15 Model of Energy and Matter Transformations

In the prior chapters of this unit, we’ve explored energy metabolism pathways in cellular respiration, lactic acid fermentation, and photosynthesis, and how they are regulated. Overall, energy and matter availability are fundamental drivers of the reactions occurring in cells, and cells often create specialized structures and compartments in which specific reactions occur. But cells also need specific enzymes to carry out most reactions of metabolism, since they would otherwise occur too slowly. Thus, another key component of determining an organism’s metabolism are the specific enzymes that are present. Extending off this idea, to change an organism’s metabolism, one could start with changing the enzymes. And do to that, remember that most enzymes are type of protein, and proteins are encoded by genes in the DNA. Once again, changing the characteristics of an organism, such as the specific metabolic reactions taking place, can be accomplished by changing the genetic information present in a organism.

There are many examples, both natural and carried out in the lab, of an organism’s DNA changing, when then altered the organism’s metabolism. There are herbicide-resistant crops, bacteria that digest plastic, and golden rice, just to name a few. In this chapter and upcoming class, we will focus on a bacterial species called Caldicellulosiruptor bescii. The name of this species has changed several times, and as of 2025 it is officially designated Anaerocellum bescii, but we’ll refer to these bacteria as Caldi for short. Caldi bacteria have a rare, natural ability to break down cellulose, and researchers wanted to produce Caldi bacteria that could additionally convert the products of cellulose breakdown to ethanol that could be used as a biofuel. In this chapter, we’ll see how and why researchers were able to accomplish this goal.

Chapter Outline

Section 15.1 Metabolic Recap

Section 15.2 Metabolic Pathways and Free Energy Changes in Fermentation

Section 15.3 Enzymes and Fermentation Pathways

Section 15.4 Metabolism to Produce Biofuels

Section 15.1 Metabolic Recap

Energetics Principles

Before getting into the details of the bacterial engineering, let’s review a few key ideas from the unit. First, processes can be classified as energetically favorable or unfavorable depending on whether the process releases energy or requires energy input. A favorable process has a negative free energy change, or negative ΔG, when the products of that process have lower free energy than the starting molecules. The opposite is true of energetically unfavorable processes. Importantly, only energetically favorable processes can occur!

However, cells are constantly carrying out process that require energy input, such as creating macromolecules, transporting small molecules, and otherwise maintaining their internal environments. For these processes to occur, cells must use energetic coupling, in which the energy released from an energetically favorable process is used to drive an energetically unfavorable process. Successful energetic coupling requires that the ΔG of the coupled processes must be negative, meaning the favorable process releases more energy than the unfavorable process requires.

Let’s recap some of the major energetic couplings we observed in the major metabolic pathways of cellular respiration and photosynthesis. In cellular respiration, glucose is oxidized to CO₂, a very energetically favorable process that cells couple to the production of ATP from ADP + P_i, which requires energy input (Figure 15.1a). In photosynthesis, plants use light energy input to move electrons, ultimately producing ATP and NADPH that are in turn used to reduce CO₂ to the carbohydrate G3P (Figure 15.1b). This G3P can be further built up into glucose (and used to create polysaccharides of glucose, like cellulose), or converted to many other types of monomeric building blocks used to create all classes of macromolecules (summarized in Figure 14.11).

Journey of Electrons through Cellular Respiration and Photosynthesis

Now that we’ve revisited the major energetic couplings occurring in cellular respiration and photosynthesis, let’s consider the journey of electrons through these metabolic pathways. Imagine a single plant cell that performs both photosynthesis and cellular respiration (Figure 15.2). In a chloroplast of that cell, light energy excites the electrons of chlorophyll in Photosystem II (PS II), resulting in oxidized chlorophyll. This leads to water being oxidized by an enzyme in PS II to replace electrons lost by chlorophyll. Thus, water is the ultimate source of electrons in photosynthesis. These electrons travel to Photosystem I (PS I), where they are again excited by light energy so they have enough energy to reduce NADP⁺ to NADPH. From here, electrons that are now part of NADPH are used in the Calvin Cycle to reduce CO₂ to triose phosphates like G3P, which can be used to produce glucose sugars. Overall, electrons that started as part of water are now part of a sugar molecule.

Sugars are a starting point for the next major pathway, cellular respiration, in which sugars are oxidized to CO₂. In coupled reactions, electrons are transferred from sugars to NAD⁺ to produce NADH. Then, the NADH is oxidized by proteins in the mitochondrial electron transport chain, and the electrons passed to O₂ to form H₂O. The start of photosynthesis and the end of cellular respiration are both water – this is the true circle of life!

Cellular Respiration and Photosynthesis Pathway Comparisons

In many ways, the pathways of cellular respiration and photosynthesis are two sides of the same coin, with the end products of one being the starting point of the other. In other ways, though, these pathways have many process and molecules in common. Fundamentally, though, both pathways are critical transformations of energy and matter, and the energetic couplings occurring in these pathways, ultimately depending on light energy input, are fundamental for life as we know it.

Section 15.2 Metabolic Pathways and Free Energy Changes in Fermentation

The in-class activity associated with this content will focus on three fermentation pathways. We encountered one such fermentation pathway in Section 13.5, lactic acid fermentation, which allows muscle cells to produce a small amount of ATP even when O₂ levels are too low for electron transport and oxidative phosphorylation to occur. Fermentation pathways are widespread, especially in single-celled organisms like bacteria and yeast, and you have likely enjoyed many products of fermentation, such as yogurt, cheese, and bread.

Here, we will explore three pathways of relevance in Caldi bacteria, two of which exist in Caldi found in the environment, and one that was introduced using genetic engineering (more to come on that one!). The common theme of all fermentation pathways is the ability to produce energy management molecules, such as ATP, in the absence of O₂. For all three pathways being discussed here, the starting point is glucose. The model in Figure 15.3 shows that glucose is converted to pyruvate using reactions that are coupled to the production of NADH. This is the same glycolysis reaction pathway that we studied before, and the repeated arrows between glucose and pyruvate are to remind us of the many steps of glycolysis. Remember too that ATP is produced by glycolysis, though it is not shown in this model.

Before delving further into specific pathways, let’s revisit ideas we have discussed previously to bring into this context. The reactions in metabolic pathways involve making and breaking covalent bonds, and we can describe individual reactions by the free energy changes occurring. As discussed in Chapter 9 and shown again in Figure 15.4, a process either results in higher or lower free energy for the system as compared to where it started. Remember that energetically favorable reaction has a negative ΔG because the products have lower free energy than reactants (Figure 15.4a), and energy is released into the environment. Conversely, the products of unfavorable reactions (Figure 15.4b) have higher free energy than the initial reactants, requiring energy to be absorbed during the process.

Let’s look at the conversion of glucose to pyruvate, which occurs by a series of reactions we discussed before (see Figure 13.5 for the detailed reactions of glycolysis). As summarized in Figure 15.3, as glucose is converted to pyruvate, these reactions also produce NADH. We learned in the Energy Metabolism chapter that the reaction of NAD⁺ to NADH is generally energetically unfavorable, so it has a positive ΔG as depicted in Figure 15.4b, and thus NADH has higher free energy than NAD⁺. The energy to create NADH in turn comes from reactions in the pathway to convert glucose to pyruvate, which is energetically favorable (similar to the process in Figure 15.4a). Thus, the glucose reactant must have higher free energy than the pyruvate product of this pathway. You can apply similar logic to other coupled reactions shown in Figure 15.3.

Section 15.3 Enzymes and Fermentation Pathways

Once pyruvate is produced, the pathway branches in multiple directions. One such pathway is to convert pyruvate to lactate, which is the same lactic acid pathway we encountered previously. However, we can see in our model that pyruvate can also be converted to acetyl-CoA, and then there are two possible outcomes: conversion to acetic acid or to ethanol. All three outcomes, lactate, acetic acid, and ethanol are possible, so what determines which products are made? With everything is this unit, energetics are everything! These reactions are all energetically favorable as written (take note of all the coupled reactions), but these reactions will only occur at reasonable rates in the presence of enzymes to catalyze reactions in these pathways. In the figure, the circled 1 indicates Enzyme 1 that catalyzes the conversion of acetyl-P + 2 ADP to acetic acid + 2 ATP. Similarly, Enzyme 2 is needed to produce lactate + 2 NAD+ from pyruvate + 2 NADH and Enzyme 3 is needed to produce ethanol in two steps from acetyl-CoA + NADH. If we can detect the product of the reaction catalyzed by the enzyme, we can use that to infer that the enzyme must be present and functioning.

Let’s revisit the lactic acid fermentation pathway once again. The enzyme that catalyzes the conversion of pyruvate to lactate is lactate dehydrogenase, or LDH. As shown in Figure 15.5a, for cells to produce this enzyme, a cell must have gene ldh in its genome, as part of its DNA, and express that gene to produce an mRNA that is translated and forms a folded and functional protein. Review Chapters 4-6 for more details on this process!

How does the presence of the enzyme affect the pyruvate -> lactate reaction? Imagine two strains of bacteria, X, and Y, and that one has the LDH enzyme and the other does not. Figure 15.5b shows a reaction-coordinate diagram to illustrate the free energy changes over the course of the reaction to convert pyruvate to lactate in each bacterial strain: the solid line shows the reaction in Strain X, while the dashed line indicates the reaction in Strain Y. From Figure 15.5b, we can see that while the reaction is energetically favorable in both strains (because the products of the reaction have lower free energy than the reactants), the energy input (activation energy) for the reaction is much greater for Strain Y than for Strain X, indicating the presence of LDH enzyme in Strain X to catalyze the reaction. Even favorable reactions can be very slow in the absence of enzymes, due to the high energy barrier. As a result, if we were to measure lactate amounts over time, we would detect lactate produced by Strain X but not Strain Y, as shown in Figure 15.5c.

The above example illustrates an important data analysis and prediction tool that we will exploit in this chapter: using the products produced by a specific bacterial strain to predict the specific enzymes present in that strain. Going back to Figure 15.3, note the final product at the end of each pathway. Reiterating the thought process from above, if a given strain produces measure amounts of lactate, it must have the gene for Enzyme 2 (LDH) in its genome. Similarly, if that strain also produces acetic acid, it also has the gene for Enzyme 1 in its genome. Both Enzymes 1 and 2 are present in Caldi bacteria found in the wild (described as Strain A in the data we will analyze in class).

To obtain the ethanol product shown in Figure 15.3, scientists had to get Caldi bacteria to produce Enzyme 3 to catalyze this reaction. How did they do that? Remember that most enzymes are proteins, and that means they’re encoded by DNA. Following the logic from above, for a cell to have a specific enzyme, the gene encoding that enzyme must be present in the cell’s genome. Furthermore, that gene needs to be expressed (transcribed and translated) and the protein needs to correctly fold into its functional three-dimensional shape. When scientists added the DNA encoding Enzyme 3, along with all the sequences to ensure correct gene expression, to the genome of Caldi bacteria, they created a new Caldi strain that we will refer to as Strain C.

In Chapters 4 and 5, we discussed specific signals that need to be present for the gene expression process to work. Review these chapters to identify DNA and RNA sequences that are required for transcription and translation to complete the pre-class activity.

Section 15.4 Metabolism to Produce Biofuels

Fossil Fuels and Biofuels

Now that we’ve recapped energetics principles and discussed some specific metabolic pathways at work on Caldi bacteria, let’s take a step back to consider why this research was done, and the problems this work was intended to help mitigate. You are likely familiar with the term ‘fossil fuels,’ which refers to coal and oil. These fuels result from a lengthy process that utilize organic material produced many millions of years ago via photosynthesis. Remember that photosynthesis uses the energy of light to capture CO₂ from the atmosphere and fix the carbon into organic molecules that help plants add biomass. This biomass accumulated, then various geologic events subjected the plant material to high heat and pressure, converting the organic material into hydrocarbons. When we use coal and oil in combustion reactions, we are taking CO₂ that was previously sequestered underground and releasing it into the environment, resulting in a net CO₂ increase in the atmosphere. In contrast, biofuels such as ethanol are derived from plants that were recently produced, and thus capturing CO₂ already in the atmosphere using photosynthesis. When these biofuels are later oxidized, the amount of CO₂ returned to the atmosphere is equivalent to the amount harvested earlier in photosynthesis, and thus this doesn’t cause a net increase in atmospheric CO₂.

One goal of biofuels science is to identify products to replace fossil fuels, which have a limited supply. A prominent example of biofuels in use today is ethanol, commonly derived from corn that is a supplement in automobile gasoline. The starches in corn kernels (Figure 15.6a) are fermented to produce this ethanol. However, there are some major challenges to using corn ethanol to replace fossil fuels. For example, it takes a lot of land to grow corn, and most of the available land that’s appropriate for growing food crops is already being used to grow food crops. Limited water and fertilizer are also problems for increasing corn production. Most importantly, there are other ways to use corn besides for ethanol production, namely as a food crop for people and livestock. Any competing use for corn products could decrease the food supply.

Given these limitations, alternative strategies for biofuel production have focused on plants that are easier to grow and are not food crops. One promising example is using switchgrass (Figure 15.6b). Switchgrass grows in many environments, is not a food crop, and contains abundant free energy in the form of cellulose. Like all plants, as switchgrass grows, it uses photosynthesis to capture light energy, which it then uses to reduce CO₂ to G3P. Further reactions convert the G3P product to glucose and finally to cellulose, a polysaccharide of glucose (Figure 15.7a).

However, there’s at least one problem with this approach. While cellulose has abundant free energy (Have you ever combusted cellulose? Perhaps in your fireplace or around a campfire?), very few organisms have the enzymes to digest cellulose and transform it into a usable and storable energy source. In fact, because cellulose is so hard to break down, it’s the most abundant macromolecule on Earth.

And yet, there are a few species of bacteria, including the Caldi bacteria mentioned earlier in this chapter, that can digest cellulose. They naturally have the enzymes and metabolic pathways needed to break down cellulose into glucose, and will readily ferment that glucose into other products such as lactate and acetic acid to meet their energy needs. However, these bacteria do not naturally produce ethanol. To change the metabolism of Caldi bacteria, scientists manipulated the genome of Caldi bacteria. By introducing DNA containing the adhE gene, scientists caused the bacteria to produce the Bifunctional Acetaldehyde and Alcohol Dehydrogenase enzyme (adhE). This enzyme takes acetyl-CoA from pyruvate oxidation and converts it to ethanol in two steps. As shown in Figure 15.7b, the new metabolic pathway in engineered Caldi bacteria starts with cellulose from switchgrass, and ends with production of ethanol biofuel.

While Figure 15.7 shows the building up and breaking down of carbon molecules in switchgrass and Caldi bacteria, respectively, it leaves out the energetics that make this possible. As your final preparation for class, use the activity below to create a summary model of switchgrass and Caldi metabolism that captures more of these details to extend upon further in class.

In this activity, boxes represent processes that may occur in several steps, arrows pointing into those boxes represent key net inputs or reactants for those processes, and arrows coming out of the boxes represent key net outputs or products of those processes. Use your notes from past chapters, along with Figure 15.3 from this chapter, and the word bank, to assist you in building this model.

Once you’ve built your model, consider how you would apply other ideas from this unit, such as anabolic and catabolic, favorable and unfavorable, oxidation and reduction, to different processes in this model. Or consider how you would describe matter and energy move and are recycled within organisms (within switchgrass or within Caldi bacteria), as well as between organisms (from switchgrass to Caldi, and from Caldi to switchgrass). The movement and cycling of matter and energy represented in these models is another circle of life!