Biomass Growth Substrate Utilization and Yield

When reduced to their barest essentials, biochemical operations are systems in which microorganisms are allowed to grow by using pollutants as their carbon and/or energy source, thereby removing the pollutants from the wastewater and converting them to new biomass and carbon dioxide, or other innocuous forms. Because of the role of enzymes in microbial metabolism, the carbon and/or energy source for microbial growth is often called the substrate, causing wastewater treatment engineers to commonly refer to the removal of pollutants during biomass growth as substrate utilization. If growth is balanced, which is the case for most (but not all) biochemical operations, biomass growth and substrate utilization are coupled, with the result that the removal of one unit of substrate results in the production of Y units of biomass, where Y is called the true growth yield, or simply the yield." Because of the coupling between biomass growth and substrate utilization, the rates of the two activities are proportional, with Y as the proportionality factor. Consequently, the selection of one as the primary event (or cause) and the other as the secondary event (or effect) is arbitrary. Both selections are equally correct and benchmark papers have been published using both substrate removal1" and biomass growth" as the primary event. The point of view taken in this book is that biomass growth is the fundamental event, and the rate expressions presented in Chapter 3 are written in terms of it. It should be emphasized, however, that rate expressions for biomass growth and substrate utilization can be interconverted through use of the yield, Y.

Because of the central role that Y plays in the relationship between biomass growth and substrate utilization, it is an intrinsic characteristic. Consequently, a clear understanding of the factors that can influence its magnitude is important. The development of such an understanding requires consideration of the energetics of microbial growth, including energy conservation and energy requirements for synthesis.

Overview of Energetics. Microorganisms require four things for growth: (1) carbon, (2) inorganic nutrients, (3) energy, and (4) reducing power. As mentioned in Section 2.2.1, microorganisms derive energy and reducing power from oxidation reactions, which involve the removal of electrons from the substrate with their ultimate transfer to the terminal electron acceptor. Consequently, the energy available in a substrate depends on its oxidation state, which is indicative of the electrons available for removal as the substrate is oxidized. Highly reduced compounds contain more electrons, and have a higher standard free energy, than do highly oxidized compounds, regardless of whether they are organic or inorganic. As described in Chapter 1, most biochemical operations are used for the removal of soluble organic matter and the stabilization of insoluble organic matter. Consequently, in this discussion we will focus on carbon oxidation by heterotrophic bacteria. Since COD is a measure of available electrons, compounds with a high COD:C ratio are highly reduced, whereas those with a low COD:C ratio are more oxidized. The carbon in methane is in the most highly reduced state possible, with a COD:C ratio of 5.33 mg COD/mg C, whereas the carbon in carbon dioxide is in the most highly oxidized state with a COD:C ratio of zero. Thus, all organic compounds will have a COD:C ratio between these extremes.

As heterotrophic bacteria oxidize the carbon in organic compounds through their catabolic pathways, they convert them to metabolic intermediates of the central amphibolic pathways that are in a higher oxidation state than either the starting compound or the biomass itself. Those metabolic intermediates are used in the anabolic pathways for cell synthesis, but since they are in a higher oxidation state than the cell material being synthesized from them, electrons must be available in an appropriate form for reducing them. Those electrons arise from the original substrate during its catabolism and are transferred to the anabolic pathways through the use of carriers such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which alternate between the oxidized (NAD

"Throughout this book, the term "yield" will be considered to be synonymous with "true growth yield."

and NADP) and the reduced (NADH and NADPH) state. Thus NAD and NADP serve as electron acceptors for catabolic reactions, forming NADH and NADPH, which act as electron donors for biosynthetic reactions. The availability of NADH and NADPH is called reducing power.

Biosynthetic reactions also require energy in a form that can be used in coupled reactions to join the amphibolic intermediates into new compounds. That energy is provided primarily by adenosine triphosphate (ATP), and to a lesser degree by other nucleotides. ATP is generated by phosphorylation reactions from adenosine diphosphate (ADP) and when the ATP is used to provide energy in biosynthetic reactions, ADP is released for reuse. ATP can be formed from ADP by two types of phosphorylation reactions: substrate level and electron transport phosphorylation. During substrate level phosphorylation, ATP is formed directly by coupled reactions within a catabolic pathway. Only small amounts of ATP can be generated in this way. Much larger amounts can be generated during electron transport phosphorylation, which occurs as electrons removed during oxidation of the substrate (and carried in NADH) are passed through the electron transport (or terminal respiratory) chain, to the terminal electron acceptor, setting up a proton-motive force." The magnitude of the proton motive force, and consequently, the amount of ATP that can be generated, depends on both the organism and the nature of the terminal electron acceptor.

An important concept to recognize about microbial energetics is that as a compound is degraded, all of the electrons originally in it must end up in the new cell material formed, in the terminal electron acceptor, or in the soluble organic metabolic intermediates excreted during growth. If a compound is mineralized, the amount of metabolic intermediates will be very small, so that essentially all electrons must end up either in the cell material formed or in the terminal acceptor. Because the yield is the amount of cell material formed per unit of substrate destroyed; because the amount of cell material formed depends on the amount of ATP generated; and because the amount of ATP generated depends on the electrons available in the substrate, the organism carrying out the degradation, and the growth environment, it follows that the yield also depends on the nature of the substrate, the organism involved, and the growth environment.

Effects of Growth Environment on ATP Generation. The electron transport chains found in most Bacteria and Eucarya share common features. They are highly organized and are localized within membranes. They contain flavoproteins and cytochromes which accept electrons from a donor like NADH and pass them in discrete steps to a terminal acceptor. All conserve some of the energy released by coupling the electron transfer to the generation of proton motive force, which drives a number of processes, such as the synthesis of ATP from ADP and inorganic phosphate, active transport, and flagellar movement. The electron transport chain in Eucarya is located in the mitochondria and is remarkably uniform from species to species. The electron transport chain in Bacteria is located in the cytoplasmic membrane and exhibits considerable variety among individual species in the identity of the individual components and in the presence or absence of sections of the chain. Nevertheless, the sequential organization of the components of the electron transport chain is determined by their standard oxidation-reduction potentials. Table 2.3 presents the potentials for the array of couples found in mitochondrial electron transport chains.2" The couples in Bacteria are similar, but not necessarily identical. The transfer is in the direction of increasing redox potential until the final reaction with the terminal

Table 2.3 The Standard Oxidation-Reduction Potentials of a Number ol Redox Couples of Interest in Hiological Systems

Ferredoxin red. oxid. NADPH, NADP NADH NAD

Flavoproteins red., oxid. Cyt. b red. oxid. Ubiquinone red. oxid.

From Kef. 211.

acceptor is catalyzed by the appropriate enzyme. When the environment is aerobic, oxygen serves as the terminal acceptor and the enzyme is an oxidase.

ATP generation is associated with the transfer of electrons down the electron transport chain through electron transport phosphorylation, although it is not directly coupled to specific biochemical reactions that occur during that transfer." Rather, the generation of ATP is driven by the proton motive force through chemiosmosis. The elements of the electron transport chain are spatially organized in the cytoplasmic membrane of Bacteria and the mitochondrial membrane of Eucarya in such a way that protons (hydrogen ions) are translocated across the membrane as the electrons move down the electron transport chain, i.e., toward more positive E,' values. In Bacteria the transfer is from the cytoplasm (inside the cell) to the periplasmic space (outside the cell); in Eucarya, from inside the mitochondria to outside. The transfer of electrons across the membrane establishes a proton gradient which causes a diffusive counterflow of protons back across the membrane through proton channels established by a membrane-bound ATPase enzyme. This proton counterflow drives the synthesis of ATP from ADP and inorganic phosphate. The number of ATP synthesized per electron transferred to the terminal acceptor depends on the nature and spatial organization of the electron transport chain because they determine the number of protons that arc translocated per electron transferred down the chain. In mitochondria, 3 ATP can be synthesized per pair of electrons transferred. However, in Bacteria the number will depend on the organization of the electron transport chain in the particular organism involved. This explains why the amount of ATP synthesized from the oxidation of a given substrate depends on the organism performing the oxidation.

In the absence of molecular oxygen, other terminal acceptors may accept electrons from the electron transport chain, and the redox potentials for them, as well as for various donors, are given in Table 2.4. " In order for ATP to be generated by electron transport phosphorylation, the oxidation-reduction potential for the donor redox couple must be smaller (more negative) than the potential for the acceptor redox couple, there must be at leasl one site of proton translocation in the electron

Table 2.4 The Standard Oxidation-Reduction Potentials of Various Acceptor and Donor Redox Couples


Fumarate/succinate + 33


NADH/NAD' -320

Lactate/pyruvate —197

Malate/oxaloacetate -172

Succinatc/fumarate + 33

transport chain between the final acceptor and the point where the donor contributes its electrons, and the associated free energy change (AG1") must exceed 44 kJ [AG"' = -2F-AE,',, where F = 96.6 kJ/(V-mol)]. Nitrate and nitrite are important terminal electron acceptors in biochemical operations performing denitrification and the bacteria capable of using the nitrogen oxides as electron acceptors are biochemically and taxonomically diverse.15 The enzyme nitrate reductase is responsible for the conversion of nitrate to nitrite. It is membrane bound and couples with the electron transport chain through a specific cytochrome b. The enzymes nitrite reductase, nitric oxide reductase, and nitrous oxide reductase are involved in the reduction of nitrite to nitrogen gas and appear to be linked to the electron transport chain through specific c-type cytochromes.2" 15 It is possible that all of the reactions are coupled to the generation of proton motive force, but the number of ATPs synthesized per electron transported is less than the number associated with oxygen as the terminal acceptor because the available free energy change is less. Consequently, bacteria growing with nitrate as the terminal electron acceptor exhibit lower yields than bacteria growing under aerobic conditions.'4"

Under strictly anaerobic conditions, i.e., when neither oxygen nor the nitrogen oxides are present, many Bacteria generate their ATP through substrate level phosphorylation associated with fermentation reactions in which the oxidation of one organic substrate is coupled to the reduction of another. The second substrate is generally a product of the catabolic pathway leading from the oxidized substrate with the result that the fermentation pathway is internally balanced, with neither a net production nor a net requirement for reducing power. Several types of fermentation reactions are listed in Table 2.5. Because ATP generation occurs only by substrate level phosphorylation and a large part of the available electrons in the original substrate end up in the reduced organic products, bacteria receive relatively little energy in this mode of growth, and thus have low yield per unit of substrate processed. As

Table 2.5 Types of

Fermentations of Various Microorganisms

Type of fermentation




Ethanol, CO.


Lactic acid

Lactic acid


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  • stefan
    Which terminal electron acceptors can have a low biomass yield?
    11 months ago

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