Rickus Biol295F Team 1

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Project
David DiGiovanni.

Craig Barcus.

Tony Czapla.

Sarah Lewis.

Clean-up of Polluted Lakes and Rivers

The earth is mostly covered with bodies of water. Human activities throughout time have polluted many of the earth’s lakes and rivers. The pollution of lakes and rivers prevents people from using the resources that the water can provide. This includes fish, other edible sea creatures, and the water itself. Some of the specific causes of this problem are factory waste water, field run off of pesticides, and waste water plants that are allowed to dump sewage into lakes and rivers. A recent issue occurred within the state of Indiana. The state of Indiana allowed BP to dump more waste into Lake Michigan. The state of Illinois does not agree with the decision made by Indiana and is trying to get the decision overturned. This legal battle will most likely cost both states millions of dollars as well as the cost to BP, the company. This issue shows the problems that water pollution causes in our society. Our overall goal of this design project is to create a solution that will lower the amount of water pollution in fresh water lakes. To achieve this goal, our solution must be a flexible solution that can be implemented in a diverse variety of water environments.

Process overview

This is the "picture" of what our process will look like.

[[Media:arsenic.doc | process overview]]

References and Resources


Potential Purdue faculty to talk to: Prof David Salt.

hyperaccumulation of arsenate in plants Nature Biotechnology 2002

A bacterial view of the periodic table: genes and proteins for toxic inorganic ions

Article related to arsenical pump-driving ATPase http://www.jbc.org/cgi/content/full/276/9/6378



go find it: A Computational and Conceptual DFT Study on the Michaelis Complex of pI258 Arsenate Reductase. Structural Aspects and Activation of the Electrophile and Nucleophile

Scope of Problem, Impact in Dollars, Environmental and Health Considerations
Scope of the Problem and Impact in Dollars Toxic levels of arsenic in freshwater around the world cause a health risk to humans and animals alike. The ability to remove arsenic from a freshwater source would improve the quality and safety of water sources. In India alone, Arsenic contamination has put 100 million people at risk of cancer and other health related problems (P. Bhattacharya et al.) Mohan et al. (2007). People are put at risk to the effects of arsenic by drinking arsenic containing water, eating water inhabitants of arsenic containing water, and simply by immersing themselves in arsenic containing water. In Bangladesh, between 35 and 77 million people are in danger of drinking arsenic contaminated water in 2000. The goal of our project is too eliminate these health risks.

There is opposing views on what levels of arsenic is dangerous and what is believed to be safe. The Environmental Protection Agency (EPA) has a maximum level of arsenic at 50 micrograms per liter. The international standard for drinking water is 10 micrograms per liter. If the EPA were to lower their standard to the international standard of 10 micrograms per liter, 4% of the nation’s water supply would be above this level. It would cost up to 1 billion dollars to bring 100% of the nation’s water supply under the 10 microgram per liter arsenic level. The cost of physically implementing our solution or even testing it depends on the solution that we create. Our initial ideas involve some sort of biological organism (algae, bacteria) that can consume the arsenic in the fresh water environment and convert it into an environmentally friendly substance. The cost of such biological organisms would depend on which one we choose. If we wish to test our design solution, there will be costs involved with purchasing arsenic and materials to construct a model environment. This will include the cost of water, fresh water vegetation and inhabitants, and physical container for the environment. There are many health considerations involved with our design project. Arsenic is a known toxin that can cause cancer if consumed regularly in concentrations above EPA standards. Our design must provide a solution that improves this situation. We must take into account the effects our solution will have on humans, plants, and animals that exist in the fresh water environment.

Environmental Conflicts and Changes

In the design of our project, we must consider other environmental conflicts and changes besides pollution. In the Great Lakes, a major contributor to the degradation of the ecosystem is climate change. This will change the amount of precipitation and the rate of evaporation which consequently will change the lake levels. Another common problem is the introduction of non-indigenous species. Boaters who use their boats on different bodies of water can introduce invasive species which can harm native organisms. Climate change, as well as the introduction of non-indigenous species should be considered in the design of our project, to insure success in reducing the amount of pollution found in fresh water.

Properties and Effects of Arsenic

Arsenic is an odorless and tasteless semi-metal. It has a number of effects on the human body. It can cause thickening of the skin along with discoloration (EPA arsenic page). Arsenic is also known to cause nausea, stomach pain, vomiting, and diarrhea as well as numbness in hands and feet, blindness, and partial paralysis (EPA arsenic page). Exposure to arsenic has been connected to cancer of the skin, lungs, kidney, bladder, liver, nasal passages, and prostate(EPA arsenic page).

Substrate Level Arsenic Reduction

Microbial remediation of Arsenic is not very common today, as more microbes actually use arsenic (V) as an energy source for respiration, creating the more toxic and more mobile AS (III). The common methods used today are coagulation-flocculation (to be explained later), ion exchange and reverse osmosis. Reverse Osmosis is rather expensive and is not used extensively. Natural sorbents such as Fe-rich oxisols have been used in the past and are one of the most widely used methods in developing countries (P. Bhattacharya et al. 2007). E. coli has been used to study microbial remediation with the insertion of Plasmid R773to reduce As (V), contributing to the natural Arsenic cycle in sub-terranian soil environments.

Conditions of the System The conditions of our system will include flow rates of arsenic, volumetric flow rates of our water system, uptake rate of arsenic from the system by our biological catalyst, uptake rate of arsenic by natural factors, input rate of arsenic over time into the system. These factors and more, such as reaction rate of arsenic reduction and other essential reactions inside the bio-catalyst must be considered. The time it takes for our solution to be implemented will be an important variable. The longer the solution takes, the more environmentally friendly it must be. A fresh water authority is not likely to implement our solution if it requires that their lake is shut down for an extended period of time, however, a solution that does not require a lake to be shut down does not have many time restraints. Sustainability

The sustainability of the system must be one of a long term solution to our problem. We have to walk a fine line between removing the desired amount of arsenic and removing too much. Since arsenic is an essential element for all life forms, we cannot remove all arsenic from the system, as it will destroy the ecosystem.

http://www.epa.gov/safewater/arsenic/index.html

Beeton, Alfred (2002). “Large freshwater lakes: present state, trends, and future.” Environmental Conservation. 29(01), 21-38. Smith, Allan H., Lingas, Elena O., & Rahman, Mahfuzar (2000). “Contamination of drinking water by arsenic in Bagladesh: a public health emergency.” Bulletin of the World Health Organization. 78(9), 1093-1103.

http://www.waterindustry.org/Water-Facts/arsenic-8.htm (Associated Press article)

Quantitative Variables
Rate of arsenic removal  (kgAs/time)

Rate of arsenic transport through plant (kgAs/time)

How much arsenic one individual plant can remove (kgAs/plant)

Rate of arsenic absorption into the cell (kgAs/time)

Rate of survival, how many plants survive after planting (plants remaining/plants planted)

Ideal number of plants per amount of As (#plants/kgAs)

[[Media:Quantitative Variables.doc | variables document]]

Process, Enzymatic and overall
For our bioremediation process of arsenic in an aqueous environment, our biological organism must be able to: survive in a high concentration of arsenic compounds, remove arsenic compounds from the environment, and store them in a non-harmful way within the biomass of the organism. We propose to use an aqueous plant to perform these functions. The plant will act as a warehouse for arsenic reduction and storage. Its functions as a warehouse will include packaging, intra-warehouse shipping, and storage. In order for our bio-warehouse to function properly, it must be genetically modified. We propose to use a lily pad as our aqueous plant.

The lily pad should be a good scaffold for our phytoremediation (remediation through plants) for a number of reasons. One of the main reasons has to do with the arsenic transport gene we hope to incorporate in our plant. The protein, arsenic reductase or arsC, is light mediated. It is obvious from looking at the anatomy of the lily pad that its massive surface area in contact with the atmosphere is ideal for the activation of this protein. Dhankher et al. (2002) reported the use of arsC and γ-glutamylcyseine synthetase (γ-ECS) in conjunction to tolerate and hyper accumulate arsenic compounds in vitro. They hypothesized that using the two proteins together will give Arabidopsis thaliana, their genetically altered plant, a better tolerance to As and the ability to reduce arsenate to arsenite and then sequester the arsenite with thiol groups. We will heavily base our method of aqueous phytoremediation using the pathways and genetic mechanisms proposed by Dhankher. (We will obviously optimize these pathways to our own substrate plant). Now that we have a basic overview of our process, let’s dive into enzymes.

ArsC has been shown by many groups to exist in many bacteria and even a yeast or two. Dhankher et al. showed that this gene could be successfully transgendered to plants using the R773 plasmid containing the gene expression for arsC. Utilizing SRS1p (soybean rubisco promoter) to enhance the proteins capability and using vacuum infiltration (details need to be learned by our group) to insert this complex into the genome of the plant, they were able to successfully translocate the gene into the genome. The arsC/SRS1p complex is used to reduce arsenate to the more motile and toxic arsenite. The arsC/SRS1p complex was detected in majority in the leaves and stems of the plant, giving us an almost perfect use of the lily pad since the majority of its biomass is in the leaves. A similar method was utilized for the γ-ECS/ACT2p complex to insert it. The γ-ECS/ACT2p complex is useful in that it catalyzes the reaction of arsenite to thiol groups present in the plant, thereby sequestering the toxic substance and giving us our way of removing arsenic from the environment. The γ-ECS/ACT2p complex is created using the existing pathway of the synthesis of glutathione. The studies done by Dhankher et al. proved that the coupling of arsC and γ-ECS provided the plant with a strong resistance and ability to transform arsenic compounds.

One of the key components to our proposal will be to determine whether or not the lily pad can be genetically modified for our uses. This will require a careful genome comparison between A. thaliana and our plant. If we can find a genetic sequence that will be able to produce our complex, we can continue on with our modeling exercise. That modeling exercise includes determining the reaction kinetics of the enzymatic reaction, the total storage ability of a plant in terms of biomass, the rate of arsenic removal from the system and so forth.

Enzymatic Reaction Kinetics

ArsC is the light mediated enzyme that reduces Arsenate to Arsenite. The kinetics of this reaction is important because the Arsenic can not be sequestered in the plant until it has made the transformation to Arsenite. Once the arsenic is in the form of Arsenite, it can then be bound to thiol groups, rendering much less toxic. We used the Michaelis-Menten equation to determine the velocity of the reducing enzymatic reaction.

V = Vmax*[s]/([s] + Km)

where

V = Velocity

[s] = concentration of substrate

Km = Michaelis Constant = [s] when V=Vmax/2

Vmax = Maximum Velocity = Kcat*[E]

Kcat is the catalytic constant and [E] is the concentration of the enzyme.

From the literature, values for Km and Kcat were determined to be 68 microM and 215 min^-1 respectively. The concentration of substrate used was the maximum level allowed by EPA standards, 50 ppm. 50 ppm was then coverted to microM and determined to be 0.362 microM.

V = Vmax*[0.362]/(68+0.362) = [E]*215*[0.362]/(68.362) = 1.138*[E] (microM/min)

This equation represents the velocity of the reaction as a function of concentration of enzyme ArsC. Further research must be done to determine the concentration of ArsC that can be acheived in our genetically modified lilly pads.

The reaction involving y-ECS to sequester the toxicity of the arsenic must also be determined. Once kinetics of this reaction is known, we will have a general idea of the kinetics of the overall process.

References

Control of glutathione and phytochelatin synthesis under cadmium stress. Pathway modeling for plants. Mendoza-Cozatl, David G. Moreno-Sanchez, Rafael. Departamento de Bioquimica, Instituto Nacional de Cardiologia, Juan Badiano 1, Seccion XVI Tlalpan, 14080 Mexico D.F., Mexico.

Om Parkash Dhankher, Yujing Li,  Barry P. Rosen,  Jin Shi,  David Salt,  Julie F. Senecoff,  Nupur A. Sashti,  Richard B. Meagher. (2002). Engineering tolerance and hyperaccumulation of arsenic in plants by combining arsenate reductase and [gamma]-glutamylcysteine synthetase expression. Nature Biotechnology, 20(11), 1140-1145. Retrieved October 17, 2007, from Health Module database. (Document ID: 1028620561).

Ross, G, J Messens, and S Loverix. "A Computational and Conceptual DFT Study on the Michaelis Complex of PI258 Arsenate Reductase. Structural Aspects and Activation of the Electrophile and Nucleophile." JOURNAL OF PHYSICAL CHEMISTRY B 108 (2004): 17216-17225. Web of Science. Purdue Libraries. 17 Oct. 2007