml by using distilled water, (Fig. 3b). B. Medium stocking beads By mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 10 ml algal suspension has algal count (4.3*106 cell/ml), and the mix was adjusted to 50 ml by using distilled water. C. Low stocking beads By mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 5 ml algal suspension has algal count (4.3*106 cell/ml), and the mix was adjusted to 50 ml by using distilled water. III.4.8. Application of Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removal III.4.8.1. Time course of ammonia removal using high, medium and low stocking bead: The artificial samples with a constant concentration of ammonia 5 mg/l were treated with both neat polymer and low, medium and high stocking beads.

44

CHAPTER III EXPERIMENTAL

(a)

(b) Figure (3): Neat polymer (a) and immobilized microalga beads (b)

45

CHAPTER III EXPERIMENTAL III.4.8.2. Time course of phosphate removal using high, medium and low stocking bead: The artificial samples with a constant concentration of phosphate 5 mg/l were treated with both neat polymer and low, medium and high stocking beads. III.4.9. Preparation of three different concentrations of Coimmobilization microalga The microalga growth promoting bacteria (MGPB) Azotobacter chroococcum and Bacillus megatherium bacterial suspension were used for co-immobilization experiments with S. quadricauda. The source of A.chroococcum and B.megatherium media suspension were obtained from soil fertility and microbiology department, Desert Research Center, the suspension was centrifuged at 2000 rpm and the residue washed twice by saline solution and re-suspended in saline solution. The systematic biotechnology was used taking fresh liquid cultures for 48 hours old from pure local strains of Azotobacter chroococcum, and Bacillus megatherium at the rate of ~108 cfu/ml according to Hill (2000) and Nautiyal (1999) respectively, (Fig. 4). Microalga growth promoting bacteria (MGPB) control was prepared by mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 2.5 ml of A. chroococcum and 2.5 ml of B. megatherium. , then the mix was adjusted to 50 ml by using distilled water. Three co-immobilization stocking beads were prepared as follow: III.4.9.1. Co-immobilization high stocking beads Co-immobilization high stocking beads were prepared by mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 15 ml algal suspension has algal count (4.3*106 cell/ml), and addition of 2.5 ml of A. chroococcum and 2.5 ml of B. megatherium.

46

CHAPTER III EXPERIMENTAL

(a)

(b)

Figure 4: A. chroococcum (a) and B. megatherium (b) III.4.9.2. Co-immobilization medium stocking beads Co-immobilization medium stocking beads were prepared by mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 10 ml algal suspension has algal count (4.3*106 cell/ml), and addition of 2.5 ml of A.chroococcum and 2.5 ml of B. megatherium, then the mix was adjusted to 50 ml by using distilled water. III.4.9.3. Co-immobilization low stocking beads Co-immobilization low stocking beads were prepared by mixing 25 ml sodium alginate 5% and 5 ml poly vinyl alcohol 5% with 5 ml algal suspension has algal count (4.3*106 cell/ml), and addition of 2.5 ml of A. chroococcum and 2.5 ml of B. megatherium, then the mix was adjusted to 50 ml by using distilled water. III.4.10. Application of co-Immobilized S. quadricauda on composite of sodium alginate and poly vinyl alcohol for ammonia and phosphate removals III.4.10.1. Time course of ammonia removal using co-immobilizing high, medium and low stocking bead:

47

CHAPTER III EXPERIMENTAL The artificial samples with a constant concentration of ammonia 5 mg/l were treated with MGPB control and different concentrations of coimmobilized microalga low, medium and high stocking beads. III.4.10.2. Time course of phosphate removal using co-immobilizing high, medium and low stocking bead: The artificial samples with a constant concentration of phosphate 5 mg/l were treated with MGPB control and different concentrations of coimmobilized microalga low, medium and high stocking beads.

48

CHAPTER IV

RESULTS AND DISCUSSION

CHAPTER IV RESULTS AND DISCUSSION CHAPTER IV RESULTS AND DISCUSSION General outlines: Water contamination might be resulted artificially due to the development of several life activities such as industries, cultivation and population growing, or naturally due to interaction with environment components such as air, soil, rocks…etc. The quick development of human activities leads to great increase in the inputs of ammonia and phosphorus into water bodies. These inputs induce eutrophication and cause deterioration in natural water quality. As such, the removal of ammonia and phosphorus from wastewater is a fundamental way to prevent eutrophication and water blooms. Among many methods used for water treatment, microalgae had attracted a significant amount of attention due to their ability to remove ammonia and phosphorus from wastewater. Microalgae can utilize nitrogen and phosphorus from wastewater for their growth. Moreover, microalgae can also fix carbon dioxide from atmosphere through photosynthesis, thus reducing greenhouse gas emission. Also, microalgal biomass can be used for biofuel production which is considered as renewable energy. This chapter studies the possibility of using microalga Scenedesmus quadricauda in three forms free, immobilized and co-immobilized microalga in small calcium alginate beads, to remove ammonia and phosphate from aqueous solutions. It also, studies different factors that may affect nitrogen and phosphate removal process This chapter will be discussed under the following items: 1- Identification and classification of the microalgal taxa under investigation. 2- Removal of ammonia and phosphate using free microalga S. quadricauda

49

CHAPTER IV RESULTS AND DISCUSSION 3- Immobilization of microalga S. quadricauda in alginate beads. 4- Enhanced biological removal of ammonia and phosphate by co-immobilization of microalga Scenedesmus quadricauda with Azotobacter chroococcum, and Bacillus megatherium in alginate beads. IV.1. Identification and classification of the microalgal taxa under investigation: Scenedesmus quadricauda microalga was identified up to the species level according to the key of freshwater algae (Hustedt, 1976; Streble and Krauter, 1978; Komárek and Fott, 1983 and Komárek and Anagnostidis, 1989).  Characterization of S. quadricauda alga:Classification: Empire: Eukaryota Kingdom: Plantae Phylum: Chlorophyta Class: Chlorophyceae Order: Sphaeropleales Family: Scenedesmaceae Subfamily: Scenedesmoidea Genus: Scenedesmus  Description: Thalli single celled or colonial, forming 2- to 32-celled, usually 4- or 8-celled coenobium. Cells arranged linearly, touching with the lateral walls. Cells 3-78 x 2-10 µm, elongate and terminal cells with long spiny projections, Scenedesmus planktonic mainly in eutrophic freshwater ponds and lakes, rarely in brackish water; reported world-wide in all climates (Guiry and Guiry, 2014), (Fig. 5).

50

CHAPTER IV RESULTS AND DISCUSSION

(a)

(b) Figure (5): Scenedesmus quadricauda at magnification power 200X (a) and 400X (b)

51

CHAPTER IV RESULTS AND DISCUSSION IV.2. Removal of ammonia and phosphate using free microalga S. quadricauda The free microalga S. quadricauda culture was used in this work to remove the ammonia and phosphate from artificial samples, and the effect of initial concentration of ammonia, time and concentration ratio of microalgae, were studied. IV.2.1. Removal of ammonia using free microalga S. quadricauda Nitrogen is a critical nutrient required in the growth of all organisms. Organic nitrogen is found in a variety of biological substances, such as peptides, proteins, enzymes, chlorophylls, energy transfer molecules (ADP, ATP), and genetic materials (RNA, DNA) (Barsanti and Gualtieri 2006). Organic nitrogen is derived from inorganic sources including nitrate (NO3-), nitrite (NO2-), nitric acid (HNO3), ammonium (NH4+), ammonia (NH3), and nitrogen gas (N2). IV.2.1. A. Effect of time and initial concentration of ammonia Although biological treatment has a lot of advantages and economical values as mentioned before, but they need more time than other used methods, this thesis is an attempt to decrease the time needed for the removal of ammonia as it will be discussed in the following results. The artificial samples with the concentrations of ammonia 1 and 5 mg/l were treated with constant concentration ratio of microalga 95% and the removal of ammonia was measured against time from zero to 5 days. The results show that at 1 mg/l initial concentration, ammonia was completely removed after 4th day and for 5 mg/l ammonia, the maximum removal percentage reached up to 91.8% at 4th day, and then nearly remained constant (Fig. 6). As the initial ammonia concentration of ammonia increased, the removal efficiency decreased with the time (Aslan and Kapdan 2006).

52

CHAPTER IV RESULTS AND DISCUSSION

5 Phosphate (mg/l)

1.35

Phosphate at 1 mg/l conc. Phosphate at 5 mg/l conc. Chlorophyll (a) (mg/l) at 1 mg/l phosphate Chlorophyll (a) (mg/l) at 5 mg/l phosphate

1.3

1.25

4 1.2

3 1.15

2

1.1

1

1.05

0

1

0

1

2

3 Time (days)

4

5

6

Figure 6: Time course of ammonia removal and development of chlorophyll (a) at algal concentration ratio (95%). ([NH3] = 1 mg/l & [NH3] = 5 mg/l) The ammonia nitrogen removal efficiencies achieved in this study were higher compared to (Valderramaet al., 2002). Obtained data in Figure (6) are in accordance with (Martinez et al., 2000) who reported that fresh water microalga Scenedesmus obliquus achieved ammonia removal from urban wastewater documenting 100% ammonium removal in 188.33 h incubation. Our results are lower than reported by (Olgúın et al. 2003), where, 96% ammonia removal was achieved by Spirulina for ammonia concentration of 27.4 mg/l. Since uptake is the main mechanism of nutrient removal by microalgae, the microalgal population growth rate directly affects the nutrient removal rate (Xin et al, 2010).

53

Chlrophyll (a) (mg/l)

6

CHAPTER IV RESULTS AND DISCUSSION Represented data in Figure (6) showed a relation between ammonia concentration and chlorophyll (a) content in microalga S. quadricauda but with lower significant level as result of narrow range of ammonia concentration studied. Higher ammonia concentration exhibit significant chlorophyll (a) increment (Aslan and Kapdan 2006; Singh and Dhar 2007). These results are disagreeing with the obtained finding that there was significant correlation between chlorophyll (a) and NH3 (and NO2) removal rate. Heidari et al. (2013) reported that there is significant correlation between NH3 (and NO2) concentrations and biomass. IV.2.1.B. Effect of algal concentration ratio The initial cell concentration of microalga is critical for removing ammonia, and higher removal efficiency can be achieved by increasing algal cell concentration, while lower cell concentrations would reduce the nutrient removal efficiency (Zhang et al, 2012). The artificial samples with 1 and 5 mg/l concentrations of ammonia were treated with different concentration ratios of microalga (35%, 55%, 75% and 95%) and the residual concentration of ammonia was measured at the 4th day to find out the most efficient algal concentration ratio. The results showed that for 1 mg/l ammonia concentration, the ammonia removal efficiency using microalga S. quadricauda increase from 91% and 98% at microalgal concentration ratios 35% and 55% ,respectively, (Fig. 7). However complete removal of ammonia was achieved at both 75% and 95% microalga concentration ratios which reflect that there is no significant increase in ammonia removal with elevating microalgal concentration ratio from 75% to 95%. Meanwhile using 5 mg/l ammonia solution, the percentage of ammonia removal at microalgal concentration ratios 35%, 55%, 75% and 95% were 62%, 74%, 84% and 94% respectively, Which shows that there is a significant increase in ammonia removal by elevating the concentration ratios.

54

CHAPTER IV RESULTS AND DISCUSSION

Ammonia removal percentage %

110

Removal Percent of 1 mg/l ammonia (%) Removal Percent of 5 mg/l ammonia (%)

100 90 80 70

60 50 35%

55% 75% Algae concentration ratio %

95%

Figure 7: Effect of algal concentration ratio on the removal percent of ammonia. ([NH3] = 1 mg/l & [NH3] = 5mg/l) Microalgae convert ammonia to amino acids and proteins through a process called assimilation that affect the ammonia concentration in the media and cause the reduction of the ammonia concentration. The chlorophyll (a) concentration is an indication for algal growth rate which increased with time by increasing ammonia removal percent and algal concentration ratio (Fig. 8). This may be due to cell growth and multiplication (Kaya et al., 1995; Tam and Wang, 2000; Cai et al., 2013). IV.2.2. Removal of phosphate using free microalga S. quadricauda Phosphorus is also a key factor in the energy metabolism of algae and is found in nucleic acids, lipids, proteins, and the Intermediates of carbohydrate

55

CHAPTER IV RESULTS AND DISCUSSION

2 A

Chlorophyll (a) mg/l

1.8

B

C

D

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 35%

55%

75%

95%

Algae concentration ratio %

Figure 8: Effect of algal concentration ratio on the developed concentration of chlorophyll (a). ([NH3] = 1 mg/l & [NH3] = 5 mg/l) (A: initial conc. of chlorophyll (a) (mg/l) at 1 mg/l ammonia, B: conc. of chlorophyll a (mg/l) after 4 days at 1 mg/l ammonia, C: initial conc. of chlorophyll (a) (mg/l) at 5 mg/l ammonia, D: conc. of chlorophyll (a) (mg/l) after 4 days at 5 mg/l ammonia)

metabolism. Inorganic phosphates play a significant role in algae cell growth and metabolism (Martinez et al, 1999). IV.2.2. A. Effect of time and initial concentration of phosphate The artificial samples with 1 and 5 mg/l concentrations of phosphate were treated with constant concentration ratio of algae (95%). The removal of phosphate

56

CHAPTER IV RESULTS AND DISCUSSION was measured against time from zero to 6 days. The results show that phosphate concentration decreases with time reaching maximum percentage of removal 86% at 4th day and 61% at 6th day for 1 and 5 mg/l phosphate, respectively (Fig. 9).

5 Phosphate (mg/l)

1.35

Phosphate at 1 mg/l conc. Phosphate at 5 mg/l conc. Chlorophyll (a) (mg/l) at 1 mg/l phosphate Chlorophyll (a) (mg/l) at 5 mg/l phosphate

1.3 1.25

4 1.2

3 1.15

2

1.1

1

1.05

0

1

0

1

2

3 Time (days)

4

5

6

Figure 9: Time course of phosphate removal and development of chlorophyll (a) at algal concentration ratio (95%). ([PO4-3] =1 mg/l & [PO4-3] =5 mg/l) The phosphorus removal efficiencies achieved in this study were higher compared to (Valderrama et al., 2002). Microalgae Chlorealla kessleri was able to uptake only 8–20% phosphorus under the light/dark cycle for PO4-P concentration of 10 mgl−1 (Lee and Lee, 2001). Although (Dumas et al., 1998) reported complete phosphorus removal by Phormidium bohneri, the initial concentration was considerably lower (0.05mg/l PO4-P) compared to the concentrations used in our study. Also (Sreesai and Pakpain 2007) reported that

57

Chlrophyll (a) (mg/l)

6

CHAPTER IV RESULTS AND DISCUSSION approximately 55% of total phosphorous removal by Chlorella vulgaris in septage effluent wastewater. Obtained data in Figure (9) were in accordance with (Kassim, 2002) who reported that a significant decrease in wastewater phosphate content during the cultivation of three different species of algae; Scenedesmus abundanse, S. quadricauda and Chlorella vulgaris. The higher phosphate reduced for the three species were in the sixth day of experiment, it reaches 0.4, 0.5, 0.9 mg/l PO 4-3-P for C. vulgaris, S. abundanse and S. quadricauda respectively for 3.7mg/l PO4-3-p initial concentration. Our results are slightly lower than reported by (Aslan and Kapdan 2006) which achieved removal efficiency 78% for initial phosphate concentration 7.7mg/l. Also, (Martinez et al., 2000) reported 98% P removal in 94.33 h of incubation with fresh water alga Scenedesmus obliquus from urban wastewater documenting. Presented data in Figure (6, 9) reveals that the removal efficiency of phosphorus was poorer compared to ammonia in all treatments, this is in agreement with the results obtained by (Hernandez et al. 2006; Ali et al. 2012). So far, there is almost no report on complete removal of PO4, the main form of inorganic phosphorus used by algae, from domestic wastewater by biological wastewater treatment with in vitro small-scale experiments (Robinson et al., 1989; de-Bashan and Bashan 1997-2003). The microalgal cells apparently became saturated faster with PO4 (Robinson et al., 1989; Valderrama et al., 2002; deBashan et al. 2004), probably due to accumulation of polyphosphates (Darley 1987). The results agree with (Wang et al., 2013) who reported that it seems that Chlorella sp. could use phosphorus at an extremely low concentration. This phenomenon was also found in another green alga Botryococcus braunii when it was cultivated in secondarily treated sewage (Sawayama et al., 1992).

58

CHAPTER IV RESULTS AND DISCUSSION Also, from results it’s clear that the growth of microalga using phosphate as a source of nutrient is illustrated by increasing of chlorophyll (a) concentration (Fig. 9). Obtained data in Figure (9) were in accordance with (Singh et al., 2011) who reported that the percentage removed depends on the initial concentration of phosphate. When initial phosphate content in medium was 7 ppm, the algae grown and removed 70% of total phosphate during the 8 days growth period. Interestingly, 3.7–4.6 mg/ L of phosphate (50–60% of TP) was removed within the first 4 days of growth. As the initial TP was

Suggest Documents