In the Classroom
Denitrification as a Model Chemical Process Gordan Grguric Marine Science Program, The Richard Stockton College of New Jersey, Pomona, NJ 08240-0195;
[email protected] One of the most difficult challenges in undergraduate chemistry education is the search for real-world examples of processes that are straightforward and predictable enough to illustrate the concepts under study. Approaches such as service learning and development of thematic interdisciplinary courses have been used to address this challenge (1–11). At the Richard Stockton College of New Jersey, we have used bacterial denitrification in closed seawater facilities (such as aquaria and mariculture systems) as a very successful “model” chemical process. By employing actual data from several seawater systems, discussion and analysis of stoichiometry, mass and charge balance, and limiting reagents gains in its value and practical applicability. This paper describes denitrification in closed seawater systems and how an analysis of the process can be used to enhance undergraduate chemistry education. Two distinct approaches to denitrification are described, from two different facilities. Each approach can be used independently of the other, depending on the type of system the instructor wishes to model. A number of possible student exercises dealing with denitrification are described afterward. These exercises can be used as practical tools to enhance students’ quantitative understanding of chemical reactions. Denitrification in Closed Seawater Systems One of the persistent problems in the aquarium/mariculture industry is the control of high nitrate concentrations. Many of the facilities host primarily heterotrophic organisms and their seawater shows increasing nitrate concentrations over time. Nitrate in these systems is primarily produced by bacterial nitrification of excreted ammonium nitrogen (12), although other processes (e.g. disinfection by ozone) can provide additional oxidation pathways from ammonium to nitrate (13). Many small aquarium and mariculture facilities control high nitrate concentrations by performing regular water changes. In a very large system, this is impractical for two reasons: the size of the tank makes it operationally difficult to replace a significant fraction of water, and the expense of preparing large volumes of artificial seawater for facilities located far from the ocean can be prohibitive (14). As a result of these considerations, bacterial denitrification was selected as a method for controlling nitrate concentrations in several large seawater aquaria. The process consists of adding organic carbon to seawater and employing facultative anaerobic bacteria (usually the Pseudomonas group) to consume nitrate in seawater. Under anaerobic conditions, these bacteria use nitrate as an oxidizing agent for organic carbon. Methanol is commonly used as a source of organic carbon, because of its relatively low cost and low sludge production compared with other organic compounds (15). Through consecutive redox reactions (eqs 1, 2), nitrate is first reduced to nitrite and then
to nitrogen gas (16 ). 3NO3᎑ + CH3OH → 3NO2᎑ + CO2 + 2H2O
(1)
2NO2᎑ + CH3OH → N2 + CO2 + H2O + 2OH᎑
(2)
The net denitrification reaction is given in reaction 3. 6NO3᎑ + 5CH3OH → 3N2 + 5CO2 + 7H2O + 6OH᎑ (3)
Batch Denitrification (the Living Seas) The Living Seas at EPCOT Center in Florida is the largest seawater aquarium in the world, with a total volume of 23.4 million liters. Most of this volume, 21.8 million liters, is in the Main Tank, which contains the aquarium exhibition. Denitrification at the Living Seas is performed on a batch basis, in a separate system. The system consists of a holding basin with a volume of 1.1 million liters and a series of biologically active denitrifying filters. The entire batch volume is taken out of the aquarium, placed into the holding basin and then recirculated through the denitrifying filters until nitrate and nitrite concentrations become undetectable. Batch run times are a function of nitrate concentration and ambient temperature and can vary from 5 to 18 days. Flow-Through Denitrification (the New Jersey State Aquarium) The largest aquarium display at the New Jersey State Aquarium, Ocean Tank, has a volume of 2.9 million liters. Denitrification of Ocean Tank seawater is performed on-line, in a flow-through system. Seawater is pumped from Ocean Tank into a 2700-liter deaeration tank, where methanol is continuously injected. Anoxic seawater flows from the deaeration tank into a 1500-liter denitrification reactor. Large colonies of Pseudomonas living on a porous medium within the reactor enable denitrification to take place. Methanol is continuously injected into this environment as well. The denitrified seawater is collected in an 800-liter overflow tank. From there, it flows back into the aquarium through the main water treatment system. Student Exercises
Analysis of Empirical Nitrate/Nitrite Data The first exercise that can be done with the students is an analysis of empirical denitrification data. Figure 1 shows inorganic nitrogen speciation during one of the early batch runs at the Living Seas. The locations sampled are before (influent) and after (effluent) the denitrifying filters. The initial influent nitrate concentration of over 8000 µM is the result of years of nitrate buildup in the aquarium seawater. Despite such high initial concentration, nitrate in the influent
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Determination of the Required Amount of Methanol The main operational question for the successful run of denitrification systems is how much methanol to employ. Using too little will result in incomplete denitrification, whereas using too much will promote sulfate reduction with the corresponding generation of highly toxic hydrogen sulfide. The total amount of methanol required can be separated into three parts, listed below. Analysis and quantification of each of these parts are excellent opportunities for students to apply their knowledge of chemical reactions, their balancing, and stoichiometric calculations. Methanol Required for Deaeration (Deoxygenation) Before facultative anaerobes can effectively use nitrate as an oxidizing agent, dissolved oxygen in the water has to be consumed. Students are asked to compute how much methanol needs to be added to achieve this. By developing and balancing reaction 4, they can relate the amount of methanol needed to the concentration of dissolved oxygen in the water. In aquaria and mariculture facilities, seawater is constantly aerated and thus the dissolved oxygen concentration is essentially at saturation, which is a function of temperature and salinity. 2CH3OH + 3O2 → 2CO2 + 4H2O
(4)
Students use tables and charts to obtain the initial dissolved oxygen concentration and then use stoichiometry of reaction 4 to determine the concentration of methanol needed. For a batch denitrification system, the absolute amount of methanol (e.g. in liters) is then computed from the total volume of the batch. For a flow-through system, the methanol addition rate (e.g. in liters per hour) is computed from the flow rate through the system. For example, the amount of methanol needed for deaeration of one Living Seas denitrification batch is about 7 liters (Table 1). Methanol Required for Denitrification Students compute the amount of methanol needed for denitrification from reactions 1 and 2. They are asked to combine and balance these reactions into a net reaction 3. Afterward, they develop an expression for the concentration of methanol needed on the basis of the nitrate concentration in the denitrification influent. The absolute amount of methanol is again computed as a total volume (for batch systems) or a flow rate (for flow-through systems). Table 1 shows the com180
9000
Ion Concentration / µM
and effluent of the denitrifying filters becomes undetectable after 6 days. The presence of nitrite as an intermediary is shown by the shape of its profile, which peaks at the same time (6 days after the start). Comparing the influent and effluent nitrite concentrations, students realize that nitrite production exceeds consumption for the first 5 days; afterwards, they observe that there is a net reduction of nitrite during recirculation. Data such as those shown in Figure 1 are given to students, whose task is to correlate them with reactions 1 and 2 and determine an empirical denitrification rate for the system. The students typically analyze data from 5–10 batch runs. They use the results to determine the time it takes to completely denitrify a batch of seawater. With this information, and using the known parameters of the system (volume, flow rates, etc.), they can predict future nitrate concentrations.
7500
6000
4500
3000
1500
0 0
2
4
6
8
10
12
14
16
Time / day Figure 1. Influent and effluent nitrate/nitrite concentrations during a batch denitrification run at the Living Seas: (䊏) influent nitrate; (䊐) effluent nitrate; (䊉) influent nitrite; (䊊) effluent nitrite.
Table 1. Amounts of Methanol Needed for the First 10 Denitrification Runs at the Living Seas Stoichiometric Batch No.
Initial [NO3᎑ ]/ µM
DenitrifiDeaeration cation CH3OH/ CH3OH/ L L
Actual CH3OH/ L
CH3OH Ratioa
1
9570
7
355
435
1.20
2
9410
7
349
484
1.36
3
8900
7
330
443
1.31
4
8120
7
301
416
1.35
5
8310
7
308
439
1.39
6
8110
7
301
363
1.18
7
7710
7
286
397
1.35
8
7360
7
273
317
1.13
9
6990
7
259
344
1.29
10
6860
7
254
357
1.37
NOTE: The following parameters were used in calculating the values in the table: denitrification batch volume of 1.1 × 106 L and initial dissolved oxygen concentration of 220 µM (the saturation value at 24 °C temperature and 30 g/kg salinity). Stoichiometry in reactions 3 and 4 was used to determine the methanol amounts needed for denitrification and deaeration, respectively. actual aThe ratio is . deaeration + denitrification
puted amounts of methanol required to completely denitrify each of the first ten batch runs at the Living Seas. The initial nitrate concentration in each of these runs is given in Table 1, so the reader can reproduce all the relevant stoichiometric calculations. Methanol Required for Bacterial Growth The denitrifying bacteria use organic carbon in methanol not only as a source of energy (through reactions 1–4), but also as building blocks for their own growth. Studies have shown that the amount of organic carbon needed for the growth of Pseudomonas is about 30% of that used to obtain energy (17 ). Therefore, the net amount of methanol required
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In the Classroom
for deaeration and denitrification has to be multiplied by 1.30 to obtain the final amount of methanol needed. It is interesting to compare the stoichiometric amounts of methanol needed for deaeration plus denitrification with the total amount actually used at the Living Seas (Table 1). The average ratio of actual to stoichiometric methanol amounts for the first ten batch runs is 1.29, indicating that on average, 29% of the methanol was used for bacterial growth. Students may be asked to perform this analysis and find the explanation for “excess” methanol on their own, which gives them an opportunity to research scientific papers in applied chemistry and extract relevant information from them. They can also compare the amounts of methanol needed for deaeration versus denitrification: in the examples given in Table 1, the deaeration amount is quite small relative to the denitrification amount. However, as the initial nitrate concentrations continue to decrease in subsequent batches, the amount of methanol required for deaeration becomes relatively more important.
By-products of Denitrification When the volume of methanol needed for denitrification has been determined, students can compute the amounts of by-products generated. These by-products include nitrogen gas, water, carbon dioxide, and hydroxide ions (reactions 1–4). For the computation of nitrogen gas released, students have to employ the stoichiometry in reaction 3 as well as the concept of standard molar volume. If a change in nitrogen gas pressure in a reactor of specified volume is to be determined, students can employ the ideal gas law. For example, the volume of nitrogen gas produced during denitrification of the first batch listed in Table 1 is 118 m3. Determining the total amount of water produced during denitrification is particularly interesting because it changes the net volume of the system. Students are asked to compare the total volume of “new” water produced to that present in the denitrification reactor (either batch or flow-through). Through this exercise, they realize that the volume of water produced is quite small relative to the total volume present in the denitrification reactor. For the first batch run at the Living Seas (Table 1), the volume of water produced through deaeration plus denitrification is 227 liters, which is less than 0.1% of the total batch volume. The final student exercise may be to determine the effect of carbon dioxide and hydroxide ions on the pH and carbonate alkalinity of the denitrified seawater. In this exercise, students have to employ their understanding of acid–base chemistry, and in particular the carbonic acid speciation. They use acid–base speciation diagrams to determine that at seawater pH (7.5–8.0), the predominant carbonic acid species is bicarbonate, and thus equilibrium for reaction 5 is far to the right. CO2 + OH᎑ → HCO3᎑
(5)
During denitrification itself, there is a stoichiometric excess of hydroxide ions produced relative to carbon dioxide (reaction 3), so the pH of the seawater would be expected to increase somewhat. Determining the magnitude of that increase on the basis of the initial pH and carbonate alkalinity can be used as an advanced exercise in carbonic acid equilibrium calculations. The students’ results can be compared to the actual measured values and data agreement or discrepancies can be discussed.
Exercise Format The denitrification exercises described above can be employed in a number of formats: as practical examples shown by and computed by the instructor, or as homework problems, to be solved by students on their own. Other formats are possible, including the use of computer classrooms for individual or team work. In our case, theoretical aspects of denitrification are discussed in the lecture, and then the actual processes in the two aquaria are described. We have class meetings in an electronic computer classroom, equipped with a demonstration podium for the instructor and a workstation for every student. During these sessions, students work individually, or sometimes in pairs, to develop, balance, and quantify the chemical reactions in question. When they have developed arithmetic expressions for computing the quantities of reactants or products of interest, they program these expressions in Excel and apply them to a particular situation. The use of spreadsheets was shown to have several advantages: their overall effect is to enable the students to repeat calculations many times, using different input data. The main benefit of such an approach is the ability to pose and quickly answer “what-if ” scenarios. This ability can be a powerful tool to increase the students’ understanding of the chemical principles at work. An additional benefit of this approach is that students become more independent self-learners through the development and use of their own models. Conclusion Seawater aquaria have long been recognized as good environments for development of analytical chemistry skills (18). Our experience shows that denitrification in these facilities is a particularly well-suited process for illustrating important theoretical concepts in chemistry to undergraduates. Concepts that students can experience firsthand include: 1. Analyzing and quantifying a series of chemical reactions based on empirical data. 2. Using stoichiometry and mass balance to determine the amounts of reactants required (e.g. methanol) and products produced (e.g. nitrogen gas, water) in a chemical reaction. 3. Employing acid–base speciation diagrams and other information to quantify the changes in pH and carbonic acid speciation in an aqueous medium.
Development and use of denitrification computer models enables our students to: 1. Verify their approach by comparing model results with empirical data. 2. Predict future nitrate concentrations in a system, under the existing denitrification conditions. 3. Suggest ways to optimize the operation of these systems.
Our surveys show that students’ interest in the course topics is stimulated when learning is based on real-life examples. It may be plausible to surmise that as a result, such learning activities have longer-lasting effects than the traditional textbook methods. Additional benefits from the described approach
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include sharing of data and results with applied chemists in industry. This provides the most direct way of educating the future chemists who will work in water treatment facilities such as drinking water and sewage treatment plants. Acknowledgments I wish to thank Christopher Coston at the Living Seas and Frank Steslow at the New Jersey State Aquarium for numerous discussions and exchange of ideas over the years. Literature Cited 1. Goulding, M. Sci. Am. 1993, 266, 114–120. 2. Juhl, L.; Yearsley, K.; Silva, A. J. J. Chem. Educ. 1997, 74, 1431–1433. 3. Wilson, A. H. J. Chem. Educ. 1998, 75, 1176–1177. 4. Krow, G. R.; Krow, J. B. J. Chem. Educ. 1998, 75, 1583– 1584. 5. O’Hara, P. B.; Sanborn, J. A.; Howard, M. J. Chem. Educ. 1999, 76, 1673–1678.
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