Maintenance Endogenous Metabolism Decay Lysis and Death

The yield values in the preceding section are those that result when all energy obtained by the biomass is being channeled into synthesis. Energy for synthesis is not the only energy requirement for microorganisms, however. They must also have energy for maintenance.'"

Cellular processes, whether mechanical or chemical, require energy for their performance, and unless a supply is available these essential processes will cease and the cell will become disorganized and die. Mechanical processes include motility, osmotic regulation, molecular transport, maintenance of ionic gradients, and in the case of some Eucarya, cytoplasmic streaming. While it might be argued that motility can be dispensed with in some microorganisms, this argument would not hold for all because some require motility to find food. Osmotic regulation is quite important in all cells, even those protected by a rigid cell wall, and pump mechanisms, such as contractile vacuoles, exist in cells to counteract the normal tendency of osmotic pressure to pump water into them. Cell membranes are permeable to many small molecules, such as amino acids, and because of the high concentrations within the cell these tend to diffuse into the medium. Active transport mechanisms operate to bring such molecules into the cell against the concentration gradient. Of a similar nature is the necessity for maintaining an ionic gradient across the cell membrane, which is closely linked to the proton motive force responsible for ATP synthesis. Maintenance of this gradient is thought to be a major consumer of maintenance energy.71 Finally, cytoplasmic streaming and the movement of materials within Eu-carya are often required for their proper functioning. They also require energy.

Chemical factors also contribute to maintenance energy needs. Microbial cells represent chemical organization and many of the components within them have higher free energies than the original compounds from which they were formed. In general, because of this organization, energy must be available to counteract the normal tendency toward disorder, i.e., to overcome entropy. The chemical processes contributing to the energy requirement for maintenance arc those involved in resyn-thesis of structures such as the cell wall, flagella, the cell membrane, and the catabolic apparatus. For example, one study1'' suggested that energy for the resynthesis of proteins and nucleic acids was an important portion of the maintenance energy requirement for Escherichia coli.

A major point of controversy in the microbiological literature has concerned the impact on the maintenance energy requirement of the rate at which a culture is growing. Early investigations"" suggested that the need for maintenance energy was independent of growth rate, but more recent research indicates the opposite. 1 Nevertheless, engineers generally consider maintenance energy needs to be independent of growth rate in biochemical operations for wastewater treatment and that is the approach that will be adopted in this book.

Given the existence of a need for maintenance energy, what energy sources can be used to supply it? The answer to that question depends on the growth conditions of the microorganisms. If an external (exogenous) energy supply is available, a portion of it will be used to meet the maintenance energy requirement and the remainder will be used for synthesis. As the rate of energy supply is decreased, less and less will be available for new growth and thus the net, or observed, yield will decline. When the point is reached at which the rate of energy supply just balances the rate at which energy must be used for maintenance, no net growth will occur because all available energy will be used to maintain the status quo. If the rate of energy supply is reduced still further, the difference between the supply rate and the maintenance energy requirement will be met by the degradation of energy sources available within the cell, i.e., by endogenous metabolism. This will cause a decline in the mass of the culture. Finally, if no exogenous energy source is available, all of the maintenance energy needs must be met by endogenous metabolism. When the point is reached at which all endogenous reserves have been exhausted, the cells deteriorate and die, or enter a resting state.

The nature of the materials serving as substrates for endogenous metabolism depends on both the species of the microorganism and the conditions under which the culture was grown. For example, when E. coli is grown rapidly in a glucose-mineral salts medium it stores glycogen.1" If those cells arc then placed in an environment devoid of exogenous substrate they will utilize the glycogen as an endogenous energy source. Amino acids and proteins show little net catabolism until the glycogen is gone. When grown in tryptone medium, on the other hand, E. coli accumulates little glycogen. As a result, endogenous metabolism utilizes nitrogenous compounds immediately. Other organisms use still other compounds, including ribonucleic acid (RNA) and the lipid poly-(3-hvdroxybutyrate (PHB).

One question that has intrigued microbiologists concerns the route of energy flow when sufficient exogenous substrate is available to supply the maintenance energy requirements of the culture. Does endogenous metabolism continue under those circumstances so that part of the energy released from degradation of the substrate is used to resupplv Ihe energy reserves being degraded by endogenous metabolism? Or, alternatively, does endogenous metabolism cease so that the energy released from degradation of the exogenous substrate goes directly for maintenance functions? The evidence is still not conclusive. Actually, although such questions are of fundamental scientific significance, they have liltle bearing on the macroscopic energy balances used by engineers to mathematically model biochemical operations. In fact, as we see in Section 3.3.2. some models avoid the entire issue by introducing the concept of cell lvsis and regrowth.

The amount of biomass actually formed per unit of substrate used in a biochemical operation, referred to as the observed yield (Y„K). is always less than Y. One reason for this is the need for maintenance energy. The more energy that must be expended for maintenance purposes, Ihe less available for synthesis and the smaller the quantity of biomass formed per unit of substrate degraded. Other f actors also contribute to the difference, however. For example, consider the effect of predation. In a complex microbial community such as that found in the activated sludge process, protozoa and other Eucarya prey on the bacteria, reducing the net amount of biomass formed. To illustrate the effect of predation. assume that the value of Y lor bacteria growing on glucose is ().(•>() mg bacterial biomass COD formed per mg of glucose COD used. Thus, if 100 mg/L of glucose COD were used. 60 mg I. of bacterial biomass COD would result. Now assume that the value of Y for protozoa feeding on bacteria is 0.70 mg protozoan biomass COD formed per nig of bacterial biomass COD used. If the protozoa consumed all of the bacteria resulting from the glucose, the result would be 42 mg/L of protozoan biomass. As a consequence, if we observed only the net amount of biomass formed, without distinction as to what it was, we would conclude that 42 mg/L of biomass COD resulted from the destruction of 100 mg/L of glucose COD. Therefore, we would conclude that the observed yield was 0.42. which is less than the true growth yield for bacteria growing on glucose. Macroscopically, it is impossible to distinguish between the v arious factors acting to make the observed yield less than the true growth yield. Consequently, environmental engineers lump fliem together under the term "microbial decay," which is the most common way they have modeled their effect in biochemical operations."'

Another process leading to a loss of biomass in biochemical operations is cell lysis.1 The growth of bacteria requires coordination of the biosynthesis and degradation of cell wall material to allow the cell to expand and divide. The enzymes responsible for hydrolysis of the cell wall are called autolysins and their activity is normally under tight regulation to allow them to act in concert with biosynthetic enzymes during cell division. Loss of that regulation, however, will lead to rupture of the cell wall (lysis) and death of the organism. When the cell wall is ruptured, the cytoplasm and other internal constituents are released to the medium where they become substrates for other organisms growing in the culture. In addition, the cell wall and cell membranes, as well as other structural units, begin to be acted upon by hydrolytic enzymes in the medium, solubilizing them and making them available as substrates as well. Only the most complex units remain as ccll debris, which is solubilized so slowly that it appears to be refractory in most biochemical operations.4^4 The arguments for how lysis results in the loss of biomass are similar to those associated with prédation, illustrated above. The yield exhibited by bacteria growing on the soluble products released by lysis is of the same magnitude as the yield associated with growth on other biogenic substrates. Consequently, if 100 mg/L of biomass is lysed, only 50-60 mg/L of new biomass will result from re-growth on the lysis products. Thus, the net effect of lysis and regrowth is a reduction in biomass within the system. In general, starvation itself does not initiate lysis, although the events that trigger it are not yet clear. Nevertheless, engineers seeking to model the decline in observed yield associated with situations in which the microbial community is growing slowly have focused on cell lysis as the primary mechanism.12 2"

The final event impacting on the amount of active biomass in a biochemical operation is death. Traditionally, a dead cell has been defined as one that has lost the ability to divide on an agar plate"2 and studies based on this definition have shown that a large proportion of the microorganisms in slowly growing cultures are nonviable, or dead.'1 "' 4 In addition, as summarized by Weddle and Jenkins,Ml a large number of studies using indirect evidence involving comparisons of substrate removal rates and enzyme activities have concluded that large portions of the MLSS in wastewater treatment systems are inactive. However, a later study,4"41 using more sophisticated techniques for identifying dead bacteria, has suggested that a very low fraction of the cells present at low growth rates are actually dead. Instead, many are simply nonculturable by standard techniques, although they are still alive. Furthermore, the more recent work4" suggests that dead cells do not remain intact for long, but rather lyse, leading to substrates and biomass debris, as discussed above. The presence of biomass debris acts to make the mass of viable microorganisms less than the mass of suspended solids in the system. Even though the predecessor of this book used a model1'' that explicitly considered cell death, it now appears that direct consideration of the phenomenon is not warranted.4"41 Rather, the fact that only a portion of the MLSS in a biological wastewater treatment system is actually viable biomass can be attributed to the accumulation of biomass debris rather than to the presence of dead cells.

In summary, as a result of several mechanisms, biochemical reactors exhibit two important characteristics: (1) the observed yield is less than the true growth yield and (2) active, viable bacteria make up only a fraction of the "biomass." One simplified conceptualization of the events leading to these characteristics is that bacteria are continually undergoing death and lysis, releasing organic matter to the environment in which they are growing. Part of that organic matter is degraded very, very slowly, making it appear to be resistant to biodégradation and causing it to accumulate as biomass debris. As a consequence, only a portion of the "biomass" is actually viable cells. The remainder of the released organic matter is used by the bacteria as a food source, resulting in new biomass synthesis. However, because the true growth yield is always less than one, the amount of new biomass produced is less than the amount destroyed by lysis, thereby making the observed yield for the overall process less than the true growth yield on the original substrate alone.

2.4.3 Soluble Microbial Product Formation

Much of the soluble organic matter in the effluent from a biological reactor is of microbial origin and is produced by the microorganisms as they degrade the organic substrate in the influent to the bioreactor. The major evidence for this phenomenon has come from experiments in which single soluble substrates of known composition were fed to microbial cultures and the resulting organic compounds in the effluent were examined for the presence of the influent substrate."' The bulk of the effluent organic matter was not the original substrate and was of higher molecular weight, suggesting that it was of microbial origin. These soluble microbial products are thought to arise from two processes, one growth-associated and the other non-growth-associated. Growth-associated product formation results directly from biomass growth and substrate utilization. As such, it is coupled to those events through another yield factor, the microbial product yield, YMl., and the biodégradation of one unit of substrate results in the production of YM1, units of products. Values of Ysl,. for a variety of organic compounds have been found to be less than 0.1."' Non-growth-associated product formation is related to decay and lysis and results in biomass-associated products. They are thought to arise from the release of soluble cellular constituents through lysis and the solubilization of particulate cellular components. Although little is known about the characteristics of these two types of soluble microbial products, they are thought to be biodegradable, although some at a very low rate. Compared to other aspects of biochemical operations, little research has been done on the production and fate of soluble microbial products and few researchers have attempted to model the contribution of such products to the organic matter discharged from wastewater treatment systems."'Ml Nevertheless, an awareness of their existence is necessary for an accurate understanding of the response of biochemical operations.

2.4.4 Solubilization of Particulate and High Molecular Weight Organic Matter

Bacteria can only take up and degrade soluble organic matter of low molecular weight. All other organic material must be attacked by extracellular enzymes that release low molecular weight compounds that can be transported across cellular membranes. Many organic polymers, particularly those of microbial origin, such as cell wall components, proteins, and nucleic acids, are composed of a few repeating subunits connected by bonds that can be broken by hydrolysis. Consequently, the microbial process of breaking particulate and high molecular weight soluble organic compounds into their subunits is commonly referred to as hydrolysis, even though some of the reactions involved may be more complicated.

Hydrolysis reactions play two important roles in biochemical reactors for wastewater treatment. First, they are responsible for the solubilization of cellular components released as a result of cell lysis, preventing their buildup in the system. Because cell lysis occurs in all microbial systems, hydrolysis reactions are even important in bioreactors receiving only soluble substrate. Second, many biochemical operations receive particulate organic material, in which case hydrolysis is essential to bring about the desired biodégradation. In spite of its central position in the functioning of biochemical operations, relatively few studies have sought to under stand the kinetics and mechanisms of hydrolysis."1:1 Nevertheless, it has important impacts on the outcome of biochemical operations and must be considered for a complete understanding of their functioning.

2.4.5 Ammonification

Ammonification is the name given to the release of ammonia nitrogen as amino acids and other nitrogen containing organic compounds undergo biodégradation. It occurs as a normal result of the biodégradation process, during which amino groups arc liberated and excreted from the cell as ammonia. The rate of ammonification will depend on the rate of nitrogen containing substrate utilization and the carbon to nitrogen ratio of that substrate. Ammonification is very important in wastewater treatment processes for nitrogen control because organic nitrogen is not subject to oxidation by nitrifying bacteria. They can only oxidize nitrogen to nitrate after it has been converted to ammonia and released to the medium.

2.4.6 Phosphorus Uptake and Release

If a suspended growth bioreactor system is configured as two zones in series with the first zone anaerobic and the second aerobic, PAOs, which possess a special metabolic capability not commonly found in other bacteria, will proliferate and store large quantities of inorganic phosphate as polyphosphate, thereby allowing phosphorus removal from the wastewater via biomass wastage. Although PAOs are often present in significant numbers in totally aerobic suspended growth cultures, they only develop the ability to store large quantities of phosphate when they are subjected to alternating anaerobic and aerobic conditions by being recycled between the two zones.'7 This follows from their unique capability to store carbon at the expense of phosphate under anaerobic conditions and to store phosphate at the expense of carbon under aerobic conditions. Two scenarios have been postulated to explain the functioning of PAOs. One was developed independently by Comeau et al." and Wentzel et al.,K" whereas the other was developed by Arun et al.: The former is referred to as the Comeau-Wentzel model whereas the latter is called the Mino model.K1 The difference between the two models is the result of the metabolic diversity among PAOs, and since it is not yet known which model is the more generally applicable, both will be presented.

Comeau-Wentzel Model. We will first consider the events occurring in the anaerobic zone. Because of fermentations that occur in sewers, much of the soluble organic matter in domestic wastewater is in the form of acetate and other short chain fatty acids. Furthermore, when the wastewater enters an anaerobic bioreactor, additional quantities of fatty acids are formed by fermentative reactions performed by facultative heterotrophs. As indicated in Figure 2.5A, acetate is transported across the cell membrane by passive diffusion (as undissociated acetic acid), but once inside, it is activated to acetyl-CoAby coupled ATP hydrolysis, yielding ADP. Although not shown in the diagram, ATP is also used to maintain the proton motive force that has been lost by transport of the proton associated with the undissociated acetic acid. The cell responds to the decreasing ATP/ADP ratio by stimulating ATP resynthesis from stored polyphosphate (Poly-P„). A portion of the acetyl-CoA is metabolized through the TCA cycle to provide the reducing power (NADH + H ) required for

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A Anaerobic B. Aerobic

Figure 2.5 Schematic diagram depicting the Comeau-Went/el model lor the uptake and release of inorganic phosphate by PAOs: A. Anaerobic conditions; B. aerobic conditions. (Adapted from Went/el et al.sl)

the synthesis of PHB. The remainder of the acetyl-CoA is converted into PHB, with about 90% of the acetate carbon being conserved in that storage polymer. Without the presence of the polyphosphate to provide energy for ATP resynthesis. acetate would build up in the cell, acetate transport would stop, and no PHB formation would occur. The hydrolysis of the polyphosphate to form ATP increases the intracellular concentration of inorganic phosphate, P„ which is released to the bulk solution, along with cations (not shown) to maintain charge balance.

When the wastewater and the associated biomass enter the aerobic zone, the wastewater is low in soluble organic matter, but the PAOs contain large PHB reserves. Furthermore, the wastewater is rich in inorganic phosphate, while the PAOs have low polyphosphate levels. Because they have oxygen as an electron acceptor in the aerobic zone, the PAOs perform normal aerobic metabolism for growth by using the stored PHB as their carbon and energy source, generating ATP through electron transport phosphorylation, as illustrated in Figure 2.5B. Furthermore, as the ATP-ADP ratio increases, polyphosphate synthesis is stimulated, thereby removing phosphate and associated cations (not shown) from solution and regenerating the stored polyphosphate in the cells. Because of the large amount of energy provided by the aerobic metabolism of the stored PHB, the PAOs are able to take up all of the phosphate released in the anaerobic zone plus the phosphate originally present in the wastewater.

The continual cycling between the anaerobic and aerobic zones gives the PAOs a competitive advantage over ordinary heterotrophic bacteria, because without the capability to make and use polyphosphate, the ordinary heterotrophs are not able to take up organic matter in the anaerobic zone. It should be noted that while most systems that remove phosphate through the use of PAOs employ aerobic zones for the regeneration of the stored polyphosphate, some PAOs can use nitrate and nitrite as alternative electron acceptors,' allowing anoxic conditions to be used as well.

Mino Model. The Mino model, illustrated in Figure 2.6. is very similar to the Comeau-Wentzel model, the major difference being the role of glycogen, a carbohydrate storage polymer. In this case, in the anaerobic zone the reducing power required for synthesis of PHB from acetyl-CoA comes from the metabolism of glucose released from the glycogen. Glucose is oxidized to pyruvate through the Entner-Doudoroff (ED) or Embden-Meyerhof-Parnas (EMP) pathway, depending in the type of PAO, thereby providing some of the ATP required to convert acetate to acetyl-CoA and some of the reducing power needed for PHB synthesis. Pyruvate, in turn, is oxidative]}' decarboxylated to acetyl-CoA and carbon dioxide, with the electrons released also being used in the synthesis of PHB. Thus, all of the acetate taken up is stored as PHB, as is part of the carbon from the glycogen. In the aerobic zone, PHB is broken down as in the Comeau-Wentzel model to provide for biomass synthesis as well as for phosphate uptake and storage as polyphosphate. In addition, however, PHB is also used to replenish the stored glycogen.

2.4.7 Overview

A diagram depicting the overall sum of the events occurring in an aerobic bioreactor receiving a soluble substrate is shown in Figure 2.7. Bacteria consume the substrate (Ssl) and grow, leading to more bacteria, with the relationship between substrate consumption and biomass growth being given by the true growth yield, Y. There

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