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Research Proposal: First assessment

Experimental Design for the Effect of Inorganic Compound in the Aerobic Granular Sludge Development

Master Student: Muhammad Anwar Alias Supervisor: Dr. Khalida Bt Muda Co- Supervisor: Dr. Azmi Aris

Outline of Presentation
Introduction

Problem Statement
Aim and Objectives of the Study Scope of the Study Significance of the Research Literature Review Methodology Expected Outcomes

Introduction
Biorranulation
Granulation is the process in which a non discrete flocculent biomass forms discrete welldefined pellets or granules which can be several millimeters in diameter (Ong et al., 2002). Involves cell-to-cell interactions that include biological, physical and chemical phenomen

Granulation aid is promoting the formation of the granules or granular biomass.

Introduction
Biogranulation can be classified as: aerobic granulation anaerobic granulation Biogranules form through self-immobilization of microorganisms. These granules are dense microbial consortia packed with different bacterial species and typically contain millions of organisms per gram of biomass.

Advantages of aerobic granules


More compact microbial structure Improved settling ability. Enable a high biomass retention. Withstand high-strength wastewater & shock loadings. Effective sludge-effluent separation Greater biomass preservation and enrichment significantly higher organic loading rates

Factors affect aerobic granulation


Organic loading rate Seed sludge Aeration intensity Settling time Granulating agent Hydraulic retention time Divalent metal ions

Problem Statement
Time consuming for developing fully complete matured granules.
During long-term SBR operation, the aerobic granular sludge process gradually loses stability under inappropriate operational conditions. Aerobic granules often showed a poor stability after several months of operation (de Kreuk and van Loosdrecht, 2004; Liu et al., 2006) which the application wastewater treatment is limited

Aim and Objectives of the Study


The aim of this study is to enhance the aerobic granulation process using inorganic compound.

1
To identify the potential soluble ionic inorganic compounds that contributes in accelerating biogranulation formation during start up period

2
To optimize the effect of inorganic compound as aid in the biogranulation development

3
To develop biogranules with the additional of inorganic compound

Scope of the Study


Apply to the existence system- develop granule Start up for aerobic granulation process/Initial stage

Parameters to study the affect

Scope

Inorganic Compound as aid

Lab scale reactor aerobic condition

Bacterial isolation from sewage sludge Synthetic wastewater

Significance of the Research

This research could be helpful for enhancing the aerobic granules development via addition of ionic organic compound as granulation aid in terms of fast formation, high durability and stability, and high removal performance. Thus, this research development can be utilized to overcome our local waste water problem especially in palm oil mill effluent, leachate and municipal waste water.

Literature Review
Aerobic Sludge Granulation Process
Sludge may be defined as the microbial biomass utilizing nutrient substrates present in wastewater. Microbial granules are characterized as dense and compact microbial aggregates with a spherical outer shape. Aerobic granules are self-immobilized spherical aggregates of microorganisms. The growth of aerobic granules were considered to be a special case of biofilm growth (Liu and Tay, 2002; Yang et al., 2004).

Literature Review

Inorganic Compound
Inorganic compounds have traditionally been considered to be of inanimate, non-biological origin Inorganic compounds, consist of molecules with a central metal atom (usually transition metal) bonded to one or more non-metallic ligand(inorganic, organic, or both) and are often intensely coloured In the form of ion, its acts as electron-transfer proteins and substrate bindings Example : MgSO4, Al(NO3)39H2O, BaCl2, Pb(NO3)2, MnSO4H2O

Literature Review
Extracellular Polymeric Substance (EPS) Also known as exopolysaccharides EPS are a complex high-molecular-weight mixture of polymers excreted by microorganisms/microbes Produced from cell lysis and adsorbed organic matter from wastewater

Literature Review
EPS is recognized as a key factor in the granulation process (Wang et al. 2006), which is mainly composed of polysaccharide and protein Previous studies shows divalent metal ionic such as Ca2+, Mg2+ and Fe2+ augmentation led to the higher production yield of EPS (polysaccharides & protein) resulted in faster granulation process and shorter starting-up period

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Literature Review
Previous studies shows that Cationic from the inorganic compound :
can neutralize the negative charge on the surface of bacteria, and promote the sludge granulation because of the positive charge The cationic could bind to the negatively charged groups on bacterial surfaces and extracellular polymeric substances (EPS), and act as a bridge to interconnect these components, and then form EPS(cationic)EPS crosslinkage could accelerate microbial aggregation, and increase the physical strength of mature granules

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Mechanism of aerobic granulation

Mishima and Nakamura (1991)

Based on experiments in an aerobic upflow sludge blanket (AUSB), the biological assemblies is similar to the anaerobic granulation process, filamentous bacteria tangled with each other to form aerobic granules.

Beun et al. (1999)

Proposed mechanism of aerobic granulation in SBR


Beun et al. (1999) proposed a model in which aerobic granulation could be started with fungi.

Negatively Charged

- Individual bacterium

+ + +

BRIDGING

SHAPING

+ - +

Extracellular polymeric substance

Positively Charged (Ionic compound)

Modified schematic representation of extracellular polymeric substance-enhanced (Liu et al., 2004).

LITERATURE REVIEW
Previous studies on sludge biogranulation process using inorganic compound
Performance on granulation development 25 days Reactor system Biomass Type of wastewater Synthetic waste water Inorganic compound used R1 augmented with 10 mg/L of Mn2+ R2 no Mn2+ Biogranule characteristic Diameter size: R1= 2.53.5 mm R2= 1.5- 2.5mm Remarks Aerobic granules first formed in reactor R1 on day 6 Ca2+-fed granules were denser and more compact, better settling and strength characteristics, and higher polysaccharide contents. The researches had indicated that an increase in granule size resulted in the Ca2+ accumulation in granules, and finally led to a reduced granule bioactivity [ Reference

SBR

Aerobic granule

Lihui et al (2011)

ASB

Aerobic granules

Synthetic wastewater

Ca2+

augmentation

Diameter: 2.8mm Wet density: 1.01 kgL-1

32 days

Tay T-L et al., (2002)

SBR

Aerobic granule

Synthetic waste water

R1 augmented with Ca2+ at 40 mg/L R2 augmented with Mg2+ at 40 mg/L

Diameter size : R1= 1.32mm R2=0.21mm

21 days

Lin Liu et al. (2010)

UASB = Upflow Anaerobic Sludge Blanket; SBR = Sequencing Batch Reactor; ASB = Sequential Aerobic Sludge Blanket; SS = Suspended Solid; VSS = Volatile Suspended Solid; MLSS = Mixed Liquor Suspended Solid; MLVSS = Mixed Liquor Volatile Suspended Solid; SV = Settling Velocity; SVI = Sludge Volume Index; SAA = Specific Acidogenic Activity; SMA = Specific Methanogenic Activity; EPS = Extracellular Polymeric Substance; CONTINUED.. COD = Chemical Oxygen Demand; TOC = Total Organic Content; NH4+-N = Ammonium

CONTINUED..

LITERATURE REVIEW
Type of wastewater Synthetic wastewater (glucose) Influent COD = 640 mg/l Synthetic wastewater (potassium nitrate; methanol) Nitrate = 120 mg/l; TOC = 120 mg/l Biomaterial used Biogranule characteristic Size: 2.9mm Specific gravity: 1.002 kg/l MLSS: 3.2 g/l SS: 7.6 g/l SVI: 25 ml/g Performance removal efficiency Effluent COD = 56 mg/l; COD removal = 91% Remarks Mg2+ augmentation significantly decreased the sludge granulation time from 32 days to 18 days. Reference

Previous studies on sludge biogranulation process using inorganic compound


Reactor system Biomass

SBR

Aerobic sludge granule

Mg2+ augmentation

Li X-M et al., (2008)

SBR

Denitrifying granules (anaerobic condition)

Mean size of biomass: 420m 2+ augmentation MLVSS/MLSS Ca ratio: 0.37 SVI: 14.36 ml g-1

NO3--N; TOC removal = nearly 100%

Liu Y-J and Sun DD, (2010)

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Literature Review
Previous studies shows that Cationic from the inorganic compound :
can neutralize the negative charge on the surface of bacteria, and promote the sludge granulation because of the positive charge The cationic could bind to the negatively charged groups on bacterial surfaces and extracellular polymeric substances (EPS), and act as a bridge to interconnect these components, and then form EPS(cationic)EPS crosslinkage could accelerate microbial aggregation, and increase the physical strength of mature granules

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Methodology

Preparation of Inorganic compound


Stock solutions having 7 types of inorganic compounds are prepared by dissolving appropriate quantities in deionized water List of inorganic compound will be used in this research:
1. Aluminium Nitrate Anhydrous 2. Barium Chloride- dihydrate 3. Lead Nitrate 4. Manganese Sulphate-1-hydrate 5. Zinc Sulphate-7-hydrate 6. Nickel Sulphate 7. Strontium Nitrate

Preparation for stock culture of bacteria


Culture of bacteria is isolated from sewage waste water sludge Bacteria is isolated on a spread plate.

Single colonies of isolated bacteria will be labeled and stored in universal bottle as stocks.
The stocks are stored at 4C and working solutions of required concentrations were obtained by appropriate dilution in deionized water prior to each experiment Culture will be grown in the Nutrient Broth to more quantity

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List of reagent used in the experiment


Reagents/ Chemicals Nutrient Agar Potassium phosphate, K2HPO4 Xylene (C6H4(CH3)2) Nutrient broth Sodium Chloride Application Spread Plate Washing buffer Surface hydrophobicity assay Bacteria culture EPS Extraction Method

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Research Framework
Objective 1
Stage 1 To identify the potential of soluble ionic inorganic compound as aid for the aerobic granulation start-up
Isolation of bacteria from sewage waste water sludge
Screening for bacteria selection in the aerated synthetic medium Response: Hydrophobicity and Autoaggregation

Stage 2

Screening for soluble inorganic compound using aerated synthetic medium and selected bacteria in room temperature Variables: Type of ionic inorganic compound (based on concentration) Response: Hydrophobicity and Auto aggregation

Stage 3

Data analysis using Completely Randomized Design (CRD) method to select the best ionic inorganic compound

Research Framework
Objective 2 To optimize the effect of inorganic compound as aid in
granulation start up using response surface method Optimize the selected ionic inorganic compound using synthetic medium with aeration under room temperature Variables: Concentration, pH and temperature Response: Extracellular polymer substance(EPS) secretion, Hydrophobicity and Auto aggregation Data analysis using Response Surface method to optimize the best condition of ionic inorganic compound for the aerobic granulation start-up

Research Framework
Objective 3
To develop biogranules with the additional of soluble inorganic compound

Operation process using according to Muda K. et al. 2009 method + (inorganic compound) i. Granular sludge properties ii. Granular sludge removal performance

Experiment Set up
Objective 1 500mL of Schott bottle will be used as reactor with a working volume of 250 mL (synthetic medium + bubbling for aeration + culture in nutrient broth,10%(v/v)) + (inorganic compound) Batch process - (R1, R2, R3, R4, R5, R6 and R7) respectively will be augmented with Al(NO3)39H2O, BaCl2, Pb(NO3)2, MnSO4H2O, ZnSO47H2O, NiSO6H20 and Sr(NO3)29H2O Medium Synthetic Waste water (according to khalida muda et al. 2011 method) Air flow rate for aeration = 1Lmin-1 pH = 6.0 8.0 Temperature = 272 0C (room temperature) Duration = 30min

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Experiment Set up
Objective 2 500mL of Schott bottle will be used as reactor with a working volume of 250 mL (synthetic medium + bubbling for aeration + culture in nutrient broth,10%(v/v)) + (inorganic compound) Medium Synthetic Waste water (according to khalida muda et al. 2011 method)

Duration = 60 min
Air flow rate for aeration = 1Lmin-1 Variable parameters:

- Temperature = 27- 30 0C
- pH= 6-8 - Concentration of inorganic compound selected from the 1st objective
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Experiment Set up
Objective 3

Operation set up will be according to Khalida Muda et al. 2011 et al

Parameter Measurements
Objective 1

Autoaggregation Assay - Turbidity Meter

Surface Hydrophobicity Assay - Optical Density Meter

Parameter Measurements
Objective 2

Autoaggregation Assay - Turbidity Meter- refer to khalida muda et al. 2011 Surface Hydrophobicity Assay - Optical Density Meter- refer to khalida muda et al. 2011 Characteristics of the Extracellular Polymeric Substance (EPS) and protein method will refer to Zhang et al. 1999 & Liu and Fang, 2002 Alginate-like exopolysaccharides (ALE) extraction and identification method will refer to Lin et al.2008

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Experimental Design for data analysis


Objective 1:For screening purpose Completely Randomized Design ( Statistical Package for the Social Science- SPSS software) Ojective 2: For optimization Response Surface Method (Design Expert)
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Gantt Chart of Research Activities


2011 NO ACTIVITY 1 Literature Review 2 To identify the potential of soluble inorganic compound as aid for the aerobic granular sludge development a. isolate mixed culture bacteria and 7 identified inorganic compound: b. Experimental work using Completely Randomized Design Method i. Factors: Type of inorganic compounds ii. Sample preparation: - the isolated bacteria grown in schott bottle with volume 250ml of synthetic medium + inorganic compound (10% v/v) iii. Parameter/ Response: coaggregation, hydrophobicity To optimize the effect of inorganic compound as aid in granulation development using response surface method a. Experimental work using Response Surface Method ii. Sample preparation: - the isolated bacteria grown in schott bottle with volume 250ml of synthetic medium +inorganic compound+aeration(bubbling) iv. Factors: Concentration of selected inorganic compounds, temperature and pH iii. Parameter: EPS, ALE, hydrophobicity and coaggregation & others to develop biogranules with additional of inorganic compound (biogranulation aid solution) a. using Muda K. et al. 2010 method + (inorganic compound) i. Granular sludge properties ii. Granular sludge removal performance Thesis Writing 4 5 6 7 8 9 10 11 12 1 2 3 4 5 2012 6 7 8 9 10 11 12 1 2 2013 3 4 5

Expected Outcomes
Expected Results

The potential soluble inorganic compounds that contributes in accelerating biogranulation formation during start up period were identified

The optimum condition for the effect of soluble inorganic compound as aid in the biogranulation development were achieved

Aerobic granules were developed with the additional of soluble inorganic compound

Expected Outcomes
Negatively Charged

Individual bacterium

- + BRIDGING

+
+

+ + +

SHAPING

+ - +

Extracellular polymeric substance

Positively Charged (Ionic compound)

Modified schematic representation of extracellular polymeric substance-enhanced (Liu et al., 2004).

The Solubility of Inorganic Compounds in Water


Negative Ions (Anions) any anion any anion + + + Positive Ions (Cations) alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+) hydrogen ion [H+(aq)] amonium ion (NH4+) any cation any cation silver (Ag+), lead (Pb2+), mercury (Hg2+), copper (Cu+), thallium (Tl+) any other cation = = = Solubility of compounds in water soluble soluble Example sodium fluoride, NaF, is soluble hydrogen chloride, HCl, is soluble

any anion
nitrate NO3acetate (CH3COO-) Chloride (Cl-), Bromide (Br-), Iodide (I-) Sulphate (SO42-)

+
+ + + + +

=
= = = =

soluble
soluble soluble low solubility (insoluble) soluble low solubility (insoluble)

ammonium chloride, NH4Cl, is soluble


potassium nitrate, KNO3, is soluble sodium acetate, CH3COONa, is soluble silver chloride, AgCl, forms a white precipitate (a white solid) potassium bromide, KBr, is soluble barium sulphate, BaSO4, forms a white precipitate (a white solid) copper sulphate, CuSO4, is soluble magnesium sulfide, MgS, is soluble

calcium (Ca2+), strontium (Sr2+), barium (Ba2+), silver = (Ag+), lead (Pb2+), radium (Ra2+) any other cation alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+), alkali earth metals (Be2+,Mg2+,Ca2+,Sr2+,Ba2+,Ra2+), and H+(aq), NH4+ any other cation alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+), H+(aq),NH4+,Sr2+,Ba2+,Ra2+,Tl+ any other cation alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+), H+(aq),NH4+ = =

+ Sulfide S2+

soluble soluble

Hydroxide OH-

+ + +

= = = =

low solubility (insoluble) soluble low solubility (insoluble) soluble

zinc sulfide, ZnS, is insoluble strontium hydroxide, Sr(OH)2, is soluble silver hydroxide, AgOH, is insoluble (forms a precipitate) ammonium phosphate, (NH4)3PO4, is soluble magnesium carbonate, MgCO3, is insoluble

Phosphate, PO43-, Carbonate, CO32-, sulphite, SO32-

any other cation

low solubility (insoluble)

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