Post on 06-Apr-2018
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Experimental Design for the Effect of InorganicCompound in the Aerobic Granular SludgeDevelopment
Research Proposal: First assessment
Master Student: Muhammad Anwar Alias
Supervisor: Dr. Khalida Bt Muda
Co- Supervisor: Dr. Azmi Aris
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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
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Introduction
Biorranulation
Granulation is the process in which a nondiscrete flocculent biomass forms discrete well-
defined pellets or granules which can be severalmillimeters in diameter (Ong et al., 2002).
Involves cell-to-cell interactions that includebiological, physical and chemical phenomen
Granulation aid is promoting the formation of thegranules or granular biomass.
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Introduction
Biogranulation can be classified as: aerobic granulation anaerobic granulation
Biogranules form through self-immobilization ofmicroorganisms.
These granules are dense microbial consortiapacked with different bacterial species andtypically contain millions of organisms per gramof biomass.
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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
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Problem Statement
Time consuming for developingfully complete matured granules.
During long-term SBR operation, the aerobic
granular sludge process gradually loses stabilityunder inappropriate operational conditions.
Aerobic granules often showed a poor stabilityafterseveral months of operation (de Kreuk andvan Loosdrecht, 2004; Liu et al., 2006) which theapplication wastewater treatment is limited
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Aim and Objectives of the Study
1To identify thepotential solubleionic inorganiccompounds thatcontributes in
acceleratingbiogranulationformation duringstart up period
2
To optimize theeffect of inorganiccompound as aidin thebiogranulation
development
3To developbiogranules withthe additional ofinorganiccompound
The aim of this study is to enhance the aerobic granulation processusing inorganic compound.
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Scope of the Study
Scope
Start up for aerobic granulationprocess/Initial stage
InorganicCompound as aid
Lab scale reactoraerobic condition
Bacterial isolation fromsewage sludgeSynthetic wastewater
Parameters to studythe affect
Apply to the existencesystem- develop
granule
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Significance of the Research
This research could be helpful for enhancing the aerobicgranules development via addition ofionic organic compound
as granulation aid in terms of fast formation, high durabilityand stability, and high removal performance.
Thus, this research development can be utilized to overcome ourlocal waste water problem especially in palm oil mill effluent,leachate and municipal waste water.
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Literature Review
Aerobic Sludge Granulation Process
Sludge may be defined as the microbial biomassutilizing nutrient substrates present in wastewater.
Microbial granules are characterized as dense
and compact microbial aggregates with aspherical outer shape.
Aerobic granules are self-immobilized sphericalaggregates of microorganisms.
The growth of aerobic granules were considered
to be a special case of biofilm growth (Liu andTay, 2002; Yang et al., 2004).
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Inorganic Compound Inorganic compounds have traditionally been
considered to be of inanimate, non-biological origin
Inorganic compounds, consist of molecules with acentral metal atom (usually transition metal) bondedto 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 proteinsand substrate bindings
Example : MgSO4, Al(NO3)39H2O, BaCl2, Pb(NO3)2,MnSO4H2O
Literature Review
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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
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Literature Review
EPS is recognized as a key factor in thegranulation process (Wang et al. 2006),which is mainly composed ofpolysaccharide and protein
Previous studies shows divalent metalionic such as Ca2+, Mg2+ and Fe2+
augmentation led to the higher productionyield ofEPS (polysaccharides & protein)resulted in faster granulation process andshorter starting-up period
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can neutralize the negative charge on the surface ofbacteria, and promote the sludge granulation because ofthe positive charge
The cationic could bind to the negatively charged groups onbacterial surfaces and extracellular polymeric substances(EPS), and act as a bridge to interconnect thesecomponents, and then form EPS(cationic)EPS cross-linkage
could accelerate microbial aggregation, and increase thephysical strength of mature granules
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Literature Review
Previous studies shows that Cationic from the inorganic
compound :
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Mechanism of aerobic granulation
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Mishimaand
Nakamura(1991)
Based on experiments in an aerobic upflow sludge blanket(AUSB), the biological assemblies is similar to the anaerobicgranulation process, filamentous bacteria tangled with eachother to form aerobic granules.
Beun et al.(1999)
Proposed mechanism of aerobic granulation in SBR
Beun et al. (1999) proposed a model in which aerobicgranulation could be started with fungi.
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Modified schematic representation of extracellular polymeric substance-enhanced
(Liu et al., 2004).
Individual
bacterium
Extracellular polymeric
substance
Positively Charged (Ionic
compound)
Negatively Charged
--
-
- -
-
-
-
-
-
-
-
-+
+
+
+
+
++
BRIDGING
-
-
SHAPING
--
-
-
-
-
-
-
-+
+
+
+
+
+
+
++
+
LITERATURE REVIEW
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Previous studies on sludge biogranulation process using inorganic compoundLITERATURE REVIEW
CONTINUED..
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;COD = Chemical Oxygen Demand; TOC = Total Organic Content; NH4
+-N = Ammonium
Reactor
system
BiomassType of
wastewater
Inorganic
compound used
Biogranule
characteristic
Performance
on granulation
development
Remarks Reference
SBRAerobic
granule
Synthetic
waste water
R1 augmented with
10 mg/L of Mn2+
R2 no Mn2+
Diameter size:
R1= 2.53.5 mm
R2= 1.5- 2.5mm
25 days
Aerobic granules first
formed in
reactor R1 on day 6
Lihui et al
(2011)
ASBAerobic
granules
Synthetic
wastewaterCa2+ augmentation
Diameter: 2.8mm
Wet density: 1.01 kgL-1 32 days
Ca2+-fed granules were
denser and more
compact, better
settling and strength
characteristics, and
higher polysaccharide
contents.
Tay T-L et
al., (2002)
SBRAerobic
granule
Synthetic
waste water
R1 augmented with
Ca2+ at 40 mg/L
R2 augmented withMg2+ at 40 mg/L
Diameter size :
R1= 1.32mm
R2=0.2
1mm
21 days
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[
Lin Liu et
al. (2010)
CONTINUED LITERATURE REVIEW
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Reactorsystem
BiomassType of
wastewaterBiomaterial used
Biogranulecharacteristic
Performanceremoval
efficiencyRemarks Reference
SBRAerobicsludgegranule
Syntheticwastewater(glucose)Influent COD= 640 mg/l
Mg2+augmentation
Size: 2.9mmSpecific gravity:1.002 kg/lMLSS: 3.2 g/lSS: 7.6 g/lSVI: 25 ml/g
Effluent COD= 56 mg/l;COD removal= 91%
Mg2+ augmentationsignificantlydecreased thesludge granulationtime from 32 daysto 18 days.
Li X-M etal., (2008)
SBR
Denitrifyinggranules(anaerobiccondition)
Syntheticwastewater
(potassiumnitrate;methanol)Nitrate = 120mg/l;TOC = 120mg/l
Ca2+ augmentation
Mean size ofbiomass: 420mMLVSS/MLSSratio: 0.37SVI: 14.36 ml g-1
NO3--N; TOC
removal =nearly 100%
-Liu Y-J and
Sun DD,(2010)
CONTINUED.. LITERATURE REVIEWPrevious studies on sludge biogranulation process using inorganic compound
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Literature Review
can neutralize the negative charge on the surface ofbacteria, and promote the sludge granulation because ofthe positive charge
The cationic could bind to the negatively charged groups onbacterial surfaces and extracellular polymeric substances(EPS), and act as a bridge to interconnect thesecomponents, and then form EPS(cationic)EPS cross-linkage
could accelerate microbial aggregation, and increase thephysical strength of mature granules
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Previous studies shows that Cationic from the inorganic
compound :
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Methodology
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Stock solutions having 7 types of inorganiccompounds are prepared by dissolvingappropriate quantities in deionized water
List of inorganic compound will be used in this
research:
Preparation of Inorganic compound
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
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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 inuniversal bottle as stocks.
The stocks are stored at 4C and working solutions of requiredconcentrations were obtained by appropriate dilution in deionizedwater prior to each experiment
Culture will be grown in the Nutrient Broth to more quantity
Preparation for stock culture of bacteria
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List of reagent used in the experiment
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Reagents/ Chemicals Application
Nutrient Agar Spread Plate
Potassium phosphate, K2HPO4 Washing buffer
Xylene (C6H4(CH3)2) Surface hydrophobicity assay
Nutrient broth Bacteria culture
Sodium Chloride EPS Extraction Method
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Research Framework
Objective 1
Stage 2
To identify the potential of soluble ionic inorganic
compound as aid for the aerobic granulation start-up
Stage 1 Isolation of bacteria from sewage waste water sludge
Screening for bacteria selection in the aerated synthetic medium
Response: Hydrophobicity and Autoaggregation
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 toselect the best ionic inorganic compound
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To optimize the effect of inorganic compound as aid in
granulation start up using response surface method
Objective 2
Optimize the selected ionic inorganic compound using
synthetic medium with aeration under room temperature
Research Framework
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 conditionof ionic inorganic compound for the aerobic granulation start-up
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Research Framework
To develop biogranules with the additional of solubleinorganic compound
Objective 3
Operation process using according to Muda K. et al.
2009 method + (inorganic compound)
i. Granular sludge properties
ii. Granular sludge removal performance
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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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Operation set up will be according toKhalida Muda et al. 2011 et al
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Experiment Set up
Objective 3
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Parameter Measurements
Autoaggregation Assay - Turbidity Meter
Surface Hydrophobicity Assay - Optical Density
Meter
Objective 1
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Parameter Measurements
Autoaggregation Assay - Turbidity Meter- refer to khalida
muda et al. 2011
Surface Hydrophobicity Assay - Optical Density Meter- referto 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
Objective 2
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Experimental Design for data analysis
Objective 1:For screening purpose
Completely Randomized Design ( StatisticalPackage for the Social Science- SPSSsoftware)
Ojective 2: For optimization
Response Surface Method (Design Expert)
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Gantt Chart of Research Activities
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Gantt Chart of Research Activities2011 2012 2013
NO ACTIVITY 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 51
Literature Review2 To identify the potential of soluble inorganic
compound as aid for the aerobic granular
sludge developmenta. isolate mixed culture bacteria and 7 identifiedinorganic compound: -
b. Experimental work using CompletelyRandomized Design Method
i. Factors: Type of inorganic compounds
ii. Sample preparation: - the isolated bacteriagrown in schott bottle with volume 250ml ofsynthetic medium + inorganic compound (10% v/v)
iii. Parameter/ Response: coaggregation,hydrophobicity
3 To optimize the effect of inorganic compoundas aid in granulation development usingresponse surface methoda. Experimental work using Response SurfaceMethod
ii. Sample preparation: - the isolated bacteriagrown in schott bottle with volume 250ml ofsynthetic medium +inorganiccompound+aeration(bubbling)
iv. Factors: Concentration of selected inorganiccompounds, temperature and pH
iii. Parameter: EPS, ALE, hydrophobicity andcoaggregation & others
4 to develop biogranules with additional ofinorganic compound (biogranulation aidsolution)a. using Muda K. et al. 2010 method + (inorganiccompound)
i. Granular sludge properties
ii. Granular sludge removal performance
5Thesis Writing
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Expected Outcomes
ExpectedResults
The potential solubleinorganic compoundsthat contributes inacceleratingbiogranulation
formation during startup period wereidentified
Aerobic granuleswere developed withthe additional ofsoluble inorganiccompound
The optimum
condition for theeffect of solubleinorganic compoundas aid in thebiogranulationdevelopment wereachieved
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Modified schematic representation of extracellular polymeric substance-enhanced(Liu et al., 2004).
Individual
bacterium
Extracellular polymeric
substance
Positively Charged (Ionic
compound)
Negatively Charged
--
-
--
-
-
-
-
-
-
-
-+
+
+
+
+
+
+
BRIDGING
-
-SHAPING
--
-
-
-
--
-
-+
+
+
+
+
+
+
++
+
Expected Outcomes
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The Solubility of Inorganic Compounds in Water
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y g p
Negative Ions
(Anions)
+ Positive Ions
(Cations)
= Solubility of compounds in
water
Example
any anion + alkali ions
(Li+,Na+,K+,Rb+,Cs+,Fr+)
= soluble sodium fluoride, NaF, is soluble
any anion + hydrogen ion
[H+(aq)]
= soluble hydrogen chloride, HCl, is soluble
any anion + amonium ion
(NH4+)
= soluble ammonium chloride, NH4Cl, is soluble
nitrate
NO3-
+ any cation = soluble potassium nitrate, KNO3, is soluble
acetate
(CH3COO-)
+ any cation = soluble sodium acetate, CH3COONa, is soluble
Chloride (Cl-),
Bromide (Br-
),Iodide (I-)
+ silver (Ag+), lead (Pb2+), mercury (Hg2+), copper
(Cu+
), thallium (Tl+
)
= low solubility (insoluble) silver chloride, AgCl, forms a white
precipitate (a white solid)
+ any other cation = soluble potassium bromide, KBr, is soluble
Sulphate
(SO42-)
+ calcium (Ca2+), strontium (Sr2+), barium (Ba2+), silver
(Ag+), lead (Pb2+), radium (Ra2+)
= low solubility (insoluble) barium sulphate, BaSO4, forms a white
precipitate (a white solid)
+ any other cation = soluble copper sulphate, CuSO4, is soluble
Sulfide
S2-
+ alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+), alkali earth
metals (Be2+,Mg2+,Ca2+,Sr2+,Ba2+,Ra2+),
and H+(aq), NH4+
= soluble magnesium sulfide, MgS, is soluble
+ any other cation = low solubility (insoluble) zinc sulfide, ZnS, is insoluble
Hydroxide
OH-
+ alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+),
H+(aq),NH4+,Sr2+,Ba2+,Ra2+,Tl+
= soluble strontium hydroxide,
Sr(OH)2, is soluble
+ any other cation = low solubility (insoluble) silver hydroxide, AgOH, is insoluble
(forms a precipitate)
Phosphate, PO43-,
Carbonate, CO32-,
l hi SO 2
+ alkali ions (Li+,Na+,K+,Rb+,Cs+,Fr+), H+(aq),NH4+ = soluble ammonium phosphate,
(NH4)3PO4, is soluble
h i l l bili (i l bl ) i b M CO i