Growth of sulfate reducing bacteria

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PART I

Stage I: Experimental Setup;

Growth of Sulfate Reducing Bacteria under best suitable conditions in anaerobic conditions;

Objectives of the Experiment

The main objectives of this research are;

  1. To understand the growth of SRB under optimized conditions
  2. To use SRB cultures for Stage II of the experiment
  3. To find out and confirm the best conditions for highest sulfate conversion rate into sulfides

Proposed experimental setup

The proposed experimental setup is according to the figure shown below.

The proposed method is based on severe facts from literature. In this basic study, it is intend only to study about growth of SRB under anaerobic conditions at pre defined conditions.

BIO FLO 110 apparatus would be used to setup the CSTR approach. The reactor is equipped with Heat controller, pH probe and Agitator, to control enough to make necessary environmental conditions to facilitate for highest sulfate reduction.

The control panel which comes with BIO FLO 110, control all pumps, pH levels and temperature levels.

Best suggested operating conditions of Fermenter

It is summarized; following conditions are to be fulfilled for best culture conditions for SRBs with highest sulfate reduction rates.

Table 1: The best conditions for complete sulfate reduction under SRB activities

Parameter/ description

Value/ Test condition preferred

Reference

For complete sulfate reduction

COD: Sulfate ( 1g of sulfate need 0.67 of COD)

0.67

(Liamleam & Annachhatre, 2007)

Temperature

30 0C- 350C

Best Growth rate of SRB

Temperature

28 0C - 32 0C

(Hao, Chen, Huang, & Buglass, 1996)

pH

7.0

(Rinzema & Lettinga, 1988)

Sulfide generation (HS-)

pH

7.2

Feed to the Fermenter

Reactor is to be fed continuously with necessary macro and micro nutrients suitable for bacterial growth. However, there is a need to control the sulfate and COD of the feed, in order to achieve complete sulfate reduction rates.

Molasses is acted as a carbon and electron donor for highest sulfate reduction activities.

The solutions are to be daily prepared using stock solutions. Sodium sulfate would be used as a sulfate source to the system, and this is to be added into the synthetic waste water feed with other nutrients. Molasses will be fed to the system as a separate source in order to keep ratio of Total COD/S ratio.

Macro/Micronutrients for SRB activities and method of feeding to the system

Synthetic waste water will be prepared according to following formulae which is modified from previous studies of (Suktrakoolvait, 2000) and (Isa, Grusenmeyer, & Verstraete, 1986).

Both above methods are used tap water as the media to mix these macro and micro nutrients. However, it is used distilled water in this study in order to minimize any complications and to avoid unpredictable results due to the quality of water source.

Table 2: Suggested composition of synthetic influent comparing with other studies

This study

(Isa, et al., 1986)

(Suktrakoolvait, 2000)

Composition

Qty.

Composition

Qty.

Composition

Qty.

Molasses(mL/L; V/V)

4.92**Variable

(5g O2/L)

HCOONa(g)

21.3

Molasses

Variable

Na2SO4 (g/L)

11.03 **

COD con(g/lit)

5.0

Na2SO4(g)

Variable

Kh3PO4(g)

3.0

Kh3PO4(g)

3.0

Kh3PO4(g)

3.0

K2HPO4(g)

1.0

K2HPO4(g)

1.0

K2HPO4(g)

1.0

CaCl2.2h3O(g)

0.3

CaCl2.2h3O(g)

0.3

CaCl2.2h3O(g)

0.3

NH4Cl(g)

1.0

NH4Cl(g)

1.0

NH4Cl(g)

1.0

FeCl3.6h3O(g)

0.1

FeCl3.6h3O(g)

0.1

FeCl3.6h3O(g)

0.1

MgCl2.6h3O(g)

0.1

MgCl2.6h3O(g)

0.1

MgCl2.6h3O(g)

0.1

Glucose;C6h32O6 (g)

0.1

Sucrose-C12h32O11(g)

0.1

Glucose;C6h32O6 (g)

0.1

Trace Elements(ml)

1.0

Trace Elements(ml)

1.0

Trace Elements(ml)

1.0

Distilled water(Lit)

1.0

Tap Water (Liter)

1.0

Tap Water(Liter)

1.0

Note1: Trace elements composition for all studies are same as in original source of this recipe according to (Isa, et al., 1986)

Trace elements solution (mg/L);NiSO4.4h3O – 500;MnCl2.4h3O;500; FeSO4.7h3)-500;ZnSO4.7h3O-100;H3BO3-100; Na2MoO4.2h3O-50;CoCl2.5h3O-50;CuSO4.5h3O-5;with one liter of tap water.

Note 2: Our study use distilled water to dilute above trace elements, and all water inputs are distilled water except initial seed sludge volume (around 1L of seed sludge).

Reactor operation

The CST reactor has two main stages for operation. These are startup operation step and steady state process.

It will take long time to start bacterial activities under a fresh environment. Therefore, it is necessary to introduce seed sludge in order to introduce enough number of SRB to initiate the process of sulfate reduction.

Reactor startup;

The CSTR reactor is to be started using seed sludge from anaerobic UASB reactor of treating “Beer processes waste water”. Initially, the seed sludge was screened by wire mesh filter to uniform size and to remove bigger dirt particles.

Further steps for reactor startup and prepeartion of media are as follows;

  • Prepare sulfate containing synthetic waste water and mixed with screened seed sludge (ratio of 1:1 v/v).
  • Then allow this mixture to be settled for 24 hours.
  • After settling, only the upper and lighter sludge fraction would be used as seed sludge.
  • Then, it is filled only 40 % of CSTR reactor with seed sludge.( VSS and TSS would be approximately having ratio of 0.4)
  • Initial pH of the reactor is around 7.2, and synthetic waste water would be fed until working volume reaches up to 5L.
  • No recirculation would be engaged at startup period

Steady state process performance;

Steady state process would be performed after 5 days of startup process. Then after it would be operated with following conditions;

  • Recirculation -
  • Hydraulic retention time (HRT) of …….
  • In this stage , it would be analyzed for sulfate reduction rate, VFA, COD:S ratio of influent and effluent, and other main parameters.
  • Further, it will be continuously assed for the performance of the reactor with above mentioned optimized conditions for SRB growth.

Analytical Methods

The analytical methods for measuring of parameters are shown below, in the table.

Parameter

Method

COD

Dichromate method

Sulfate

Modified Turbidimetric method

Sulfide

Iodometric titration

VFA

Titrimetric method and Gas Chromatography

Alkalinity

Titrimetric method

pH

pH meter

TSS/VSS

Filtration method

Gas Production

Water Displacement

Gas composition

Gas Chromatography

Calculations and measured parameters

COD

From lab experiment, it was found that the COD of molasses is 1.016 * 106 mg/L(3563mg COD/5g of Molasses)

The total COD of molasses which is to be used in this experiment has COD value of; 1016 g O2/Liter

SO42- in 1 N Na2SO4 solution;

(Molar mass= 142.04 g/Mol)

= 96.03 g SO42-/ Lit;

(0.00102 N Na2SO4 solution ; 1ml = 100 µg SO42)

Therefore, to make an approximate ratio of COD:Sulfate =0.67 at initial feed tank,

COD= 5,000 mg/L = 5 g O2 /L

SO42- = 7,463 mg/L = 7.46 g/L

Hence; for this molasses,

I. the amount to mix with 1 L distilled water = (1000ml/(1016g/5g/L)=4.92 mL/L

= 4.92 mL/L of Molasses to be diluted with 1 L of water

= Density of molasses= 1.426 Kg/L

Therefore equivalent value to mix in g= (1.426 *1000 g/1000 ml)*4.92 mL = 7g of molasses

II. 142.04g/ (96030/7463) = 11.03 g Na2SO4/ Liter solution should be fed to the system.

*In above formulae**variable= The feed rate of molasses will be varied depending on total sulfate amount to the system which is mixed with trace element solutions, and other macro-nutrients.

Seed Sludge;

Seed sludge from “beer industry” is used to start the reactor. It is to be used 1 L of seed sludge. (Recommended MLSS is about 5000 -6000 mg/L).

The measured quality of seed sludge is;

Seed sludge analysis

MLSS =(34134 +24450)/2 mg/l = 29292 mg/L
MLVSS =(20362+ 17500 )/2 mg/l = 18931 mg/L

Above is the value for two separate tests and average of them.

COD and sulfur mass balance; refer saworpak

Molasses:

COD of molasses: 3.563 g/ 5g (Laboratory results on 11/3/2011).

The calculation is;

Density of molasses = 1.426 Kg/L; Measured in laboratory = (35.34g/25mL)

Therefore, COD in 1 L of molasses= (3.563 g/5g)*1426g O2/L= 1.016*106 mg O2/liter.

CSTR design concepts

Initial volume of seed sludge??

Molasses: Sulfate??

Notes: Day 1 initial operations:

1. COD: Sulfate ratio = 0.67

Only external influent is molasses, hence dilution factor of the synthetic feed is;

A. Molasses = ??

B. Na2SO4 = ???

Molasses fermentation reaction;

C12h32O11 þ h3O→4CH3CHOHCOOH:

Actual Experimental setup with recirculation

Working volume of CSTR ?

Volume of recirculation tank/

Keep in 4 C to analyze later??? reference

Parameters to check

  1. COD of Molasses:
  2. Influent and effluent sulfates
  3. VFA;

Parameter

Frequency

Method

References

Sulfate from culture of SRB bacteria.

Daily or HRT

Modified Turbidimetric assay

(Kolmert, Wikström, & Hallberg, 2000)

CSTR Kinetic Co-efficient;

In this section, it will decide and calculate the coefficients for SRB growth in the CSTR reactor.

% Pump in

Qin L /day

Detention time (Ө)(days)

Detention time (Ө)(Hrs)

1

0.14

38.89

933.33

2

0.29

19.44

466.67

3

0.43

12.96

311.11

4

0.58

9.72

233.33

5

0.72

7.78

186.67

6

0.86

6.48

155.56

7

1.01

5.56

133.33

8

1.15

4.86

116.67

9

1.30

4.32

103.70

10

1.44

3.89

93.33

11

1.58

3.54

84.85

12

1.73

3.24

77.78

13

1.87

2.99

71.79

14

2.02

2.78

66.67

15

2.16

2.59

62.22

16

2.30

2.43

58.33

17

2.45

2.29

54.90

18

2.59

2.16

51.85

19

2.74

2.05

49.12

20

2.88

1.94

46.67

Experimental Results

1. Seed sludge analysis;

MLVSS/MLSS ratio of seed sludge used = 17500 /24450 = 0.7157

--------------------------------------------------------------------------------------------------

PART II

Methodological explanations and procedures for parameter analysis

A. COD measurements

There are two types of CODs to measure.

  1. Particulate COD and
  2. Soluble COD.

The sample is to be filtered through 0.45 micron GFC filter, for soluble COD. Rest of the procedure is same in both methods as follows;

Experimental methodology (CLOSED REFLUX METHOD)

Notes and attention: Use dry pipettes when taking acids, and need to pay extra attention on following;

  1. Standardize should not be keep in oven
  2. Remember to add , INDICATOR before titrate
  3. Release cap of COD bottle just after VORTEXING

STEP 1: Add amount of sample to COD bottle, diluting enough to measure COD between 40- 400 mg/L. Total volume with dilution is 10 mL.

Note: Preparation of blank and standardize; Need two blanks and one standardize containing 10 mL of MQ/Distilled water.

STEP 2: Add 6 ml of digestion solution to each sample and blanks.

STEP 3: Add 14 mL of Acid Solution (h3SO4 + Ag2SO4);

In this step, if it shows a green colour, that indicates the requirement of more diluted sample. So, do not proceed further, at such situations.

STEP 4: Mix well using VORTEX and keep in Oven (150 0C) for 2 hours.

STEP 5: Allow cooling and performing titration step. If urgent, it can be cooled using a water bath too.

STEP 6: Titration;

  1. Pour the solution in to a Elementary flask (100 mL).
  2. Add Ferroin indicator - two drops
  3. Titrate using FAS solution as the titrant.
  4. Stop titration when color changes from BLUE GREEN to REDDISH BROWN
  5. Note: Use TFE covered magnetic stirring bar for better mixing

STEP 7:

Find out the Morality using Blank and standardize, and perform the COD calculation according to the given equation.

(Dilution factor is important to convert value to mg COD/L criteria.)

Sample & reagent quantities

Vessel size

Sample mL

Digestion Sol

Sulfuric acid reagent

Total Volume

25*150 mm

10.0

6.0

14.0

30.0

Calculations:

COD , mgO2L=A-B×M×8000mL of sample

Where, A = mL of FAS used for Blank

B = mL of FAS used for sample

M = Morality of FAS

M = Morality of FAS=mL of Digestion solution×0.10mL of FAS

Quick materials to the COD test

1. Requirement of chemicals to perform X samples

Solution

Amount/sample

(mL)

Stock solution(mL)

Amount of samples(Nos)

Digestion

6

500

80

h3SO4 + Ag2SO4

14

1000

70

FAS

10

750

70

Indicator

0.1 mL

100 mL

1000

Therefore prepare, above amounts initially and order chemicals accordingly

2. Equipment list

Description

Qty.

COD Bottles

08

TFE covered Magnetic stirring bars

08

Elementary flask

08

Volumetric pipettes 1 mL, 5 mL, 14 mL, 0.5 mL, 10 mL

02 each

Stirrer machine

01

50 mL burette

01

3. Chemicals and Reagent preparation

Chemical / Reagent

Qty of solution

Method of preparation for given Qty

1. Ferroin indicator

100 mL

Dissolve 1,10 phenanthroline monohydrate (1.485/5 g)

And (695/5)mg Fe.SO4.7 h3O in distilled water and dilute to 100 mL .

2. Digestion solution

1L

Add to about 500 mL distilled water with 4.903 g of K2Cr2O7, primary standard grade, previously dried at 1500C for 2 hrs,(use beaker to dry in oven before weighing)

Add 167 mL of Conc. h3SO4 and 33.3 g HgSO4.

Dissolve all above , allow to cool in room temperature and dissolve up to 1000 mL in volumetric flask

3. Sulfuric acid reagent(h3SO4 + Ag2SO4 Reagent)

1L

Add 10.12 g of Ag2SO4 reagent or technical grade, crystals or powder to 1 L of h3SO4 . Let stand 1- 2 days to dissolve Ag2SO4 , and mix thoroughly.

4. Standard Ferrous Ammonium Sulfate (FAS) solution (0.1 M approx.)

1 L

Dissolve 39.2 g Fe(NH4)2(SO4)2.6h3O in distilled water.

Add 20 mL conc. h3SO4,

Cool and dilute to 1000 mL

Note: Standardize this solution daily as follows.

o Pipette 6 mL digestion solution in to a small beaker

o Add 10 mL of reagent water to substitute for sample

o Add 14 mL of Sulfuric acid reagent

o Cool to room temperature, and add 1-2 drops diluted Ferroin indicator

o Titrate with FAS titrant

H2SO4; molar weight 98 g/mol; density= 1.84 g/cm3

---------------------------- End of COD --------------------------------------

B. Sulfate measurements:using Modified TurbidimetricAssay(MTA);

Range of sulfate = 0 – 4 Mm( 0 – 400 mg/L)

Key Notes: * Colored and Turbid samples should be filtered and remove colour and suspended solids.

Refer method 9038 for Color correction.

Main steps of the experiment;

Chemicals ;

1. Crushed barium chloride ( BaCl2);

STEP 1:

  1. Add 1mL of sample (Dilute if necessary) with 1 mL of conditioning reagent in a test tube. Then thoroughly mixed.
  2. Crushed BaCl2, about 60 mg should be added to above mix and then

C. MLSS(Mixed Liquor Suspended Solid)/TSS and MLVSS(VSS)

Both are just terms used, and followed same standard method. MLVSS below is the extended of the same test, finding the volatiles. Therefore, SS and VSS should be calculated in one test procedure.

Step1:Filter paper + Evaporation disk in Oven at 550 oC for 1 Hr

Step 2: Allow cooling in desiccator for 30 minutes

Step 3: Get weight of F+ED = M1

Step 4: Filter the sample using required dilution if any,

Step5: Keep 1050C for 1 Hr, and keep in desiccator for 30 min

Step 6: Get weight M2

Step 7: Keep at 5500C for 20 minutes, and keep in desiccator for 30 minutes

Step 8: Get weight M3

Calculations:

TSS=SS = (M2–M1)*1000mg/ Sample size (mL)* 1000 = mg/L

VSS = ( M2 – M3) *1000/ Sample size(mL)*1000(L) = mg/L

D. VFA (Volatile Fatty Acids)

VFA and Alkalinity Estimation Protocol for Leachate sample

Chemicals table

Chemical name /Morality

Qty

Method

1. O.1 N h3SO4

1000 mL

Get 2.77 96% Con. Sulfuric acid and dilute to 1000 mL

2. 0.1 N NaOH

1000 ml

Weight 4 g of solid NaOH and diluted upto 1 L

  1. Take Sample Volume of 10mL in a 250 mL beaker.
  2. Measure the initial leachate pH of the sample with pH meter.
  3. Bring the pH of sample down to 4.3 with addition of 0.1 N h3SO4 and measure the volume of acid consumed for alkalinity calculation.
  4. To measure VFA in the same sample, bring its pH down to 3.5 by further adding the same acid.
  5. Keep the sample for distillation in a distillation apparatus and wait 3 minutes after sample getting boiling.
  6. Allow the sample temperature to reach around 60-700C
  7. Start adding 0.1N NaOH to increase the pH to 7.0 and measure the volume of NaOH used for calculating VFA.

Calculations

V mL of acid consumed X Normality of the acid used X 50000

Alkalinity (mg/L) = ------------------------------------------------------------------------

V mL of Sample taken

V mL of Alkali consumed X Normality of the alkali used X 50000

VFA (mg/L) = ------------------------------------------------------------------------

V mL of Sample taken

** Find the Normality of Acid/Alkali titrating against one.

Do this step before doing sample, and use same value.

Need to use indicator for this step: as follows,

E. Alkalinity; Use above method as it is simple and handy

Calculation;

Alkalinity , mg/L as CaCO3 = A*N*50000/mL of sample

A = mL standard acid used and

N = Normality of standard acid

Sample: Do not filter, dilute, concentrate or alter the sample

Sample Volume: 20 mL or marginal is good; the sample size to be defined and can be adjusted by changing morality of titrant.

Preparing of standard acidic solution:

  1. Alkalinities < 1000 mg as CaCO3/L ; use 0.01 N HCL solution
  2. Alkalinities > 1000 mg as CaCO3/L ; use 0. 1 N HCL solution as titratnt

Procedure:

  1. Select sample size and normality of titrant
  2. Adjust sample to room temperature
  3. Discharge sample to a elementary flask using pipette(Keep pipette tip near flask bottom)
  4. If free residual chlorine is present, add 0.05 mL( 1 drop) of 0.1 M Na2S2O3 solution
  5. Add 0.2 mL of indicator( 5 drops) and titrate
  6. Titrate to pH 4.5 or lower
  7. Color is changed over a white surface to a persistent color of the equivalent point

Indicator Solution

Bromcresol green indicator: (pH 4.5 indicator); Dissolve 100 mg bromcresol green sodium salt in 100 mL distilled water.

Chemicals (Alkalinity test)

  1. Bromecresol green sodium salt - 100 mg
  2. Na2S2O3 - 20g
  3. HCl

F. pH

pH calibration for testing equipments;

pH = 4.01 à Dissolve 10.12 g of KHP( KHC8H4O4) in MQ water ,and dilute to 1 L

pH = 6.86 à Dis 3.387 g Kh3PO4, and 3.533 g Na2HPO4 in distilled water à1 L

pH = 10.01à Dis. 2.092 g NaHCO3 and 2.640 g Na2CO3 in distilled water à 1L

G. Sulfides; Using IDOMETRIC Method

Keynotes:

Step 1: Reagent preparation ;( follows the table below)

Reagent / Chemical

Method of preparation

1. HCl – 6N

Add 180 mL of Con.HCl in to distilled water and dilute to 1 L

2.

H. Gas production:

Gas production is measured by Gas Counter equipment. It is calibrated to have (=25 mL* 2) 50 mL of Gas per one unit of the electronic counter fitted on the unit.

The mechanism works using water displacement technique.

I. Gas composition

To know the composition of gas that the method used is GAS Chromatography.

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