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Culturing Pythium irregulare for the Production of omega-3 fatty acid and Biomass Yield and COD/TSS Correlation

 

Brad Kairdolf

Introduction

Pythium irregulare is a filamentous fungi which can be grown under aerobic conditions.  The organism is useful because it produces an intracellular product, omega-3 fatty acids.

This compound is important in a healthy human diet and has been proven to be lacking in many people’s diet.  The compound can be extracted and purified and then possibly used as a dietary supplement. 

The goal of this project is to determine the biomass yield (YB) and a COD/TSS correlation for Pythium irregulare.  Biomass yield is an important parameter to ascertain for a microorganism because it can be used to calculate the concentration of substrate that must be provided in order to produce a given amount of biomass, and COD can be a quick method in determining the biomass concentration in a reactor, particularly in a suspended culture. 

For this experiment, Pythium irregulare will be cultured and the substrate and biomass will be measured as well as COD of a solution of biomass that has been blended. 

Reactor

In this experiment, Pythium irregulare was grown aerobically in 250 mL Erlenmeyer flasks.  Since Pythium irregulare is a filamentous fungi, the growth of the organism can be considered as fixed film for the purposes of this project. 

There were no influent or effluent flows for this reactor, thus it is a batch reactor. 

Glucose is used as the carbon source, energy source and the electron donor for this microorganism.  Since this reactor is aerobic, oxygen is used as the terminal electron acceptor. 

As stated earlier, Pythium irregulare produces a very useful intracellular product, omega-3 fatty acids. 

System Diagram

Materials and Methods

Materials:

Glucose

KH2PO4

Yeast extract

Potato dextrose agar of Pythium irregulare

Autoclave

250 mL Erlenmeyer flask

Laminar flow hood

Ethanol

Votive

Spatula

.45 micron filter

Digital spectrophotometer

Polytron

Freezer

Centrifuge tube

Tweezers

Aluminum weigh tins

Digital scale

Plastic Buchner Funnel

Quarter inch rubber tubing

Vacuum pump

Drying oven

Micropipette

DNS reagent

COD vials

Glucose standard solution

COD standard solution

HPLC

Media Preparation:

Add the following compounds to 1 Liter of water and mix thoroughly until dissolved. 

10 g glucose

1 g KH2PO4

5 g yeast extract

Pour approximately 50 mL (measure the exact volume) of the media into 250 mL Erlenmeyer flasks.  Place a foam cap on each flask and autoclave. 

Inoculation:

Turn on laminar flow hood and clean surface with ethanol and kimwipes.

The Pythium irregulare to be used has been cultured on a potato dextrose agar plate.  

Flame the spatula to be used and then allow it to cool.  Using the flat end of the spatula, cut a 1 cm from the agar plate.  Remove the foam and flame the opening of the Erlenmeyer flask.  Place the square from the agar into the flask.  Flame the end of the flask again and replace the foam cap.  Place the inoculated flask in the shaker and incubate the flask for 5 days at 20 oC. 

Analysis:

After 5 days, remove the flasks from the incubator.  Filter the biomass and measure the glucose concentration using Total Reducing Sugars (TRS) test (a new calibration curve must be prepared as well).  Also test substrate with HPLC.  Weigh the biomass and then place half of the biomass on a filter and place in the oven to dry.  Place the other half in a plastic tube and place in the freezer. 

Add approximately 30 mL of water to the tubes containing the biomass and then shear the cells using a Polytron.  Perform a Chemical Oxygen Demand (COD) test on both the oven dried and the frozen biomass samples.  Then filter 20 mL of the solution through a .45 micron filter and dry and weigh the filter to determine the Total Suspended Solids (TSS) of the solution.  Dilute both samples and repeat the COD/TSS tests. 

Results

To determine the glucose concentration of the substrate, a TRS test was performed.  To obtain the correct correlation between concentration and absorbance, standards were tested. Table 1 shows the calibration data for the TRS test.

 

Table 1:  TRS Calibration Data

Standard Conc. (mg/L)

Abs.   (540 nm)

0

0

500

0.37

1000

0.645

This data was then plotted in order to obtain the correlation factor.

Figure 1:  TRS Calibration Curve

Table 2 shows the absorbance values (in replicates) for the reactor substrates after the 5 day incubation period was completed.  Also shown are the mean, standard deviation and coefficient of variation data for the duplicate samples.

Table 2:  Susbstrate Concentration data (using TRS)

Sample ID

Abs.   (540 nm)

Conc. (mg/L)

MEAN

Standard Dev.

Coeff. Of Variation

1a

0.262

394.578

393.072

2.129

0.541

1b

0.26

391.566

2a

0.305

459.337

460.090

1.064

0.231

2b

0.306

460.843

3a

0.271

408.132

391.566

23.428

5.983

3b

0.249

375

4a

0.256

385.542

392.319

9.584

2.442

4b

0.265

399.096

5a

0.252

379.518

386.295

9.584

2.481

5b

0.261

393.072

After the substrate concentration was determined, biomass was calculated by filtering and weighing.  This was then used to determine the biomass concentration by dividing the biomass by the volume in the reactor.  Finally, biomass concentration and substrate utilized was used to determine the biomass yield (YB) for Pythium irregulare.

Table 3:  Biomass and Biomass Yield data

Clean Filter Weight (g)

Filter weight with Biomass (g)

Biomass weight (g)

Volume (L)

Conc. (g/L)

Biomass Yield  DXB/DS

2.652

2.863

0.210

0.047

4.474

0.447

2.576

2.914

0.339

0.067

5.055

0.506

2.540

2.781

0.241

0.052

4.627

0.463

2.493

2.832

0.340

0.040

8.497

0.850

2.493

2.697

0.204

0.064

3.189

0.319

Next, a COD/TSS curve was developed.  First, a calibration curve was obtained by performing the COD test on known COD standards.  The data is shown below in Table 4.

Table 4:  COD Standard Curve data

COD Standard (mg/L)

Abs.   (600 nm)

0

0

250

0.115

500

0.229

750

0.345

The data was then plotted to determine a correlation between absorbance and COD.

Figure 2:  COD Standard Curve

This correlation was then used to determine the COD of the biomass samples.  Table 5 shows the absorbance readings at 600 nm and the resulting calculated COD along with the mean, standard deviation and coefficient of variation for the replicates.

Table 5:  Biomass COD data

ID

Abs. (600 nm)

COD (mg/L)

Mean (mg/L)

Standard Dev.

Coefficient of Variation

1

0.091

198.08446

193.731

6.1567852

3.178008005

2

0.087

189.37745

3

0.184

400.52242

525.6857

177.00757

33.67175149

4

0.299

650.84893

5

0.061

132.78189

138.2238

7.6959815

5.567769931

6

0.066

143.66565

7

0.078

169.78668

196.9961

38.479908

19.5333365

8

0.103

224.20549

  TSS was calculated next to compare the data to the data for COD.  Clean filters were used to filter deionized water and were placed in the oven in order to determine the correction for the filters.  The data and resulting correction is shown in Table 6.

Table 6:  Oven dried filter Correction

Filter weight (g)

Filter weight after drying    (g)

Correction (g)

Mean Correction (g)

0.0791

0.0778

0.0013

0.0016

0.0798

0.078

0.0018

The TSS test was performed by filtering 20 mL of the solutions used for COD through a .45 micron weighed filter.  The filters were then placed in the oven to dry.  After they were completely dried, the filters were weighed again and the Biomass weight was calculated.  The data and resulting calculations are shown in Table 7.

Table 7:  TSS data

ID

Clean Filter weight (g)

Filter weight after correction (g)

Filter weight with Biomass (g)

Biomass Weight   (g)

TSS (mg/L)

Mean TSS

Standard Dev.

Coefficient of Variation

1

0.081

0.079

0.078

0.0010

50

52.5

3.536

6.734

2

0.081

0.079

0.078

0.0011

55

3

0.083

0.081

0.078

0.0032

160

172.5

17.678

10.248

4

0.084

0.082

0.078

0.0037

185

5

0.079

0.078

0.077

0.0003

15

17.5

3.536

20.203

6

0.079

0.078

0.077

0.0004

20

7

0.080

0.078

0.077

0.0008

40

45.0

7.071

15.713

8

0.079

0.077

0.076

0.0010

50

The mean COD data was then correlated to the mean TSS data.  The correlation is shown in Table 8 and the resulting regression curve is shown in Figure 3.

Table 8:  TSS/COD Correlation data

COD (mg/L)

TSS (mg/L)

0

0

138.2

17.5

193.7

52.5

197

45

525.7

172.5

Figure 3:  COD/TSS Correlation curve

The regression was used to obtain a correlation equation for COD and TSS.

Finally, the substrate was tested using an HPLC.  The calibration data and duplicate runs are shown in the tables and figures below.

Figure 4:  HPLC calibration

Table 9:  HPLC calibration data

Figure 5:  First HPLC run of substrate

 

Table 10:  First HPLC run data

Figure 6:  Second HPLC run for substrate

 

Table 11:  Second HPLC run data

Discussion

The correlation curve that was obtained in this project can be very useful in the design of a Pythium irregulare reactor.  Although the reactor used was a lab scale reactor considered to be fixed film, there have been efforts in designing both batch and CSTR reactors for Pythium irregulare that are suspended.  This correlation provides a quick and easy method for calculating biomass, which can then be used to calculate the necessary parameters need for a CSTR reactor (hydraulic retention time and substrate concentration) 

References

Drapcho, Caye.  Biological Reactor Design lecture notes.  2002