LECTURE 25. BIOREMEDIATION

I. Bioremediation

Definition - exploiting microbes to degrade wastes and xenobiotics

A. Xenobiotic - a (foreign = xeno) synthetic compound not normally found in nature. Examples include:

1. Pesticides

a. Herbicides

b. Insecticides

c. Fungicides

2. Detergents

3. Plastics and various other synthetic polymers

The structures of some xenobiotic compounds are shown in Figure 17.48. Note that aromatic rings, often substituted with chlorine atoms, are common. Table 17.7 shows the length of time these substances persist in the environment. However, these times are not fixed - environmental factors will influence these rates of degradation.

B. Oxic Degradation of Hydrocarbons

1. Microbes that utilize hydrocarbons:

a. Pseudomonas spp. - predominantly

b. Mycobacterium

c. Nocardia

d. Some yeasts and molds


2. Long chain aliphatic hydrocarbons are converted into fatty acids (Figure 16.47) by oxidation of a terminal carbon (to an alcohol, then an aldehyde, then a carboxylic acid). The fatty acids thus generated are activated with CoA and simply fed into the normal pathway for fatty acid degradation - beta oxidation (Figure 16.46). These reactions yield 2-carbon acetyl-CoA units that can be consumed in the citric acid cycle.

Which of the following (a, b, c, d, e) would be recalcitrant to oxic degradation?

Generally, hydrocarbons that have additional groups (e.g. -CH₃ , -Cl atoms) at the position destined to become the beta-carbon in the beta oxidation pathway will be recalcitrant to oxic degradation. However, a hydroxyl group is favorable at this position since the pathway normally generates a beta alcohol anyway. A double bond between the alpha and the beta carbons is also favorable.

3. Oxic degradation of aromatic hydrocarbons

• Complex aromatic compounds are first converted to a "starting substrate" such as catechol (Figure 16.48a);
• Formation of catechol may involve the introduction of oxygen atoms by a monooxygenase (Figure 16.48b);
• A dioxygenase breaks open the aromatic ring of catechol, producing cis,cis-muconate, an unsaturated dicarboxylic acid (Figure 16.48);
• This product is then oxidized to acetyl-CoAs by the aforementioned beta oxidation path. Substitution of aromatic rings (e.g. with -CH₃ or -Cl) interferes with the oxic degradation of the aromatic ring.

For instance, the herbicides 2,4-D and 2,4,5-T (Figure 17.48) are aromatic rings, respectively containing two and three chlorine substitutions. Compare the persistence of these compounds in the environment (Table 17.7). The additional chlorine atom makes 2,4,5-T five-fold less degradable compared to 2,4 D.

C. Anoxic Degradation of Xenobiotics

1. Recall that in anoxic environments, since oxygen is not available as an electron acceptor, microorganisms utilize alternates such as nitrate, sulfate and ferric iron. Organisms view xenobiotics as another electron acceptor choice.

2. Some of the xenobiotics include herbicides, industrial solvents etc.

3. A warning - some partially degraded materials may be even more toxic than the starting materials. 4. Two broad strategies for anoxic degradation:

a. Reductive dechlorination e.g. the substrate 3-chlorobenzoate can be dechlorinated by reduction of the chlorine atom, producing benzoate and chloride anion:

• Note that in this reaction carbon is not reduced. The chlorine atom is reduced from 0 to -1.
• What organisms? Reductive chlorination is practiced by organisms that utilize alternate electron acceptors such as the denitrifiers and sulfate reducing bacteria (e.g. Desulfomonas).
• Ultimate source of reducing power? Electrons ultimately came from the oxidation of substrates normally utilized by these organisms (e.g. molecular hydrogen, acetate etc. for the sulfate reducers).

b. Reductive ring cleavage e.g. the substrate benzoate can be converted to a saturated dicarboxylic acid, one end of which is linked to CoA (Figure 16.49). There are three principal steps in the conversion:

(i) Activation - ATP energy is expended in order to attach CoA to the carboxylic acid group of benzoate, producing a highly reactive mixed anhydride.

(ii) Reduction - hydride units from NADPH are utilized to reduce the carbons within the aromatic ring, producing hexanyl-CoA.

(iii) Ring cleavage - carbon 2 of the hexanyl ring is oxidized (to an alcohol, then a ketone, then a carboxylic acid), leading to the cleavage of the ring system. Note the source of the oxygen atoms in this anoxic reaction is water, the remaining electrons and protons of which are used to reduce NAD⁺.



The saturated dicarboxylic acid product is then oxidized to acetyl-CoAs by the beta-oxidation pathway.

D. Some Remaining Problems

1. Microorganisms Although microorganisms help in bioremediation, they are not especially in a hurry. Relevant organisms are being studied to induce them to metabolize waste faster.

2. Humans

a. Population - The human population is increasing. People are in a hurry. If not for the recycling activities of microorganisms, we would "drown in our own wastes." However, it is difficult for microbes to keep up with the amount of waste that humans generate.

b. United States plastics industry. The plastics industry produces about 70 billion pounds of plastic per year, about half of which ends up in landfills. Many of these plastics contain synthetic structures that cannot be degraded by microbes (Figure 17.49a).

c. "Developing" world. Developing countries aspire to meet the "standards" of consumption of the United States. In other words, the rate of waste production by third world nations is increasing.

d. Europe has been crowded since the seventeenth century. Since there is no room for more landfills, European nations, especially Germany and Switzerland, have agressive recycling programs to lessen the load of waste.

E. Some Imaginative Solutions

1. Collect, treat, bury/reuse. The idea is to use biodegradable polymers in the construction of plastics so that plastic waste can be microbially treated for purposes of degradation. The materials remaining can be buried, or preferably reused.

a. Photobiodegradable plastics. Some newer plastics contain polymers that change structure when illuminated with UV radiation, forming biodegradable materials.

b. Starch-linked biodegradable plastics. Starch has been incorporated into the structure of some plastics. When treated with starch-degrading bacteria, these plastics release glucose and smaller polymers that can be microbially degraded.

c. Bacterial plastics.

A number of microbes naturally produce biodegradable polymers that are suitable for the plastics industry. For example, the bacterium Alcaligenes eutrophus produces the polyester poly-beta-hydroxybutyrate (PHB) (Figure 3.51) as a storage reserve for excess carbon. The monomeric unit of PHB is beta-hydroxybutyrate.

(i) Lengths: Polymeric derivatives can be prepared containing monomers of longer lengths than the four carbon butyrate backbone. For example, beta-hydroxyvalerate contains five carbons and beta-hydroxycaproate contains six carbons. Alternatively, mixtures of different monomers may be used to generate more structurally complex co-polymers.

(ii) Functional groups: The monomers contains two functional groups - a hydroxyl group and a carboxylic acid group.

(iii). What determines strength, flexibility and crystallanity of polymers?

• Type of repeating units.
• The media and type of bacteria used to produce the polymer determines the structure of the polymer.

(iv). What determines cost?

• Growth media - the media used to grow polymer-producing bacteria is very cheap. It consists of carbon and energy sources such as glucose and acetate.
• The "productivity" of each cell or the amount of plastic made per cell = percent of cell weight. For the sake of cost efficiency, industry strives to find conditions that maximize the amount of plastic that can be made per cell. e.g. In England, Imperial Chemical Industries have developed a system that induces a genetically engineered strain of A. eutrophus to polymerize large amounts of the substrate glucose - 80% of the dry weight of the cells is plastic polymers.

Thus microbes are entering polymer science!