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I. Introduction to Biogeochemical Cycles
Nature recycles. The atoms within organisms (C, H, O, N, S, Fe, traces of other metals) came from inorganic, non-living matter. The same atoms will exit living organisms as inorganic matter, thus completing a cycle. Microorganisms are crucial to this recycling.
A. Some Important Implications and Observations
1. A balance must be achieved if the nature of the planet, and of life as we now it, are to continue.
An imbalance in a biogeochemical cycle would have dramatic consequences on a global scale. For instance, the advent of cyanobacteria made the atmosphere oxygenic and greatly affected the biology of this planet. This was a truly historical event.
2. Role of microorganisms? Microorganisms are intimately involved in the recycling of biological matter. Some materials that higher organisms depend on would not exist without microorganisms. For instance, molecular nitrogen is converted to ammonia only by certain species of bacteria. As a result, all the nitrogen atoms found in all living things started out in bacteria. Equally important, without microorganisms to recycle their wastes, "higher" organisms would be killed.
3. Reservoirs? Reservoirs are sources of atoms. Not all reservoirs can be tapped by biological organisms - some forms of atoms cannot be utilized by incorporation into organic molecules. In addition, materials that can be recycled do not necessarily make up the largest reservoir available. For instance, although the largest carbon reservoir is within rocks in the Earth's crust, this source is biologically useless since organisms do not perform reactions capable of utilizing it.
4. Types of reactions. The reactions that organisms perform to interconvert inorganic and organic molecules invariably involve oxidation-reduction, i.e. "redox", chemistry:
a. Synthesis of more complicated molecules usually involves fixation and/or reduction. This requires an electron donor and an energy source.
b. Degradation of molecules usually involves their oxidation (equivalent to burning) to obtain the energy that they contain. This requires an electron acceptor, the most efficient of which is molecular oxygen.
5. Sources and sinks of electrons. Organisms adapt to use the best electron sources (donors) or sinks (acceptors) available. When the "favorite" is used up, gene expression is altered to permit the use of the next best source or sink. This situation is analogous to carbon utilization wherein glucose is favored but other sugars can be utilized when glucose is lacking.
6. Proteins. Enzymes are needed to catalyze the biological reactions of the biogeochemical cycles. Classification of microorganisms is in part determined by the presence or absence of these key enzymes. For instance, the presence of the enzyme RubisCO indicates that the organism fixes carbon by the Calvin Cycle.
7. Suppose a required reaction is unfavorable? Enzymes are only capable of speeding up the rates (forward and reverse) of chemical reactions. Enzymes cannot make thermodynamically unfavorable reactions favorable. As a rule, unfavorable reactions are driven by coupling them to favorable reactions. For example,
(i). A + B <-----> C + D
(ii). C <-----> E
Suppose reaction (i) is unfavorable [i.e. at equilibrium, there is more (A + B) than (C +D)], and reaction (ii) is favorable (i.e. at equilibrium, there is more E than C). By rapidly converting C to E when it forms, there is no opportunity for the first reaction to go in "reverse." In other words, removal of the products of the first reaction by the second reaction will drive the first reaction.
Coupling of reactions occurs:
a. within an organism.
b. between organisms. In other words, the waste products of one organism may be the food for another. This is known as syntrophy (= "to eat together").
II. Introduction to the Carbon Cycle
Most of the carbon within organisms comes from the carbon dioxide (CO2) in the air.The atmosphere is 0.03 mol % in CO2. However, the greatest physical reservoir of carbon is not atmospheric carbon dioxide but instead is located in the Earth's crust and is not easily accessible to biological organisms. Figures 17.27 and 17.28 show the reservoirs of carbon and the transactions that take place in the carbon cycle.
A. What Reactions Involving the Carbon Cycle Must Balance in Nature?
1. Carbon fixation. Photosynthetic carbon fixation is responsible for the vast majority of the carbon fixed in nature. It involves the use of light energy to "fix" atmospheric carbon dioxide into sugars (and subsequently into other biological molecules):
CO2 + H2O + energy -----> (CH2O)n + O2
Photosynthetic carbon fixation may be done in both oxic and anoxic environments. In the reaction above, use of water as an electron donor results in the formation of oxygen. This is the chemistry done by oxygenic organisms such as algae, cyanobacteria, and green plants. Alternate electron donors are used by photosynthetic carbon fixers, such as the phototrophic green or purple sulfur bacteria, living in anoxic environments.
2. Respiration via the TCA cycle.
(CH2O)n + O2 -----> CO2 + H2O + energy
Respiration involves the oxidation of carbohydrates to obtain energy. Respiration may be done in both oxic and anoxic environments. In the reaction above, oxygen serves as the electron acceptor, resulting in the formation of water. This is the chemistry done by all organisms living in oxic environments including plants, animals and microorganisms. Note that this reaction is the reverse of photosynthetic carbon fixation in oxic environments. Alternate electron acceptors are used by organisms that do respiration in anoxic environments (since oxygen is not available). Fermentation is a related process done by microorganisms in anoxic environments wherein energy is obtained from the partial oxidation of organic molecules.
3. Respiration by the methanogens (= "methane generators").
CO2 + 4 H2 -----> CH4 + 2 H2O + energy
In this reaction, the electron donor is hydrogen, and carbon dioxide serves as the electron acceptor. Methane can also be generated from other methyl compounds, including methanol and acetate. The methanogens are strict anaerobes.
Generally, when oxygen is not used as the electron acceptor in respiration, it is likely that the organism:
a. is living in an environment where oxygen is not available,
b. or cannot live in an environment containing oxygen.
Since more energy can be extracted when oxygen is used as the final electron acceptor in respiration, organisms that do not use oxygen could not compete with others that do.
4. Respiration by the methanotrophs (= "methane eaters").
CH4 + 2 O2 -----> CO2 + 2 H2O + energy
a. Methane is oxidized, i.e. used as the electron donor. Oxygen serves as the electron acceptor; it is reduced to water. Other substrates may be oxidized by some methylotrophs (see Table 19.8), of which the methanotrophs are a subset.
b. Where do the methanotrophs live?
The problem is that the methanotrophs need the "waste" methane produced by the methanogens as well as oxygen -- but the methanogens are strictly anaerobic. The solution is to live at the "boundaries" of oxic and anoxic environments, where both methane and oxygen are obtainable.
c. The diagnostic enzyme that indicates the presence of a methanotroph is methane monooxygenase; this enzyme is used in the first step of the oxidation of methane wherein methanol is produced.
B. Some Imbalances in the Carbon Cycle Caused by H. sapiens
1. Coal formation occurs very slowly, on a geological time scale. On the other hand, the burning of fossil fuels by man happens very fast, and the rate of utilization of these resources is increasing. As a result, the amount of carbon dioxide in the atmosphere is increasing.
2. Plants growth has been stimulated by the "green revolution" and by this additional carbon dioxide in the atmosphere. In addition, deforestation of land has accelerated in this century, producing yet more decaying plants. Plant decay releases methane and even more carbon dioxide into the atmosphere.
3. Agricultural ruminants, such as cattle, eat grasses and grains (grown on deforested lands that are no longer available to fix the increasing carbon dioxide in the air!). Together with symbiotic microorganisms, they digest the grasses and grains, forming more carbon dioxide and, in addition, methane.
As a result of these and other events, the levels of carbon dioxide and methane in the atmosphere have increased. For instance, there has been a 2% increase in the atmospheric methane in the last few years alone.
Why is this noteworthy? Much of the infrared radiation (IR) that comes to Earth from the sun does not reach the surface of the planet because it bounces away. Gases like carbon dioxide and methane trap IR and prevent it from radiating away from the Earth. This is known as the greenhouse effect. Without this phenomenon, the average temperature of the planet would be -18oC. As a result of the increasing levels of CO2 and CH4 in the atmosphere, more IR is being "captured" and not bouncing away from the planet. The Earth is getting warmer! Increased levels of methane are especially dangerous because this gas absorbs five times more IR than carbon dioxide. Methanotrophs oxidize methane, but they are simply not efficient enough to use up methane in the concentrations found in the atmosphere. Current research is focused on manipulating these organisms to increase their affinity for methane and their rates of methane utilization.
III. Details of the Carbon Cycle
A. Overview of Carbon Fixation (see the handout)
Carbon fixation involves the incorporation ("fixation") of carbon from carbon dioxide into organic molecules and its subsequent reduction to the level of an alcohol.
1. The enzyme that affixes the CO2 to a pre-existing carbohydrate is usually considered the key diagnostic enzyme in a carbon cycle. For example, ribulose 1,5 bisphosphate carboxylase (= rubisCO) is indicative of organisms that use the Calvin cycle.
2. The overall process is reductive. It involves successive activations of carbons using energy from ATP for attack by:
a. CO2, i.e. the fixation step,
b. Hydride ions (equivalent to H-), supplied by biological reducing agents such as NADPH, NADH.
c. The reduction steps are usually as follows:
(i) A carboxylic acid COO- is reduced to the level of H-C=O, an aldehyde;
(ii) The aldehyde H-C=O is reduced to the level of H-C-OH (i.e. CH2O), an alcohol.
B. Requirements for Carbon Fixation
1. Carbon dioxide (CO2), a substrate
2. A sugar (or some other carbon compound) on which to affix the carbon trapped from the atmosphere
3. Enzyme(s) for the fixation reaction
a. ATP (or sometimes CoA) is used to activate the carbons for subsequent reactions,
b. NADPH or NADH (or sometimes reduced feredoxin) provides reducing power. They contribute the hydrides that are used to reduce carbon. A hydride is a pair of energetic electrons and a proton that may be viewed as H-.
5. Energy source
a. The best source is sunlight.
(i). In oxic environments, the electrons that are used to reduce carbon are ultimately derived from the light-powered splitting of water.
H2O -----> 1/2 O2 + 2 H+ + 2 e-
This is done by the green plants, algae and cyanobacteria.
(ii). In anoxic environments, obviously oxygen is not generated; instead the electrons that are used to reduce carbon ultimately are derived from the light-mediated splitting of hydrogen sulfide.
H2S -----> So + 2 H+ + 2 e-
This is done by the green and purple sulfur bacteria.
b. Less efficient methods do not use sunlight as an energy source. For instance, some microorganisms [chemoautotrophs, or chemolithoautotrophs, (= "make their foods from chemical compounds")] get their energy from the oxidation of inorganic compounds. For example, Thiobacillus ferrooxidans oxidizes Fe2+ -----> Fe3+ + e-. It then uses the energy from this reaction to generate a proton gradient that powers carbon fixation.
6. A cycle of enzymatic reactions to incorporate carbon into the organism's carbohydrate pool.
C. The Calvin Cycle (consult the handout, Figures 16.19 and16.20)
1. Two enzyme are diagnostic of this cycle:
a. Phosphorybulose kinase,
b. Ribulose 1,5 bisphosphate carboxylase, abbreviated rubisCO. This is the most abundant protein on earth!
2. Immediately following the fixation reaction, every mole of the unstable intermediate produced is spontaneously hydrolyzed to 2 moles of phosphoglyceric acid.
3. Although only one of the two moles of phosphoglyceric acid thus formed has the fixed carbon, both are reduced to glyceraldehyde-3-phosphate.
This reduction of 2 moles of phosphoglyceric acid to 2 moles of aldehyde (i.e. glyceraldehyde-3-phosphate) may be regarded as being equivalent to two successive reductions of 1 mole of acid to 1 mole of alcohol. This is done in the sugar rearrangements that follow.
4. For every turn of the cycle, 1 fixed CO2 is reduced to 1 H-C-OH, (i.e. CH2O). Thus six turns of the cycle may be viewed as generating a six-carbon sugar from 6 CO2.
D. Aerobic Degradation of Carbon
1. The oxidative degradation of sugars is done by all aerobic organisms and involves the TCA cycle. Since this reaction is not special to microorganisms, it will not be discussed further.
(CH2O)n + O2 -----> CO2 + H2O
2. The oxidative degradation of methane by the methanotrophs has already been discussed.
CH4 + 2 O2 -----> CO2 + 2 H2O
E. Anaerobic Degradation of Carbon
Anaerobic degradation of carbon is strictly done by microorganisms. This is responsible for most of the biological CO2 and CH4 released to the atmosphere.
1. Anaerobic respiration involves the complete oxidation of organic substances:
(CH2O)n + Xox -----> CO2 + Xred
Alternative electron acceptors, denoted "Xox", commonly used are nitrate (NO3-), sulfate (SO42-), elemental sulfur (So), or ferric iron (Fe3+) ions.
2. Anaerobic decomposition of organic substances to carbon dioxide and methane (Figure 17.29) is a collaborative effort involving many different reactions and species of microorganisms. This is also called interspecies hydrogen transfer. These reactions occur in the gut (to a limited extent), sediments, soils, and the rumen.
a. Complex polymers (e.g. cellulose, starch, proteins) are broken down to monomers by fungi and by cellulolytic bacteria such as Bacteroides succinogenes.
b. The monomeric subunits (e.g. sugars, amino acids) thus produced are broken down by enterics and other fermentative bacteria such as Clostridium butyrium, producing organic acids [e.g. butyrate (i.e. CH3CH2CH2COO-), propionate (i.e. CH3CH2COO-], alcohols, etc.
c. Further fermentations are done by the syntrophic bacteria Syntrophomonas sp. and Syntrophobacter sp. that produce acetate (i.e. CH3COO-), carbon dioxide (i.e. CO2), and molecular hydrogen (i.e. H2).
d. The carbon dioxide, molecular hydrogen, and acetate are used by methanogens to produce methane (i.e. CH4).
3. Some important points.
a. Suppose we stop after the reactions of the fermentative bacteria? Organic acids and alcohols would accummulate. The organisms would drown in their own wastes. Many would stop growing or die.
b. What is the rate-limiting step? The rate limiting step in the anaerobic decomposition of carbon is the fermentation of organic acids by the syntrophs. These reactions are thermodynamically unfavorable. They are driven by removal of the hydrogen and acetate products by the methanogens using reactions that are thermodynamically favorable.
c. What do the syntrophs do for the:
(i) Fermentative bacteria? Syntrophs remove the wastes of the fermentative bacteria.
(ii) Methanogens? Syntrophs feed the methanogens.
d. Where do the methanogens get:
(i) Their reducing power? From the H2 produced primarily by the syntrophs and to a lesser extent by the fermentative bacteria.
(ii) Their carbon? From the CO2 and acetate produced primarily by the syntrophs and to a lesser extent by the homoacetogens and the fermentative bacteria.
In brief, although these degradative reactions occur in groups of different organisms, they are tightly interrelated in that the waste of one group of bacteria becomes the food for another. Consequently, if any single reaction in the path is deficient or overefficient, many organisms starve to death or are killed by the accumulated wastes.
e. Energetics of syntrophy
|(i) 2 butyrate + 4 H2O ---------->
4 acetate + 4 H2 + 2H+
(ii) 4 acetate + 4 H2O ----------> 4 CH4 + 4 HCO3-
(iii) 4 H2 + HCO3- + H+ ----------> CH4 + 3 H2O
|Delta Gi = 2 x (+48.2 kJ) = 96.4 kJ
Delta Gii = 4 x (-31 kJ) = -124 kJ
Delta Giii = 1 x (-136 kJ) = -136 kJ
|Net reaction: 2 butyrate + 5 H2O ----------> 5 CH4 + 3 HCO3- + H+||Delta Gtotal = -163.6 kJ|
Reaction (i) is a thermodynamically unfavorable fermentation done by the syntrophs. The products of this reaction are used by the methanogens as substrates in the favorable reactions (ii) and (iii). The overall free energy change for this series of reactions is negative, indicating that the net conversion of butyrate to methane and bicarbonate is favorable. Note the energetic coupling between the favorable reactions of the methanogens and the unfavorable reaction of the syntrophs.