Anaerobic Respiration

The last few sections have talked extensively about aerobic respiration. What defines it as aerobic is its use of oxygen as the terminal electron accepter. Since this is very similar to the type of respiration that humans use, our bias is obvious. Now let me fill you in on a little secret. Microbes are capable of using all sorts of other terminal electron accepters besides oxygen. Below we talk about a few examples of anaerobic respiration. The one thing that they all have in common is the use of an electron transport system in a membrane and the synthesis of ATP via ATP synthase. In both nitrate reduction and sulfate reduction there are two types of pathways, assimilatory and dissimilatory. Assimilatory pathways are methods for taking a nutrient in the soil, moving it into the cell and using it for biosynthesis of macromolecules. Dissimilatory pathways use the substrate as a place to dump electrons and generate energy. Here we examine dissimilatory pathways. Assimilatory pathways will be explained in the context of cell biosynthesis.

Nitrate reduction

Some microbes are capable of using nitrate as their terminal electron accepter. The ETS used is somewhat similar to aerobic respiration, but the terminal electron transport protein donates its electrons to nitrate instead of oxygen. Nitrate reduction in some species (the best studied being E. coli) is a two electron transfer where nitrate is reduced to nitrite. Electrons flow through the quinone pool and the cytochrome b/c₁ complex and then nitrate reductase resulting in the transport of protons across the membrane as discussed earlier for aerobic respiration.

N0₃^- + 2e^- + 2H⁺N0₂^-+ H₂0

Figure 1 - The reaction for nitrate reduction. N0₃^-, nitrate; N0₂^-, nitrite

This reaction is not particularly efficient. Nitrate does not as willingly accept electrons when compared to oxygen and the potential energy gain from reducing nitrate is less. If microbes have a choice, they will use oxygen instead of nitrate, but in environments where oxygen is limiting and nitrate is plentiful, nitrate reduction takes place.

Denitrification

Nitrite, the product of nitrate reduction, is still a highly oxidized molecule and can accept up to six more electrons before being fully reduced to nitrogen gas. Microbes exist (Paracoccus species, Pseudomonas stutzeri, Pseudomonas aeruginosa, and Rhodobacter sphaeroides are a few examples) that are able to reduce nitrate all the way to nitrogen gas. The process is carefully regulated by the microbe since some of the products of the reduction of nitrate to nitrogen gas are toxic to metabolism. This may explain the large number of genes involved in the process and the limited number of bacteria that are capable of denitrification. Below is the chemical equation for the reduction of nitrate to N₂.

N0₃^-N0₂^- NO N₂O N₂

Figure 2 - The reduction of nitrate to nitrogen gas. NO, nitric oxide; N₂O, nitrous oxide; N₂, nitrogen gas.

The advantage for the cell of carrying out a complete reduction of nitrate is two fold. The nitrate ETS serves as a place to oxidize NADH and free it to be used in catabolism of more substrate. Denitrification take eight electrons from metabolism and adds them to nitrate to form N₂ versus just two for nitrate reduction alone. Also, donation of electrons from NADH through the cytochrome b/c₁ complex and eventually to nitrous oxide (N₂O) reductase provides another opportunity to pump protons across the membrane. The figure below presents a schematic of the spacial arrangement of the denitrification enzymes in the membrane

Figure 3 - Denitrification in the membrane. NADH dehydrogenase complex (DH), nitrate reductase (NAR), nitrite reductase (NIR), NO reductase (NOR), and N₂O reductase (N₂OR)

Nitrate reduction has been extensively studied in bacteria due to its significance in the global nitrogen cycle. Denitrification removes nitrate, an accessible nitrogen source for plants, from the soil and converts it to N₂ a much less tractable source of nitrogen that most plants cannot use. This decreases soil fertility making farming more expensive. Intermediates of denitrification, nitrous oxide and nitric oxide, are gases and will sometimes escape the cell before being completely reduced. These compounds, when in the atmosphere, contribute to the greenhouse effect and exacerbate global warming. The use of high nitrate fertilizers in modern agriculture makes matters worse. For more information, there is an extensive review of denitrification available on line.

Sulfate reduction

The disimilatory reduction of sulfate seems to be a strictly anaerobic process as all the microbes capable of carrying it out only grow in environments devoid of oxygen. Sulfate (SO₄^-2 is reduced to sulfide (S^-2), typically in the form of hydrogen sulfide (H₂S). Eight electrons are add to sulfate to make sulfide

acetate + SO₄^-2 + 3H⁺ + 2CO₂ + H₂S + 2H₂O

Figure 4 - The reduction of sulfate to sulfide during growth on acetate.

The electron potential and energy yield for sulfate reduction is much lower than for nitrate or oxygen. However, there is still enough energy to allow the synthesis of ATP when the catabolic substrate used results in the formation of NADH or FADH. Substrates for sulfate reducers range from hydrogen gas to aromatic compounds such as benzoate. The most commonly utilized are acetate, lactate and other small organic acids (lactate, malate, pyruvate and ethanol are some examples). These compounds are prevalent in anaerobic environments where anaerobic catabolism of complex organic polymers such as cellulose and starch is taking place.

Biochemistry of sulfate reducers

Sulfate reducers take these growth substrates and metabolize them to acetate. The reducing power generated travels down an electron transport chain eventually reducing sulfate to hydrogen sulfide and generating energy using ATP synthase.

Figure 5 - Pathway of sulfate reduction when grown on lactate. Lactate (in blue) is oxidized to acetate (in red) and the electrons remove eventually end up reducing sulfate (in blue) to sulfide (in red). Note that the energy gained in the process by SLP - converting acetyl phosphate to acetate - is used up to activate sulfate in the first step of sulfate reduction. Energy for metabolism is only generated via an electron transport chain.

Recent work in bioremediation of anaerobic sediments has resulted in the isolation of many novel sulfate reducing species capable of metabolizing environmentally intractable compounds including TNT, PCP and benzoate. It is becoming apparent that this group of bacteria are very important in recycling carbon to CO₂ as part of the global carbon cycle in anaerobic environments. For a look at some recent research on sulfate reducing bacteria, check out the "Journal of Bacteriology"

Carbonate reduction

Several groups of microbes are capable of using carbonate (CO₂) as a terminal electron accepter. Carbonate is a poor choice to leave your electrons with due to its low reduction potential and energy yields from CO₂ reduction are low. However, carbonate is one of the most common anions in nature and its ready availability makes it a tempting target.

Several groups of microbes have evolved mechanisms to take advantage of carbonate. The most important group among these is the methanogens. Methanogens are Archaea and comprise one phylogenetic group that is very closely related. Methanogenesis seems to be highly conserved and have deeps roots in the phylogenetic tree of life. It must have evolve early on and practitioners of methanogenesis cannot mess with the genes too much, lest they die.

HC0₃^- + 4H₂ + H⁺ CH₄ + 3H₂O

Methanogens use compounds that contain very high energy electrons as their electron donors and in the process convert CO₂ to methane (CH₄). Above is shown the use of hydrogen as the source of electrons. They are the only group of microbes that produce a hydrocarbon as major end product of their metabolism.

Another group of carbonate reducing microbes are the homoacetogens. They utilize hydrogen as the electron source and use it to reduce CO₂ to acetic acid.

HC0₃^- + 4H₂ + H⁺ CH₃-COO^- + 4H₂O