PEDRO: Welcome to week two. This week we are going to be covering some important principles of biodegradation. As you know, natural attenuation relies on several mechanisms that proceeded without human intervention to decrease the concentration and toxicity or priority pollutants. And these mechanisms include dilution, volatilization, sorption, and perhaps the most important one, biodegradation. SPEAKER: So this week, we'll learn when biodegradation can or cannot occur and what conditions are conducive to faster degradation of two common classes of priority pollutants, hydrocarbons and chlorinated solvents. PEDRO: So at the end of this week, you will have improved understanding of the thermodynamic and biogeochemical constrains that could hinder biodegradation and, perhaps more importantly, of the environmental conditions and factors that favor these redox reactions. SPEAKER: I'm going to turn it over to Pedro for the first four videos and then you'll see me later on in the week. PEDRO ALVAREZ: Let us begin with our review of what is required for biodegradation to proceed. The first requirement is that organisms that can make enzymes capable of degrading the target pollutants must exist. And these target pollutants, often called xenobiotics or synthetic compounds that are alien to life, are degraded to an impressionable extent whenever bacteria can transform them into intermediates or substrates to common metabolic pathways. So what this means is that the greater the similarity in the structure between the xenobiotic and the natural light diet of these bacteria, or the building blocks that they are made up of, then the higher the likelihood that extensive biodegradation will occur. Of course, these specific degraders must also be present at the contaminated site of interest. It doesn't do you any good if these bacteria exist in a lab in China when you need them to be degrading the target pollutants at the site that you are managing. Also, the compound has to be accessible, or bioavailable, to the bacteria that is going to degrade it, and this bioavailability could be considered within a biochemical or a physical chemical context. What I mean by that is that for the pollutant to be able to enter inside the cell, sometimes bacteria need to make selective gates, or porings, to allow it to come in. And from the chemical sense, I mean that if you need to break up a carbon-carbon bond, this bond has to be exposed and not occluded by larger chlorine atoms that could essentially exert steric hindrance. But most often when we talk about bioavailability, we are referring to a physical context. And here we need to recognize that for biodegradation to proceed, bacteria usually attack the contaminants when they are in the water phase. And for them to get to the water phase, they may need to dissolve from organic oily phases, or diffuse out of micropores or nanopores, or desorb from surfaces. And then they need to be transported to the cell surface. And any of these processes could be rate limiting, especially for hydrophobic compounds. So consideration of this potential mass transfer limitations is important. Now, these compounds are going to be degraded often by inducible enzymes. These enzymes are not always expressed because bacteria do not carry excess baggage. And to be induced, the inducer-- often the target compound-- has to be present in sufficiently high concentrations. For example, for toluene you need about 50 micrograms per liter in order for it to be able to induce these compounds. It's also important to recognize that some enzymes have very relaxed substrate specificity and are capable of attacking pollutants that do not serve as substrates and do not support their growth or provide energy. I am referring to cometabolism, which is a fortuitous transformation where an enzyme acts on a substrate and the compound that is being cometabolized does not provide any metabolic benefit to the organism. However, this is quite common, by the way, for chlorinated solvents, for pesticides, and for 1,4- dioxane. And because there's no growth resulting from cometabolism, these cometabolic reactions are not expected to accelerate and are much slower than metabolic reactions. The good news is that some of these byproducts could be utilized by other bacteria that eat the table scraps of cometabolism by an association known as cometabolic commensalism. And this could result in the eventual mineralization of the target compound. Another important requirement is that the environment has to be conducive and favorable to the proliferation of the specific degraders and for the functioning of their enzymes. And this essentially means seven subconditions. The first one being that suitable substrates and nutrients have to be present. For example, it's often desired that the carbon to nitrogen to phosphorus ratio be on the order of 30 to 5 to 1. In addition, we have to remember that life originated in water, and the presence of water is essential for biodegradation. So when the pollutants occur in the unsaturated zone, we must try to find an optimum water content, which is on the order of 80% of the soil field capacity. And a very important requirement is the availability of suitable electron acceptors for oxidation reactions, as is the case for the biodegradation of hydrocarbons and of suitable electron donors for the reduction of oxidized pollutants, such as hexavalent chromium or chlorinated solvents. We are going to learn more about the feasibility of electron transfer and the suitability of different electron acceptors and electron donors in our next lecture. Temperature also can play an important role in the sense that often biodegradation rates double or even triple when the temperature increases by 10 degrees Celsius. And the pH also has to be adequate, hopefully near neutral, so that the degree of protonation of enzymes and their function-- their three dimensional structure which is affected by the pH-- is at an optimum level. Also, it's important that toxic substances are either absent or controlled. And usually one factor that may hinder biodegradation is the presence of heavy metals that are toxic to bacteria. Fortunately, many of these can be precipitated with sulfites and their sulfate reducing conditions, which reduces toxicity. But sometimes for natural attenuation to proceed, you need to wait for the target pollutants when these are present in very high potentially inhibitory concentrations. You need to wait for these pollutants to dilute and to volatilize to levels that are no longer toxic. And finally, it is important to have absence of easily degradable substrates that could be preferentially degraded, as is the case, for example, for gasoline spills that contain ethanol. Ethanol is like candy to bacteria, and bacteria will preferentially utilize it instead of the target benzene, toluene, and xylenes that are of greater regulatory concern. And finally time. Time is important. Without engineered enhancements, benzene half-lives in aquifer, for example, could be on the order of 100 days. And what is important here is that the number, that is the ratio of the degradation rate to the migration rate, be greater to one. In that event, the plume will no longer expand. In some cases, it will be receding, and therefore natural attenuation will be an appropriate option for managing contamination. So, there are certain heuristic relationships that are worth considering. First, biodegradability tends to increase with solubility because the more soluble compounds are more bioavailable. Also, highly branched compounds are relatively recalcitrant because these tertiary and quaternary carbons are very difficult to degrade by beta oxidation, which is a common metallic pathway. Also, for hydrocarbons, those that contain chains with 12 to 20 carbon atoms are very easily degradable. But if they have more than 40 carbons, if their molecular weight exceeds 500, they become very resistant to biodegradation because microorganisms cannot get them inside the cell bodies. For polycyclic aromatic hydrocarbons that are very hydrophobic and sorb strongly to soil, they are poorly bioavailable. And also they are poor enzyme inducers, and they tend to be recalcitrant when they have more than three rings. Also, unusual substituents that are not common building blocks of life-- such as chlorine atoms and other halogens, nitro groups, cyanide, and so on-- they increase the recalcitrant of a molecule. On the other hand, when you have functional groups that are commonly found in the normal diet of organisms, such as alcohol groups and carboxylic acids, those make the molecule much easier to be biodegraded. So, to summarize. The key points here that microorganisms with the required catabolic capacity must be present in sufficient numbers, and must exert their biodegradation capabilities so that you can observe significant biodegradation rates. And finally, environmental and thermodynamic conditions must be favorable to sustain the expression of these biodegradation capacities.