P Removal Varying Aerobic Time

A Study on Optimizing Biological Phosphorous Removal by Changing Aerobic Operating Times

Phillip Dixon and Juan Diaz-Robles CEE 453 Laboratory Research in Environmental Engineering December 8, 2004 Cornell University Ithaca, NY 14850

2 Table of Contents ABSTRACT...... 3

INTRODUCTION...... 4

OBJECTIVES...... 5

MATERIALS AND METHODS...... 5

SETUP...... 5 Figure 1. Reactor Set-up...... 6 Figure 2. Refrigerator/ Influent Flow Set-up...... 6 Influent solution:...... 7 Colorimetric Wet Chemistry Technique:...... 7

PROCEDURES...... 7 Figure 3. Wastewater, Tap Water, and Sludge in the Reactor...... 8 Figure 4. Colorimetric Test Set-up...... 9

RESULTS AND CONCLUSIONS...... 10

Figure 6. Fraction Phosphorous Removed with Standard Deviations...... 10

SUGGESTIONS...... 13

APPENDIX A...... 14

APPENDIX B...... 15

Table 1-1. Reagents...... 15 Reagents...... 15 Table 1-2. Equipment list...... 15

REFERENCES...... 17

3 ABSTRACT

Phosphorus (P) is a nutrient that is essential for plant growth. Phosphorous pollution in water bodies causes eutrophication, a condition where high nutrient concentrations stimulate blooms of plant growth (algae), which then lead to fish kills and decreased water quality. Consequently, one of the main concerns of wastewater treatment facilities’ is to avoid having their effluent water contaminated with phosphorous (Lake Champlain).

Phosphorous removal is accomplished in wastewater treatment by exposing the wastewater to an aerobic/anaerobic sequence in a biological reactor. As a result, high levels of phosphorus removing microorganisms will accumulate. These organisms release phosphorous in the anaerobic phase, but take it up in the aerobic phase, and then die off, taking the phosphorous out of the water as biomass in the form of activated sludge.

For this experiment, a bench scale wastewater treatment plant was built and then the aerobic operating time was varied while the anaerobic operating time was held constant. The operating times were varied using a computer process controller. The computer also monitored the dissolved oxygen concentration in the water throughout the entire length of the experiment. Samples of the effluent plant’s water were taken two times a day for a period, which spanned over three weeks.

No published research could be found that directly related to this experiment. The results from this experiment suggested that 4.5 hours aerobic operating time produced the most phosphorous removal with a 1.5 hour anaerobic operating time. According to another group that was varying anaerobic time while holding aerobic time constant at 6 hours it was found that 0.75 hours of anaerobic operation produced the most phosphorous removal. Therefore, as researchers we recommend that a 4.5 hours aerobic time be used with a 0.75 hour anaerobic time to get the optimum phosphorous removal in a wastewater treatment facility for the conditions that were examined in this experiment. However, further research should be conducted to validate our results.

4 INTRODUCTION

A nutrient essential for plant growth is phosphorous. A high concentration of phosphorous in treated wastewater can lead to eutrophication. Eutrophication is a condition where high nutrient concentrations stimulate blooms of algae that interfere with the health and diversity of a water body. Specifically, algal blooms increase the water’s turbidity, clouding it, and blocking sunlight from penetrating into its depths, which eventually cause underwater grasses to die. These grasses provide food and shelter to aquatic creatures. Therefore, as the grasses disappear the aquatic creatures die off. Another example of how eutrophication interferes with a water body is when algae die and decompose dissolved oxygen concentrations are used up. Dissolved oxygen is essential for most aquatic creatures to live, therefore without it they die off (fish kills) (US EPA).

One of the main concerns of wastewater treatment facilities is to avoid having phosphorous contamination in their effluent water because this water is then discharged into different types of water bodies, many of them natural in which eutrophication is not desired. Treatment facilities can remove phosphorous from water by exposing the wastewater to an aerobic/anaerobic sequence in each cycle of the biological reactor. The aerobic/anaerobic sequence will cause the biological reactor to select microorganisms that accumulate high levels of intercellular phosphorous. The phosphorous removing microorganisms release phosphorous in the anaerobic phase to produce energy to take up fermentation products. In the aerobic phase the phosphorous removing bacteria produce energy by oxidizing the stored fermentation products while simultaneously taking up phosphorous. Phosphorous accumulates intercellularly within the phosphorous removing bacteria. Therefore, phosphorous is removed from solution in the form of biomass as activated sludge (Wisconsin Department).

For this experiment, it was calculated what aerobic operating time, 3, 4.5, or 6 hours produced the most phosphorous removal while the anaerobic operating time was held constant at 1.5 hours. Therefore, a bench scale wastewater treatment plant was built and a phosphorous concentration of 6.9 mg/L (a concentration that is ideal from phosphorous removing microorganisms) was pumped into the reactor as a generic wastewater. Then samples were periodically collected while aerobic operating time sequences were changed. After enough samples had accumulated the concentration of phosphorous in each sample was measured in the spectrophotometer using a colorimetric wet chemistry technique. The sample concentrations were then compared to the influent concentration to determine how much was removed in the reactor.

5 OBJECTIVES

The proposed research’s objective was to find the effect on phosphorous removal when aerobic operating times were changed while maintaining a constant anaerobic operating time. The next part of the objective was to determine what aerobic operating time, 3, 4.5, or 6 hours, produced the best wastewater phosphorous removal.

MATERIALS AND METHODS Setup The reactor for this experiment was a 6 L container that was filled to the 4 L mark with wastewater and tap water. The reactor rested on a stirrer on top of the laboratory bench. The reactor was connected to an input flow of tap water and concentrated wastewater by a ¼ inch tube that ran out of a 1.05 L container in the refrigerator and through a peristaltic pump at 380 mL/min. The 1.05 L container contained concentrated wastewater at a concentration of 20 times the required value of phosphorous; this was done to decrease the need for storage space. The tap water coming in to the reactor came from a 3/8-inch hose that ran from a local faucet in the lab. In the refrigerator, the tap water tube and the wastewater tube were combined into one ¼ inch tube to decrease the need of excess tubing that went to the reactor.

Also, running into the reactor was an input air gas tube that was used to aerate the wastewater. The gas tube ran out of an output valve on the laboratory workbench. The amount of gas entering the reactor was controlled by two other valves, and then finally connected to a port at the bottom of the reactor. At another bottom port of the reactor was a pressure sensor, which was calibrated to tell what the volume of solution was in the reactor using a process controller pressure sensor program. Inside the reactor was a small mixing bar that periodically mixed the wastewater solution. Running out of the reactor was a tube that ran to a valve that controlled whether the wastewater was allowed to flow or not into a 50 mL holding container. This container was located in a drain that captured the treated wastewater, which was collected to sample at a later date. When the holding container became full it was allowed to overflow into the drain. The entire reactor set-up can be seen in figure 1 and the refrigerator/ influent flow set-up can be seen in figure 2. The plant was controlled by software titled “Process Controller”. Specifically, the software used rules, states, and set points to determine what should be happening in the reactor.

6 Figure 1. Reactor Set-up

Figure 2. Refrigerator/ Influent Flow Set-up

7 Reagents:

Influent solution: Starch: 84.40 mg/L Casein: 125.00 mg/L

Sodium acetate (C2H3O2Na3H2O): 31.90 mg/L Capric acid (C10H20O2): 11.60 mg/L Ammonium chloride (NH4Cl): 75.33 mg/L Potassium phosphate (K2HPO4): 38.8 mg/L Sodium hydroxide (NaOH): 175.00 mg/L Glycerol (C3H8O3): 12.00 mg/L

Colorimetric Wet Chemistry Technique: Stock A H2SO4: 13.6 mL E-pure H2O: 80 mL Stock B Ammonium Molybdate: 4 g E-pure H2O: 90 mL Stock C Ascorbic Acid: 0.9 g E-pure H2O: 40 mL Stock D Antimony K tartate: 0.3 g E-pure H2O: 80 mL Procedures

To find how much phosphorus was being removed from the biological reactor phosphorous-concentrated wastewater (6.9 mg/L phosphorous) was pumped into the reactor and then the concentration of phosphorous in the effluent was measured. Specifically, to do this, 1.2 L of concentrated sludge was left in the 6 L reactor to have enough bacteria to remove phosphorous, and then 140 mL of concentrated wastewater and 2.66 L of tap water were pumped into the reactor using a peristaltic pump. All of the solutions inside the reactor can be seen in figure 3. As the solutions were added to the reactor they were both simultaneously mixed together using the magnetic stirrer. All of the solutions in the reactor produced a total volume of 4 L. The next stage was to put the reactor into an anaerobic stage for 1.5 hours with the stirrer left on. When the anaerobic operation stage was completed the next stage was to aerate the wastewater at a constant rate with the stirrer on. For this experiment the aeration times were ranged from 3 to 4.5 to 6 hours. The next stage in the reactor sequence was to let the solution settle. The settling time for this experiment was 1 hour, which allowed the sludge to settle to the bottom of the reactor, out of solution. Following the settling stage was the drain stage. The system was allowed to drain into a 50 mL holding container in the drain. The reactor

8 was allowed to drain until it reached a total volume of 1.2 L, which was when the process controller began the whole cycle again at the concentrated wastewater fill stage.

The reactor was operated as a batch reactor, treating and draining a single volume of wastewater at a time. Samples were collected twice a day from the holding container in the drain (see Appendix A for sample collection schedule), poured into a 15 mL bottle, capped, and put into a refrigerator for storage. The samples were measured at the end of the experiment using the colorimetric wet chemistry technique (see figure 4 for an example of the set-up) and a spectrophotometer (see figure 5 for a picture of the spectrophotometer) to determine the phosphorous concentrations in the effluent of the reactor. A detailed explanation of the colorimetric wet chemistry technique used can be found in Appendix B.

Figure 3. Wastewater, Tap Water, and Sludge in the Reactor

9 Figure 4. Colorimetric Test Set-up

Figure 5. Spectrophotometer

10 RESULTS AND CONCLUSIONS

The major objective in this experiment was to design a reactor that could remove a considerable amount of phosphorous from a generic wastewater influent. All of the states in the cycle of the reactor were kept constant with the exception of the aerobic operating time. This was done to determine the best operating time between 3, 4.5, and 6 hours. A fraction of phosphorous removed was obtained from the samples concentration and influent concentration. This fraction was plotted against time to depict how the reactor was removing phosphorous from the wastewater. This can be seen in figure 6 shown below.

Fraction P Removed

40% d e

v 20% o m e r

P 0% n o i t c a -20% r F

-80% 0 5000 10000 15000 20000 25000 30000 35000 Time (min)

Aerobic 6 hours Aerobic 3 hours Aerobic 4.5 hours

Figure 6. Fraction Phosphorous Removed with Standard Deviations

As can be seen in figure 6 the standard deviations for each point are very large, which means that the phosphorous concentrations for the samples were not very accurate. In the process of measuring the phosphorous concentrations in the spectrophotometer some hardships were found along the way, these were:

1. The mixing of the stock solutions for the stock phosphorous standards had to be made very precisely, in other words the amount of each stock solution used for the stock phosphorous standard had to be measured accurately. 2. This mixing had to be done in a very clean container (washed with E-pure water) to prevent contamination of the combined reagent.

11 3. The concentration of phosphate in the sample and the influent were considerable high to be measured, using the previously prepared standards, by the spectrophotometer. Therefore, the samples and the influent were diluted by a factor of 20, so that the spectrophotometer could measure the sample concentrations to values within the limits of the standards. 4. The standards used in the spectrophotometer, measured phosphate. Since what we wanted was the concentration of phosphorous measured, the concentration of phosphate measured in the spectrophotometer had to be converted in terms of phosphorous. Also, potassium phosphate dibasic (K2HPO4) was used for the influent phosphorous in the reactor, and potassium phosphate monobasic (KH2PO4) was used as the reagent containing phosphorous for the standards in the spectrophotometer. These two different chemicals produced different concentrations of phosphorous in solution (K2HPO4 = 78 mg P/L and KH2PO4 = 100 mg P/L). This could have made a difference in the measurement of the phosphate concentrations in the samples.

According to figure 6 the reactor showed to remove phosphorous at one time and “produce” phosphorous at another time when no parameters were changed. Theoretically, the reactor was supposed to only remove phosphorous and not produce it, but in the reactor in this experiment this was not the case. In figure 6 the data points that showed a positive fraction of phosphorous removed depicted an actual phosphorous removal from the wastewater and the negative fraction of phosphorous removed depicted a “production” of phosphorous. Some hypotheses of why there was a production of phosphorous were:

1. Settling time could have acted as an anaerobic phase in which the microorganisms would have released phosphorous. 2. The time the effluent sample was left to sit in the 50 mL container located in the drain would have acted also as an anaerobic phase in which the microorganisms would have released phosphorous. 3. There were times that the bacteria in the sludge were drained from the reactor and ended up in the 50 mL container. These bacteria would have released even more phosphorous because it was in an anaerobic phase.

Due to these factors it was impossible to create a correlation between the fractions of phosphorous removed and the time that had proceeded past the previous collection.

The results suggest that there was the most phosphorous removal when the aerobic operating time was set at 4.5 hours, this can be seen in the last trend of the 4.5 hour series depicted in figure 6. Since, the data stops at the middle of this trend, the suggestion that the 4.5 hour aerobic operating time was the best for phosphorous removal over the 6 and 3 hour operating times, can not be considered conclusive.

According to another group that a collaborative effort was being made with and who varied anaerobic time while holding aerobic time constant at 6 hours, it was found that 0.75 hours of anaerobic operation produced the most phosphorous removal. Therefore,

12 as researchers we recommend that a 4.5 hours aerobic time be used with a 0.75 hour anaerobic time to get the optimum phosphorous removal in a wastewater treatment facility for the conditions examined in this experiment. However, further research should be conducted to validate our results.

13 SUGGESTIONS

The following were found throughout the experiment. The combined regent for the colorimetric wet chemistry technique has to be made in a clean container in a very precise way. The samples to be measured in the spectrophotometer have to be diluted by at least a factor of 10. Different pipettes should be used for each solution mixed in the cuvets and their tips should also be changed between each sample. The samples from the effluent of the reactor have to be collected very shortly after the reactor has been drained. The samples have to be collected without particulates of sludge. The samples have to be maintained at a temperature less than 4 ºC before they are measured in the spectrophotometer.

14 APPENDIX A

Notes Anaerobic Aerobic Hours 1.5 6 0.75 3 3 4.5

Date Sampler Time 1 (min) Time 2 (min) 11-Nov Laura 0 495 12-Nov Phil 1155 1755 13-Nov Sarah 2703 3220 14-Nov Phil 4165 4680 15-Nov Laura 5465 5885 16-Nov Juan 6925 7540 17-Nov Sarah 8338 X 18-Nov Laura 9910 10690 19-Nov Juan 11110 11590 20-Nov Phil 12015 13125 21-Nov Laura 14055 14500 22-Nov Sarah 15420 15790 23-Nov Juan 16790 17185 24-Nov Phil X X 25-Nov X X 26-Nov X X 27-Nov X X 28-Nov Phil X 24720 29-Nov Phil 25255 25825 30-Nov Laura 26845 27480 1-Dec Juan 28205 28695 2-Dec Sarah 29728 30244 3-Dec Phil 31060 X

15 APPENDIX B

Colorimetric Wet Chemistry Technique (taken from CEE 453 Phosphorous Measurements)

Reagents A Sulfuric acid solution, 4.9 Table . Reagents N: Add 136 mL concentrated H SO to 800 mL E-pure water. Description Supplier Catalog 2 4 number Cool and dilute to 1 L with E- concentrated Fisher Scientific

pure water. H2SO4 B Ammonium molybdate (NH4 )6 Fisher Scientific Mo7O24•4H2O solution: Dissolve 40 g of (NH4)6 C6H8O6 Fisher Scientific Mo7O24•4H2O in 900 mL E-pure K(SbO)C4H4O6• Fisher Scientific water and dilute to 1 L. Store at ½H2O 4°C. sodium lauryl sulfate C Ascorbic acid: Dissolve 9 g of KH2PO4 Fisher Scientific ascorbic acid (C6H8O6) in 400 mL E- pure water and dilute to 500 mL. Store at 4°C. Keep well covered. Table . Equipment list Prepare fresh monthly or as needed. D Antimony potassium tartrate: Description Supplier Catalog # Dissolve 3.0 g of 100-1095 µL Fisher Scientific 13-707-5 pipette K(SbO)C4H4O6•½H2O in 800 mL E- 10-109.5 µL pipette Fisher Scientific 13-707-3 pure water and dilute to 1 L. Store at Disposable cuvets Fisher Scientific 14-385-942 4°C. Cuvet holder Fisher Scientific 14-385-939 Combined color reagent: Combine UV-Vis Hewlett-Packard 8452A spectrophotometer Company the following solutions in order, mixing (but do not entrain air as oxygen oxidizes the ascorbic acid) after each addition: (Prepare fresh weekly. Store at 4°C) Stock A, (4.9 N H2SO4) 50 mL Stock B, (Ammonium molybdate solution) 15 mL Stock C, (Ascorbic acid solution) 30 mL Stock D, (Antimony-tartrate solution) 5 mL Water diluent solution: Add 4.0 g sodium lauryl sulfate and 5 g NaCl per L of E-pure water. Stock phosphorus standard: Dissolve 0.4394 g of Potassium phosphate monobasic (KH2PO4) (dried at 105°C for one hour) in 900 mL E-pure water. Add 2 mL of concentrated H2SO4 and dilute to 1 L. 1.0 mL = 0.100 mg P (100 mg P/L).

16 Standards Preparation Method  Use 1000 g P/L stock.  Use a digital pipette and prepare 1 mL of each standard.  Use E-pure water to dilute the 1000 g P/L stock.

Reagent Addition for Samples and Standards  Pipette 50 L sample into a disposable microcuvet using a 100 L digital pipette (make sure there are no solids in the sample) and then 950 L of E-pure water using a 1 mL digital pipette.  Add 160 L-combined reagent and mix thoroughly by swirling (by sucking solution into pipette and squirting back out multiple times).  After at least 10 minutes but no more than 30 minutes, measure absorbance of each sample using a reagent blank as the reference solution.

Samples and Standards to Prepare  Reagent blank to be used as reference samples.  Prepare 6 standards containing 0 (reagent blank), 50, 100, 200, 500, 800, 1000 g P/L.  Prepare samples.

Spectrophotometer Method  Use sample cuvettes. (Make sure to orient all cuvettes with the arrow on the left because the cuvettes are not symmetrical and have different absorbance when turned 180.) Use a wavelength close to 820 nm for analysis.  Use the reagent blank as the reference sample for all samples.  Use units of g P/L.  Fill in the general description (in the spectrophotometer software) with your NetID and a description of the type of samples.

Samples Analysis  Measure the reference using a reagent blank (E-pure water + color-reagent). Analyze the reagent blank as a sample and verify that the absorbance deviates less than 0.004 AU (absorbance units) from zero. If the absorbance deviates more than 0.004 AU reanalyze the reference sample.  Analyze 6 standards as standards using the spectrophotometer and save the data.  Analyze samples collected from the effluent of the reactor.

17 REFERENCES CEE 453 Phosphorous Measurements. “Phosphorus Measurements.” December 7, 2004. http://ceeserver.cee.cornell.edu/mw24/cee453/

Wisconsin Department of Natural Resources. “Wastewater Characterization for Evaluation of Biological Phosphorous Removal.” April 29, 2003. http://www.dnr.state.wi.us/org/water/wm/ww/biophos/1intro.htm

US EPA. “Eutrophication.” September 8, 2003. http://www.epa.gov/maia/html/eutroph.html

Lake Champlain Basin Program. “Phosphorus Pollution.” December 3, 2004. http://www.lcbp.org/phospsum.htm