O H opo

iNuçleicj

Continues

Figure 6.8

Partial structure of a DNA strand.

Figure 6,9

Representation of a portion of the DNA double-helix structure.

Two forms of DNA are of interest. One is chromosomal DNA, which is the essential form that contains all the basic information required for normal cell metabolism, growth, and reproduction. The other is DNA contained in separate, relatively small, self-replicating circular fragments called plasmids. Plasmids contain genetic information that conveys additional capabilities to an organism such as resistance to i toxic heavy metals such as mercury or to drugs such as penicillin. A plasmid may j also contain genetic information for a key enzyme that allows an organism to initiate a reaction with an organic compound that would otherwise be non-biodegradable! by the organism. Another interesting property of plasmids is that they can be ex-: changed between bacterial species, sometimes resulting in a particularly good com-: bination of enzymes in one organism, allowing it to degrade a new organic chemi-j cal that is introduced into the environment. This is an important "adaptive"; mechanism for bacteria. It is known that some environmentally significant reactions; such as degradation of halogenated organic compounds are encoded on plasmids.'3 : Much of the information contained in DMA and plasmids is unique to a specific organism, a specific degradation pathway, or production of a specific product. Scientists now have the capability of separating and identifying the components (i.e., enzymes, genes, DNA, plasmids, etc.) responsible for these activities. With this capability comes the potential for manipulating, controlling, and understanding various biological reactions. Some specific examples are given next.

Example Applications

New genetic tools (sometimes referred to as "molecular biology tools") are being developed and introduced rapidly. They are having wider application in helping to understand and solve environmental contamination problems. The environmental engineer and scientist is well advised to keep abreast of new developments.

Genetic Engineering Genetic engineering, sometimes called recombinant-DNA technology, involves the recombination of DNA from several sources. One successful application of genetic engineering is in the production of human insulin with genetically engineered bacteria. The genes involved in the production of insulin in the human pancreas were characterized, separated, recombined, and introduced into the bacterium Escherichia coll This process is termed gene cloning. The E. coli then can produce large quantities of insulin precursors that are easily converted to human insulin by chemical means.

Another example involves the enzyme toluene dioxygenase described in Sec. 6.13. The genes responsible for production and activity of this enzyme, along with its ability to biodegrade chlorinated organic compounds such as TOE, were isolated from the bacterium Pseudomonasputida F1 and placed into an E. coli strain.'4 This l3B. E. Ritt mann, B. F. Smets, and D. A. Stahl, The Role of Genes in Biological Processes: Part 1, Env. Sei. Tech., 24: 23-29 (1990).

"*G. j. Zylstra, L. P. Wackett, and D. T. Gibson, Trichloroethyiene Degradation by Escherichia coli Containing the Cloned Pseudomonas putida Fl Toluene Dioxygenase Genes. AppL and Env. Micro., 55:3162-3166(1989).

chapter 6 Basic Concepts from Biochemistry

genetically engineered E. coli was then shown to degrade TCE as long as an inducing substrate (toluene or isopropyI-j3-D-thiogalactopyranoside in this case) was present. There are indications that enzyme activity such as this can be induced by means other than chemical addition (e.g., temperature).

Genetic engineering has resulted in at least one patented bacterium for use in bioremediation. A. M. Chakrabarty developed a genetically engineered Pseudomonas strain by cloning four different plasmids into the organism, giving it the ability to biodegrade a wide variety of petroleum products.13 Successes like this and the other ones described lead some engineers and scientists to believe that genetic engineering holds significant promise for remediating many ha2ardous and industrial wastes and that we can develop "designer genes" for the removal of specific pollutants.

The use of genetic engineering is not without controversy. There is concern with release of genetically altered organisms into the environment. A current example is the controversy surrounding use of genetically engineered corn. The bacterium Bacillus thuringiensis produces toxins that will kill some types of insects. These insecticidal genes have been cloned into several varieties of corn with the goal of protecting the corn without having to apply insecticides. One major concern is the development of insects that are resistant to these naturally produced toxins. There is also concern with gene transfer to other strains of corn and perhaps other plants. Finally there is concern about the potential human and environmental health effects of long-term exposure to these genetically engineered plants.

Molecular Tools These tools are being used to assess both bacterial community structure (i.e., molecular ecology—what types of bacteria are present and in what numbers) and biodegradation capability (what types of compounds can be degraded by the organisms present). This field is advancing at a very rapid pace with methods becoming more standard and commercially available. The following is meant to be a brief introduction to some of the more common techniques being used today. All are based on the ability to extract, concentrate (amplify), and detect DNA and RNA that is specific to the organism or degradation capability of interest. Much more detail is contained in the references given at the end of this chapter.

Several methods in current use are based on the sequence of bases contained in the 16S segment of ribosomal RNA. Portions of the base sequence in 16S rRNA are common to most life, while other portions are unique to a given organism. Gene probes, today commonly called oligonucleotide probes, are single strands of DNA or RNA containing perhaps 15 to 25 bases that, when exposed to the complementary sequence in the target 16S rRNA (or DNA), hybridizes (combines) to form a compound that can be measured. Experimental conditions are controlled such that the probe DNA does not combine with cells that don't contain the complementary sequence of 16S rRNA because the sequences don't match. Detection is typically accomplished by attaching an appropriate label (e.g., radioisotope, fluorescent compound) that can be measured analytically. With some of these techniques, the RNA

lsP. J. VanDenmark and B. L. Batting, 'The Microbes: An Introduction to Their Nature and Importance," Benjamin/Cummirigs, Menlo Park, CA, 1987.

and DNA must be extracted from the microbial population. Commercially available kits are often used. In one technique, fluorescence in situ hybridization (FISH), the probe is tagged with a fluorescent molecule and added to "intact" cells. Here, the probe is contained within the cells, which are fixed and viewed using a fluorescence microscope. In this fashion, die cells of interest can be viewed in contrast to other cells within the community being studied. For example, methanogenic bacteria t made to fluoresce could be distinguished from other members of an anaerobic bac- j terial community converting a carbohydrate to methane.

Another technique involves extraction of DNA from a bacterial community fol- ■ lowed by amplification using the polymerase chain reaction (PCR). This process is j somewhat analogous to enriching for bacteria by adding a specific substrate (e.g., ac- ! etatej. In general this is done because, at least with environmental samples, bacterial : numbers will be low. With PCR, one objective might be to produce many more . copies of genes that code for 16S rRNA. These can then be hybridized with oligonu- ; cleotide probes that are specific for organisms of interest. For example, a general probe might hybridize with 16S rRNA of all bacterial life in the community. More specific probes can target specific strains of bacteria (e.g., strains of methanogenic or sulfate-reducing bacteria). In this fashion, we can leam about the variety of bacteria in the community and perhaps how the community changes with time. A limitation of this technique is that it works only for those bacteria that have been isolated and for which the ribosomal DNA has been sequenced, It is generally believed that only a few percent of the different microbial strains on earth have been isolated.

A promising modification that allows quantification of bacterial density (or, alternatively, the number of copies of a specific gene of interest) is real-time quantitative PCR (RTQ-PCR). A specific sequence in the 16S ribosomal DNA of a target organism [or, alternatively, a gene responsible for specific enzyme activity (e.g., toluene dioxygenase)] is amplified using PCR and a special fluorescent oligonucleotide probe. During PCR, the intensity of fluorescence increases and can be measured and related to bacterial density (or degradative gene density).

Another promising method for quantification of bacterial numbers or, more specifically, the number of copies of a specific target gene is competitive PCR. A competitor DNA is designed so that it can be amplified by PCR using the same primers as used to amplify the target DNA of the cells. Primers are compounds added to react with specific sequences of DNA or RNA. Then a known amount of a competitor DNA is added to the DNA extracted from cells. A series of samples is prepared with different amounts of competitor added to each. Then, primers are added, and the target and competitor DNA are amplified together. The amplified target DNA and competitor DNA are then separated by electrophoresis, and the amount of each present is quantified and compared. What is sought is a particular sample in which the amplified amounts of target DNA and competitor DNA are the same. Here, the number of copies of target DNA is equal to the known amount of competitor DNA added to that sample. The procedure is relatively rapid and precise.

There are techniques being developed that allow study of communities containing bacteria that have not been isolated and sequenced. In one such technique, community DNA is extracted and selectively amplified using PCR. Primers are used for chapter 6 Basic Concepts from Biochemistry

this selective amplification and are chosen to amplify for some general or specific function. For example, primers can be very general such as for the 16S rRNA of all bacteria or very specific such as for the 16S rRNA of the dehalorespiring bacteria Dehalococcoides ethenogenes. Primers can also be designed to select for genes encoding specific enzymes such as toluene dioxygenase. One way the amplified DNA can be analyzed is by using denaturing gradient gel electrophoresis (DGGE). Here, electrophoresis is used to separate amplified DNA based on differences in the base sequences. Distinct bands form, which can represent, for example, different species of bacteria present in a community. It is also possible to remove these bands from the gel for additional analysis. For example, after further amplification, the base sequence can be determined and compared with computer databases containing 16S rRNA sequences for known bacterial species. Such analyses are useful for identifying bacteria.

These methods have been successful in detecting a wide variety of microorganisms such as Salmonella, Legionella, sulfate-reducing bacteria, methanogens, and dehalorespirers.16 They are finding increasing use in assessing community structure and biodégradation capabilities17,18 and in detecting and quantifying specific bacterial strains.19

immunochemical Techniques Immunochemical techniques are special types of molecular biology techniques that involve immunization. The two most common examples are immunoassays and immunochemical probes. Immunoassay is a terni used to describe assays that detect and quantify specific chemicals such as pesticides and PCBs. Immunochemical probes are used to detect specific microorganisms, genes, or enzymes (or enzyme activity).

These methods take advantage of the fact that the immune systems of living organisms respond to the introduction of foreign organisms or chemicals. The general procedure involves immunizing an animal, usually a mouse or rabbit, with a target organism (e.g., methanogenic bacteria), gene, or chemical. These foreign substances, called antigens, cause the production of specific proteins called antibodies by the animal's immune system, There are many antibody producing cells in the animal and the suite of proteins produced, called polyclonal antibodies, meaning clones from many different cells, may respond to many different antigens. Screening procedures are then used to isolate the antibodies that are most useful. In many cases it is important to have monoclonal antibodies, that is, antibodies produced by mB. G. Olson, Tracking and Using Genes in the Environment, Env. Sci. Tech., 25: 604-611,1991.

I7S. JT. Flynn, F. E. Loffler, and J. M. Tiedje, Microbial Community Changes Associated with a Shift from Reductive Dechlorination of PCE to Reductive Dechlorination of eii-DCE and VC, Env. Sci. Tech., 34: 1056-1061,2000.

!SF. E. Laffler, Q. Sun, J. Li, and J. M. Tiedje, 16S rRNA Gene-Based Detection of Tetrachloroethene-Dechlorinating Desulfuromonas, and Dehalococcoides Species, Appl. & Env. Microbiol., 66: 1369-1374, 2000.

"K. R. Hristova, C. M. Lutenegger, and K. M. Scow, Detection and Quantification of Methyl tert-Butyl Ether-Degrading Strain PM1 by Real-Time TaqMan PCR, Appl. & Env. Microbiol., 67: 5154-5160, 2001.

one type of cell that are specific for a specific antigen. These antibodies are usually obtained from cells taken from the animal's spleen. Since it is very difficult to culture these cells, they are fused with tumor cells to create special organisms called hybridomas, which can easily be grown in mass culture to produce relatively large quantities of the specific monoclonal antibody.

Assay procedures involve reacting the antibodies with environmental samples containing the target organism, gene, enzyme, or chemical. In some cases we are just interested in whether the target species is present while in other cases we are interested in the concentration of the target species. A common method of detection is by enzyme linked immunosorbent assays (ELISAs). Here an enzyme is linked to the antibody and its activity is detected and calibrated by the release of a colored prod- ; uct that can be measured spectrophotometrically (Chap. 12). Other methods of de- ! tection include tagging antibodies with radioactivity or fluorescent compounds. : Immunochemical probes have been developed for organisms such as methanogens; and sulfate-reducing bacteria. Major potential advantages of immunochemical tech-: niques are ease of use, low cost, and specificity. For example, immunochemically based home pregnancy tests are widely used.

In the future immunoassay methods may make it possible for homeowners to accurately and inexpensively determine the concentration of chemicals such as atrazine in their drinking water. There are several commercially available immunoassay kits in common use today. Examples include kits for atrazine, cyanazine, alachlor, metolachlor, and 2,4-D, among others.20 Detection limits are reported to be as low as 0.04 ¡xgtL for some compounds. These kits are semt-quanti-tative and are currently used primarily as a screening tool; that is, the test kit will determine if the concentration is above or below the detection limit. If an accurate determination of concentration is desired, standard, approved analytical methods (e.g., extraction followed by gas chromatography) must be used. Such methods are described in Part 2 of this text.

6.15 I BIOCHEMISTRY OF HUMANS

Since environmental engineers and scientists are concerned with the disposal of human wastes, it is important that they be familiar with the major changes that organic matter, taken as food, undergoes in its passage through the body.

Carbohydrates

Much of the carbohydrates consumed by humans is utilized by the body. The remainder, consisting of undigestible matter, is eliminated in the feces. Most of the rejected carbohydrate matter is cellulose and other higher polysaccharides for which the human body does not provide enzymes to accomplish its hydrolysis, or for which the detention time in the intestine is too short to complete hydrolysis. The

J0M. Vanderlaan, B. E. Watkins, and L. Stanker, Environmental Monitoring by Immunoassay, Env. Sei. Tech., 22: 247-254 (1988).

short detention time is aggravated by improper chewing of food and by diarrhetic conditions.

The carbohydrate matter that is assimilated into the blood stream is used for energy, stored as glycogen (animal starch) in muscle tissue and the liver, or converted to fat and stored as fatty tissue. The carbohydrates that are oxidized to produce energy are converted to carbon dioxide and water. The carbon dioxide is carried away, by the blood, from the cells where it is formed. The blood is buffered to such an extent that it can carry considerable amounts of C02 and release it to the air in the lungs, in accordance with the principles of Henry's law.

The human body contains a remarkable mechanism for controlling the amount of sugar (glucose) in the blood stream. If excessive amounts accumulate, the excess is released into the urine. This is a part of the kidney function. Persons with diabetes suffer from improper metabolism of sugar. As a result, blood sugar exceeds the amount acceptable to the kidney (renal threshold), and the excess is separated in the kidneys and escapes in the urine. The urine of diabetics shows the presence of glucose consistently. If the carbohydrate intake of a diabetic exceeds the capacity of the kidneys to excrete sugar, blood-sugar levels build up to a point at which the person may pass into a coma.

Fats

Crude fatty materials contain certain substances that are not hydrolyzed in the human alimentary system. These materials and some of the undigested fats are passed in the feces. Fats are hydrolyzed to a considerable extent by lipase in the stomach. Further hydrolysis occurs in the intestine, where the reaction is facilitated through the emulsifying properties of the bile salts. The fatty acids that enter the blood stream are oxidized to produce energy or are stored in fatty tissue for future use. The end products of oxidation are principally carbon dioxide and water, but some ketones, principally acetone, are formed. The carbon dioxide is expelled by the lungs. The ketones are excreted in the urine. Ketones are found in unusual amounts in the urine of diabetics and people suffering from faulty fat metabolism.

Proteins

Hydrolysis of proteins is started in the stomach and continues in the intestine. Amino acids, when released by hydrolysis, are absorbed into the blood stream. Fractions that are not completely hydrolyzed are excreted in the feces. The amino acids are used mainly for the building and repair of muscle tissue, and in these capacities they become fixed in body tissues.

The end products of protein metabolism that require excretion as waste products result principally from two processes; the "wearing" of muscle tissue and oxidation of amino acids to obtain energy. Deamination of amino acids precedes their use as energy sources. The ammonia is released principally as urea, but small amounts of NB^ are normally present. Excretion is by way of the urine. The major function of the kidneys is to separate waste nitrogen compounds from the blood. That protein metabolism involves a variety of complicated processes may be de duced from the considerable number of nitrogenous compounds present in urine. Creatine, creatinine, uric acid, hippuric acid, and traces of purine bases are normally present, in addition to urea and ammonium ion.

Vitamins

Vitamins are very potent organic substances that occur in minute quantities in nat- j ural foodstuffs. They must be supplied in the diet of animals if they are not synthe- j sized naturally within the animal from essential dietary or metabolic precursors, j Some function as precursors of enzymes; with others, the function is not well un-; derstood. In general, they exert a hormone-like or enzymatic action in the control of! specific chemical reactions in the animal body, and the absence or lack of a sufficient supply of certain ones leads to the development of vitamin-deficiency diseases, e.g., beriberi, rickets, pellagra, and scurvy.

A wide variety of vitamins are known. They are generally classified into two i groups, the fat-soluble and the water-soluble, as shown in Table 6.8.

The role of vitamins in biological processes employed by environmental engineers and scientists has not been explored. Several of the vitamins are recovered from industrial wastes, particularly those from the fermentation industry, and their i economic value has been an important factor in helping to solve the waste-disposal problem in the distilling industry. Activated sludge has been found to be a rich source of vitamin Bl2. Methanogenic bacteria have also been shown to produce vitamin B12, and this vitamin has been shown to facilitate reductive dehalogenation of halogenated aliphatic hydrocarbons through cometabolism. Environmental engineers and scientists should thus be informed on the subject of vitamins and their significance.

Table 6.8 I Classification and function of some important vitamins

Vitamin

i Good sources , \

Pat-soluble:

A

Butter, liver oils

Eye health

D

Liver oils, egg

Ca metabolism, i.e.,

antirachitic

E

Cottonseed oil, cereals

Prevents sterility

K

Green plants, egg yolk

Clotting of blood

Water-soluble:

Antiberiberi

B[ Thiamine

Pork, whole wheat, peanuts

B2 Riboflavin

Eggs, liver, cereals, milk

General health

Nicotinic acid

Meat, whole wheat, yeast

Antipellagra

Bi Pyridoxine

Egg yolk, liver, yeast

Skin tone

Biotin

Egg yolk, liver, yeast

Skin tone

Pantothenic acid

Egg yolk, liver, milk

Skin tone, growth

Folic acid group

Green leafy vegetables

Antianemia

Inositol

Fruits, vegetables

Hair, growth

Bl2 Cobalamtn

Liver, activated sludge

Antianemia

C Ascorbic acid

Citrus fruits, apples

What role do enzymes play in living organisms?

What terms are used to describe enzymes with respect to (a) where their action occurs, and (b) the nature of the reaction that they control? 63 How does the environmental engineer make use of temperature and pH relationships of biochemical reactions in the design and operation of biological waste treatment facilities? (¡A Explain how the enzyme methane monooxygenase might be used by environmental engineers and scientists.

6.5 Disposal of the large quantities of bacteria produced during waste treatment is one of the most significant {and expensive!) problems in environmental engineering. Explain why this particular problem might be minimized by anaerobic rather than aerobic waste treatment?

6.6 (a) How many acetic acid molecules are produced during the complete beta oxidation of a stearic acid molecule? (b) How many hydrogen atoms are removed from stearic acid during the beta oxidation of part («)?

6.7 (a) Show how n-octane is degraded step by step first by omega oxidation (discussed in Chap. 5) and then by beta oxidation. Balance ail reactions.

(b) How many acetic acid molecules are produced during the complete beta oxidation of rc-octane?

(c) How many hydrogen atoms are removed from n-octane during this beta oxidation?

6.8 Use of methanol has been proposed to rid a wastewater of nitrate by biological denitrification to N2.

(a) Write a balanced overall equation for nitrate removal with methanol, using/,(m9s) and assuming nitrate serves as the nutrient source for bacteria,

(b) If the nitrate (NOJ) concentration in the wastewater equals 100 mg/L, what concentration of methanol must be added for complete nitrate removal?

(c) What percentage of the nitrate-nitrogen is used for cell synthesis?

6.9 Acetic acid is a common fatty acid in wastewaters.

(a) Write a balanced overall equation for aerobic oxidation of acetic acid, assuming ammonia is available as a nitrogen source and that/5(mM) applies.

(b) How many grams of oxygen are required to oxidize 100 g of acetic acid?

(c) How many grams of bacteria are produced per gram of acetic acid metabolized? Answers: (a) 0.125CH3COO" + 0.0295NHj" + 0.102502 = 0.0295QHAN +

0.00?C02 + 0.0955H20 + 0.0955HC03~; (£>) 44.5; (c) 0.45

6.10 The biological oxidation of Fe2 + in mine drainage waters can result in red discoloration in streams from subsequent Pe3+ precipitation.

(a) Name the type of organism responsible for this process.

(b) Is this reaction spontaneous? Show calculations to justify your answer.

• (c) Write a balanced overall equation for biological oxidation of FeI+, assuming fx max) and the presence of ammonia. {d) How many grams of oxygen are required per gram of Fe2+ oxidized?

6.11 Denitrification of wastewaters can be obtained through autotrophic biological reactions.

(a) Write a balanced equation for denitrification of NO; to N2 using S203~ (which is oxidized to S04~), using /l(max) and assuming the bacteria use ammonia as a nitrogen source for cell synthesis. b) How many grams of Na2S203 would need to be added to the wastewater per gram of NO; reduced?

Answers: (a) 0.125S20|" + 0.04C02 + 0.01NHÎ + O.OiHCOJ + O.iôNOj" + 0.055H20 = 0.25SO'f + 0.01C5H702N + 0.08Na + 0.09H+; (b) 1.99

6.12 Answer the following regarding the biodégradation of phenol.

(a) Construct a half reaction for the oxidation of phenol to C02(g).

(b) Calculate AG0' for this reaction, given that the free energy of formation for phenol(£¡7) is —47.5 kJ/mol.

(c) For anaerobic conditions and pH = 7, is the conversion of phenol to CH4(g) thermodynamically favorable?

(d) For anaerobic conditions and,/; = 0.10, construct a balanced reaction for the conversion of phenol to methane.

(<?) For a waste containing 1000 mg/L of phenol, what volume of methane is produced with 98 percent phenol utilization?

6.13 Biological nitrification is a process where ammonium (NH4 ) is converted to (NOf) by aerobic bacteria. .

(a) Write a balanced overall reaction for nitrification using/s(fflai).

(b) How much dissolved oxygen is required to remove 25 mg/L of NH4 -N?

(c) What concentration of bacteria is produced?

(d) What concentration of alkalinity (as CaC03) is consumed?

Answers: (a) 0.13NH4h + 0.22502 + 0.005HCOJ + 0.02C02 = 0.125N03~ + 0.005C3H702N + 0.25H+ + 0.12H20; Q>) 98.9; (c) 7.8; (d) 175

6.14 Approximately 2500 dry tons of dewatered wastewater treatment plant sludge (chemical formula is Ci0Hl{,O3N) have been disposed in a sanitary landfill. Assuming anaerobic conditions, how many cubic feet of methane can be formed from the biological degradation of this sludge? You will have to assume an/, to work this problem; justify your assumption.

6.15 At midnight on January 1,1992, an employee of the M. T. Head Waste Disposal Corporation empties 2 million gallons of an industrial wastewater containing 1000 mg/L of benzoate (benzoic acid) and 500 mg/L propionate (propionic acid) from a tank truck into Innocent Lake. The lake has 500 million liters of water containing 7.5 mg/L dissolved oxygen and no nitrate. Assuming no atmospheric reaeration takes place, that sufficient ammonia-N is available for bacterial growth, and that the lake is completely mixed, will Innocent Lake go anaerobic due to the biological activity stimulated by discharge of this wastewater?

Answer: yes

6.16 Recently anaerobic "dehalorespiring" bacteria have been found that will use tetrachloroethene (PCE) as an electron acceptor in support of growth. These organisms have been shown to use molecular hydrogen (H2) as an electron donor for growth,

(a) Develop a balanced half reaction for the reduction of PCE(aç) to ethene(a#) and calculate AG0' for your half reaction. You will neEd to use data contained in App. A.

chapter 6 Basic Concepts from Biochemistry

(£i) Assuming/5 = 0.05 (comment on why this is a reasonable choice for fs), determine how much H2fe) is required to convert 50 g of PCE to ethene. (c) Calculate the mass (g) of bacteria produced. (¡2) Does the pH increase, decrease, or remain constant as a result of this transformation?

6.17 Perchlorate (CIO4) is an oxyanion that has been found in some groundwaters especially near defense department sites, it has been used as a solid rocket fuel ("oxygen source"). Recently, several bacteria have been isolated that can use perchlorate as an electron acceptor for growth, reducing CIO* to CI". Typically, perchlorate degradation by these organisms requires addition of a carbon and energy source. One such compound that can serve both purposes is acetate.

(a) Is the combination of perchlorate as an electron acceptor and acetate as an electron donor theraiodynamically favorable? Justify your answer. (f>) If so, and if you wanted to develop a balanced biochemical reaction (that is, R), what value would you use for/, and why? Consult Table 6.5 in determining your answer.

(c) Using your value for/, calculate how many mg/L of acetate will be required to biodegrade 50 mg/L of CIO4

6.18 Given the following six pairs of compounds, for each pair, which of the two compounds is likely to be the easiest to degrade. Why? Be specific!

6.19 An organic chemist has developed a new pesticide with the hypothetical structure shown. Based on your expertise of degradation pathways, suggest a likely means (pathway) whereby this compound could be completely mineralized. Include or or

RCOOCH2R' or RCH5OCH2R' RCHjCHO or RCH2COCH3

RCH2C(CH3)2CH2COOH or RCH2CH(CH3)CH2COOH

appropriate environmental conditions (e.g., aerobic, anaerobic). Balance your reactions to the extent possible.

6.20 Biotransformation of 1,1,1-trichloroethane (CH3CCl3)in anaerobic biological systems i can be accomplished by oxidation or reduction.

(a) Construct a half reaction for the reduction of CH3CC13 to inorganic chloride.

(b) In a system fed acetate, construct a half reaction that could produce the electrons :: required for part (a). :;

(c) Add half reactions from parts (a) and (b) to yield a balanced reaction for reductive | dechlorination of CH3CC13 in ait anaerobic biological system fed acetate. \

(d) Construct a half reaction for the oxidation of CH3CCi3 to carbon dioxide and inorganic chloride. \

(<?) For an anaerobic biological treatment system, propose a half reaction that will use the electrons generated in part (d). •

(/) Add half reactions from parts (d) and (e) to yield a balanced reaction for the .; oxidation of CH3CC13 in an anaerobic biological system.

6.21 Rank the following organic compounds in order from easiest to oxidize to most resistant to oxidation. Provide justification for your answer.

(a) Carbon tetrachloride

(¿0 Dichloromethane ;

(c) Hexachloroethane

(d) Trichloroethene

(e) Vinyl chloride

6.22 A contaminated groundwater contains the following six xenobiotic chemicals: chlorobenzene, dichloromethane, hexachloroethane, pentachlorophenol, tetrachloroethene, and vinyl chloride. Propose a bioremediation scheme, based on terminal electron acceptor or acceptors to be used, that will remove all compounds to the maximum extent feasible.

6.23 Explain the difference between DNA and RNA,

6.24 Why are plasmids of importance to environmental engineers and scientists?

6.25 What is a genetically engineered microorganism? How might an environmental engineer make use of such an organism?

6.26 Explain in general how oligonucleotide probes work. What is a limitation of using such probes?

6.27 Explain how FISH might be used to determine whether a specific microbial species is present in a bacterial community.

6.28 What is the polymerase chain reaction?

6.29 Explain how DGGE might be used by environmental engineers and scientists.

6.30 What are monoclonal antibodies?

6.31 What is an immunoassay? How do environmental engineers and scientists use this

technique?

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