Bioremediation is the use of biological processes to overcome environmental problems. Enzyme activity is a biological process that can be used to modify chemicals that are harmful to the environment, converting them to inert molecules. Two areas of interest wherein enzymes can be used as bioremediators are with the clean-up of petroleum spills and herbicide run-off.
Specific enzymes can be harvested from microorganisms that produce them in order to be used for bioremediation. A benefit of using an enzyme is that it has a singular function: to act upon a target substrate molecule. In the case of bioremediation the target is a pollutant. An enzyme will catalyze the reaction, detoxifying the pollutant, thousands of times a second. The enzyme, in most cases, is just a protein and will be otherwise innocuous. Other methods of bioremediation utilize bacteria, fungi, algae and plants. These organisms are either identified for their special useful biochemical activity or genetically modified to achieve a desired action.
Releasing live organisms as a method of bioremediation has a host of possible problems. Laboratory simulations can be effective but knowing all of the consequences of introducing a new organism into an ecosystem is not possible. Once released, particularly in the case of a microorganism it is impossible to re-capture and destroy the organism should unintended effects occur. Consider releasing a strain of bacteria to clean-up a pollutant. Although the bacteria may be effective, its actions also may be disruptive to other microorganisms. In this case there would be no viable way of recovering and destroying every released bacterium, particularly if they were to enter a water way. Live organisms are also not proving to be as effective in the field as they are in the lab. Often it is the case that the microorganism performs well in laboratory conditions but once released the productivity is reduced. Living organisms have a specific acceptable range of temperature, pH, oxygen, and other factors that they thrive in. The environment may not consistently have these conditions. Organisms also need other nutrients to survive and if they are not present in the location in need of bioremediation this will pose a problem.
Bioremediation of Triazine Herbicides
Atrazine, the most commonly used triazine herbicide, has been in use since 1958. The chemical is synthesized and distributed by Syngenta, an international company. Atrazine is used in the production of corn, sorghum, and sugarcane, around the globe, to increase crop yields. Atrazine is most effective against broadleaf plants. Atrazine is a Restricted Use Pesticide (RUP) meaning that it can only be used by registered professionals. Atrazine is used heavily by corn farmers in the US, but is also used as roadside weed control and golf course turf management. The action of the chemical is to block the photosynthetic pathway ultimately killing the affected organism. There are triazine resistant crops that have been developed, one of which is canola. Corn is naturally resistant to the chemical. Other triazine pesticides include methoxytriazines (atraton), methylthiotriazine (ametryn), propazine, and simazine.
Triazine herbicides are of particular interest as environmental pollutants because of their wide-spread use, persistence, and mobility. Atrazine remains stable and active for 4 to 57 weeks after application. It has been found in soils, groundwater, lakes, and even oceans. Atrazine is a threat to lakes, wetlands, and reefs. The chemical disrupts photosynthetic action of corals, phototrophic bacteria, freshwater algae, and mangrove trees. These are all non-target organisms of the application process. The product is applied to a crop, but run-off carries the atrazine to a multitude of down-river destinations. The Great Barrier Reef off the coast of Australia is one of the un-intended targets.
An enzyme that metabolizes atrazine was first discovered by chance. A bacterium, Pseudomonas sp. ADP, was isolated from an herbicide spill. This strain of Pseudomonas was dubbed ADP because of it metabolized atrazine as a nitrogen source. This bacterium used atrazine as it sole nitrogen source, metabolizing it remarkably fast, at a rate of "9 x 10
cells per ml degraded 100 ppm of atrazine in 90 minutes"
. The enzyme responsible for the metabolism was isolated and named atrazine dechlorinase (AtzA), an atranine dechlorinase. The enzyme dechlorinates atrazine transforming it from an effective herbicide to an innocuous non-herbicide molecule. The gene that codes for this enzyme has since been isolated. With this isolated gene scientists have been able to produce transgenic E. coli, as well as plants, in order to field trial them as bioremediation devices. For reasons discussed above it is more promising to use the enzyme alone for bioremediation. Currently this is an area of research that is gaining ground. The enzyme has been improved upon; triazine hydrolase (TrzN), is a recent iteration that shows promise because it can act on a broader range of triazines and not just the chlorinated ones.
Fostering Relevance, Connections, and Questions
Herbicide pollution is of paramount importance to this generation. "Organic" is a common household word that students will have some familiarity with because it used in daily conversations and advertisements. High school students are at the age where they are starting to make decisions about their lives and bodies and it is not uncommon to meet a student that chooses to eat organic foods. This is the connection that helps to garner buy-in from the students, because they are already invested in the concept. Organic foods are not grown with synthetic herbicides such as atrazine.
Studying genetically modified organisms, especially the methods of creation and the use of them in the food industry, is required state content. One can easily put together a mini curriculum that focuses on both genetic engineering and the subsequent use of pesticides. Genetically modified foods (GMOs) are developed to be resistant to pesticides and herbicides. Although this may not result in the increased application of pesticides, the fact remains the same: the pesticides are used and therefore released into the environment affecting non-target organisms. A unit that connects bioremediation methods with production of GMOs will be much richer and more interesting than a unit that focuses just on one of the two topics.
Students need to be convinced of the relevance of what they are being taught. Connecting content objectives, such as enzymatic action, to current events is an effective way to demonstrate importance to the students. An added benefit is that these are also controversial topics and will elicit increased interest from high school students who are eager to define themselves.
Bioremediation of PAHs
Polycyclic aromatic hydrocarbons (PAHs) are semi-volatile organic compounds (SVOCs). These compounds are present in crude oil that has spent time in the ocean and is thus a concern when oil spills occur. PAHs come from other sources as well such as the incomplete combustion of gas, coal, garbage, wood, and from motor vehicle exhaust. There are a number of PAHs the Environmental Protection Agency (EPA) will test for, following an oil spill and clean-up efforts.
PAHs are environmental pollutants that may also be detrimental to humans. It is possible that they cause cancer, adversely affect reproduction, and disrupt the immune system. "The Department of Health and Human Services (DHHS) has determined that some PAHs may reasonably be expected to be carcinogens. Some people who have breathed or touched mixtures of PAHs and other chemicals for long periods of time have developed cancer. Some PAHs have caused cancer in laboratory animals when they breathed air containing them (lung cancer), ingested them in food (stomach cancer), or had them applied to their skin (skin cancer)"
Gaseous PAHs contaminate soils and aquifers as they deposit from the atmosphere. The widespread contamination, magnitude of the pollution, and effect on humans makes remediation of PAHs a very important and necessary activity. Bioremediation with enzymes may be the answer.
A phenoloxidase called laccase can be harvested from Trametes versicolor, a species of mushroom. This enzyme catalyzes the oxidation of PAHs when a mediator is present to act as an electron shuttle. Not only is the enzyme needed to remediate PAHs but enzymes mediator is also required. Mediators have been developed to couple with the enzyme. Oxidation of the PAHs degrades the molecule into inert products.
Fostering Relevance, Connections, and Questions
Oil spills are current hot topics. Unfortunately oil spills are all too common as well. Your students will have questions about these tragedies. What better way to harness their innate interest than to focus on a learning objective that must be taught? Teaching about enzymatic bioremediation of PAHs can be coupled with an ecology unit. Teaching about enzymes in this tangible, meaningful, forum will motivate students to take more interest in the topic. Students want to connect their learning to something that they care about. The environmental damage caused by oil spills interests students because they are such devastating events. This will be in contrast to teaching enzymes through the state embedded task wherein one uses pectinase to produce apple juice from apple sauce: an activity that is so obscure and strange that I have a hard time justifying the loss of class time it takes to perform the activity.
If students are interested they will ask questions. If students are asking questions they care about the content; they have a desire to understand. If students are asking questions they are acting like scientists, this is the point of science instruction, is it not?
As science instructors we are failing our students if our focus is so narrow that it presents only content. We must teach students what science is and the impetus of science is posing questions. Presenting students with real-world problems in the context of the content that they will be held responsible for garners their interest effectively. Encouraging students to study the application of biological phenomena to solve human problems will bridge the gap between their interests and the state requirements. Once they are hooked and interested in the class the questions will come. It is our job to define and support the question process, validating the action for the students. We validate the action of question posing by allowing our students to investigate their questions and essentially act as scientists.
Question Formation Technique
Just One Change: Teach Students to Ask Their Own Questions
by Dan Rothstein and Luz Santana. Rothstein and Santana describe a deliberate and easily replicable technique that a teacher can employ at the beginning of a unit. There is a well-described question formation technique described in this book that will be useful for generating questions from your students. The hidden benefit is that activities such as these foster student interest in the class because students are given a voice. With the questions generated you structure the class instruction to answer their questions, creating instant purpose and relevance for the students. This allows the students to have a say in how the unit is framed and gives them control of what they will be learning.
Penny Enzyme Activity
This is a great hands-on activity that is engaging, active, fun, and cheap. Students learn the concepts of enzyme structure, active site, competitive inhibitors, temperature effects, denaturation, reaction rates and substrate-enzyme relationships.
The premise is that a penny is the substrate and the hand is the enzyme. The reaction is the picking up of one penny from a bowl and placing it on the table "heads" up. The fingers are the active site. Depending on the length of the class one can modify this activity in many ways. I use the activity in a 90 minute block.
The students are paired, one being the enzyme and the other counting and validating the enzymatic activity. The supplies per pair are: plastic bowl, 100 pennies, masking tape, access to ice, access to clock with a second hand, and 100 paper clips. The students will run multiple trials for each of several scenarios in order to model enzymatic activity. The data that are collected can be used to determine averages, create graphs, and calculate rates. The data can also be aggregated for the whole class for analysis.
The following reaction simulations can be run in fifteen-second trials. The reaction is the picking up of one penny from the bowl and placing it on the table "heads up."
1. "Normal Enzyme Activity" - this is to collect baseline data
2. "Denatured Enzyme" – the student must tape his/her four fingers together
3. "Competitive Inhibition" – the students add 100 paper clips to the bowl of pennies
4. "Temperature Effect" – the student first cools his/her hand with ice for a minute