Environmental Pollution Control Microbiology - Chapter 3 ppt

Chapter 3 BACTERIA GROWTH Under the proper environmental conditions bacteria follow definite patterns of growth that are highly reproducible. The small size of bacteria and their simplicity of metabolism allow bacteria to reproduce at a rapid rate, compared to higher organisms. Bacterial growth is best examined in completely soluble substrates under optimum environmental conditions. Growth can be measured by turbidity, by direct counts in liquid media, or by plate counts on solid media. Turbidity is useful over a moderate range of bacteria growth. Turbidity can be determined as optical density at 600 nm in a single beam spectrophotometer. Turbidimetric measurements work best for growth studies in dilute nutrient media. As bacteria growth increases significantly, the errors in turbidimetric measurements increase at a rapid rate. The primary error comes from several cells located along the same light path, giving the impression of only a single cell in the turbidimetric measurement. Direct bacteria counts require dilution to reduce the number of bacteria to a reasonable level for accurate counting. Staining is normally used to permit easy observation of the cells. Fluorescent dyes have been used to permit direct counting of specific bacteria in mixed culture systems. Plate counts on solid media have proven to yield the best results and have been widely accepted for measuring bacteria growth. Experience has shown that serial dilutions to produce between 30 and 300 colonies per plate provide the best results. There is no single universal media that permits growth of all bacteria. Standardized protein and carbohydrate media have been used for isolation and growth of most common bacteria. Specialized bacteria require both specific nutrients and the proper environment. Membrane filters have been used for concentrating bacteria from dilute solutions for either direct counting or growth on specific media. Since the bacteria have definite masses, the total mass of bacteria has also been used to Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. measure growth. There is no one recommended method for evaluating bacteria growth. You use the method best suited to your particular study. Initially, bacterial growth patterns were made on batch-fed systems. A small population of bacteria was introduced into a nutrient solution and counts were made at regular intervals until the bacteria stopped growing and began to die off. Since a single pure bacterial culture was used, the numbers of bacteria produced good results. Mixtures of bacteria created problems since the different bacterial species did not grow at the same rate or use the same amount of substrate per cell. It was not always possible to distinguish the different bacteria, one from the other. Continuous flow systems were developed to examine equilibrium populations under uniform environmental conditions and produced sufficient microbial mass to allow mass units as the measure of growth. Use of the mass of bacteria allowed the study of mixed bacteria populations and pure bacterial cultures on a common basis. The mass of bacteria were separated from the liquid by vacuum filtration through pre-weighed, glass fiber filters, having maximum pore sizes of 0.2u, dried in a 103°C oven, and reweighed in an analytical balance. The change in microbial mass was determined from the weight difference. Combustion in a muffle furnace at 550°C results in loss of the organic solids, leaving the microbial ash as the weight difference. Analytical technique is very important in obtaining valid mass data. BATCH-FED GROWTH PATTERNS Initial bacteria growth patterns were observed in concentrated nutrient solutions under sterile conditions to prevent the growth of extraneous bacteria. A small sample of a pure bacteria culture was inoculated into the sterile liquid and allowed to grow over time. At regular time intervals samples were removed aseptically and plated in solid media for growth and counting. The solid media consisted of a concentrated nutrient solution with agar as the solidifying agent. Agar is a purified polysaccharide from marine algae that is not metabolized by most bacteria. Agar has the unique characteristic of remaining solid until the temperature is raised above 100°C and then not solidifying until the temperature drops below 40°C. When solid media is sterilized, the agar melts and mixes with the concentrated nutrients. When the liquid agar solution cools to about 40°C, a one ml bacteria sample, having between 30 and 300 bacteria, is added to a sterile petri dish with about 10 ml of the liquid agar solution and rapidly mixed. As the temperature drops below 40°C, the agar solidifies. The bacteria are incubated at the desired temperature for growth for a period of 24 to 48 hours. Individual bacteria produce a colony that can be seen with a low-power magnifying glass and counted. Each distinct colony represents the growth from a single bacterium. The bacteria counts can be graphically plotted against time to yield the bacteria growth curve. A typical Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. bacteria growth curve is shown in Figure 3-1. The bacteria growth curve is a complex curve with several distinct phases. The first phase of growth has been designated as the NUMBERS OF BACTERIA Lag Accelerating Death Log Death Death TIME Figure 3-1 TYPICAL BACTERIA GROWTH CURVE Lag Phase. During the lag phase the bacteria do not increase in numbers, but are adapting to metabolism of the new substrate. Once the bacteria have adapted to the substrate, they begin Log Growth. During log growth the bacteria are metabolizing at their maximum rate, doubling at a fixed interval designated as the Generation Time. Bacteria continue in the log growth phase until metabolism becomes limiting. The number of bacteria per unit volume often limits continued growth at the log rate. Metabolic end products, accumulating in the liquid around the bacteria, can slow the rate of metabolism by applying backpressure shift from log growth. As metabolism slows, growth shifts to Declining Growth. Eventually, the numbers of bacteria reach a maximum and enter the Stationary Phase where the bacteria numbers remain constant for a long period of time. As the bacteria begin to die, they enter a period of Accelerating Death. The rate of dying soon reaches the Log Death rate. Finally, the rate of dying slows as the bacteria reach the final Death Phase. The graph shown in Figure 3-1 is a generalized graph of the numbers of bacteria using both numbers and time on linear scales. Plotting the growth data on semi-log graph paper with the numbers of bacteria shown on the semi-log scale and time on the linear scale, the log growth and the log death phases will plot as straight lines. Figure 3-2 shows the semi-log plot of the bacteria growth pattern. The declining growth phases and accelerating death phases tend to be compressed with the log Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. scale plot while illustrating the full ranges of the log growth and log death phases. LOG NUMBERS OF BACTERIA Declining Stationary AcceteraBng TIME Figure 3-2 SEMI-LOG PLOT OF BACTERIA GROWTH CURVE In concentrated nutrient substrates the initial growth will be aerobic until the dissolved oxygen (DO) has all been used. Metabolism shifts from aerobic to anaerobic with accumulation of end products that ultimately limits metabolism. Aerobic conditions can be maintained by growing the bacteria in low nutrient concentrations or in thin liquid layers in Erlenmeyer flasks rather than in test tubes or bottles, which have a small surface area to volume ratio. Using thin layers of liquid media in Erlenmeyer flasks on a shaking apparatus can insure aerobic conditions during the growth cycle. Completely anaerobic conditions require purging the media with nitrogen to strip the oxygen, as the first step, and then growth in an anaerobic jar or in an anaerobic chamber. Oxygen can also be removed chemically to produce an atmosphere of nitrogen. Jacques Monod was one of the first bacteriologists to quantitatively examine the growth of bacteria in dilute organic solutions. His research was published in France in 1942. Because of World War II, it was 1949 before Monod could publish his research in English. Monod's original study is considered a classic in the microbiological literature. Monod used turbidity as the measure of bacterial growth and converted turbidity data to weight of bacteria. By growing bacteria in different concentrations of a simple organic substrate, Monod found that the maximum quantity of bacterial growth was directly proportional to the initial organic concentration. Since the plot of cell mass against organic substrate concentration passed through zero, it was concluded that the bacteria did not require any maintenance energy. Another part of his study showed that the total mass of Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. bacteria produced in a sample that was agitated was the same as a sample that was not agitated. The agitated sample reached its maximum concentration faster than the unagitated sample. The same growth was also taken to suggest that the bacteria did not require maintenance energy during growth. Monod's failure to observe maintenance growth, i.e. endogenous respiration, effectively put a damper on this important concept of bacterial growth for many years. The most significant aspect of Monod's research was establishing the fact that the rate of bacteria growth was a function of the substrate concentration up to a specific concentration where the rate of growth became a constant with increased substrate concentrations. An important part of Monod's work was the introduction of quantitative relationships to predict the rate of microbial growth. He developed a series of equations that could be used to predict the amount of bacteria mass, produced from the metabolism of specific amounts of organic nutrients. Monod's equations started with the previously observed relationship for log growth where the rate of growth was a function of the microbial mass and substrate nutrients were always in excess. The basic log growth relationship for bacteria, based on numbers of bacteria, has been expressed as follows. dN/dt=u,N (3-1) where: N = number of bacteria t = time, hrs. u. = specific growth rate, 1/hr. Solving Equation 3-1 for the number of bacteria results in Equation 3-2. N = N 0 (e^') (3-2) where: N = number of bacteria at time t. N 0 = number of bacteria at initial time, t = 0. The numbers of bacteria increase very rapidly during log growth. TYPICAL CALCULATIONS: E. coli at 37° C has a specific growth rate of about 3/hr If we started with one (1) E. coli, in one hour we would expect N = (l)(e (3X1) )= 20 bacteria. Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. In 10 hrs, the number of bacteria would be N = (1) (e (3X10) ) = 1.07 x 10" bacteria. If E. coli continued to grow at a log rate, it would quickly reach an overwhelming number. It had long been observed that microbial growth slowed when the nutrient substrate became limiting. The shift from log growth to declining growth intrigued Monod. Since the bacteria were growing as fast as they could, Monod recognized that the specific growth rate, u, shifted from being a constant to being a variable that was related to the remaining substrate concentration. He found that the value of (j, could be expressed in terms of substrate concentration, Equation 3-3. Monod determined that Umax for E. coli occurred above 25 mg/L glucose with the value of K s being 4 mg/L glucose. The maximum specific growth rate, \^ mm , was a constant that could be measured in an excess of substrate during log growth. The saturation constant, K s , was measured experimentally as S)) (3-3) where: \\ mai = maximum specific growth rate, 1/hr. S = substrate concentration, mg/L K s = saturation constant, substrate concentration when U, = O.SlVax, mg/L. the substrate and the specific growth rate decreased. Substitution of these data in the initial equation showed \i. would be O.Sd^w for 25 mg/L glucose. At 50 mg/L glucose, |i would be O^u^. Yet, Monod's data showed a constant growth rate at 25 mg/L glucose and higher. The differences in the measured data and the calculated results reflect the limitations of the empirical equation developed by Monod. The Monod equation should be recognized as an approximation of the data and not as a precise equation to predict the total range of data. The data gave the best results for the equation when the substrate concentration was close to K^. As the data approached both ends of the equation, the errors increased. It is important to understand the limitations of published equations if they are to be properly applied. When equations become common and are published over and over, the limitations of the equations tend to be overlooked. A second equation, Equation 3- 4, developed by Monod showed that the amount of bacteria growth was related to the substrate metabolized. dx/dt = Y(dS/dt) (3-4) Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. where: dx/dt = rate of bacteria mass growth, mg/L/hr Y = yield factor, mg bacteria mass/mg substrate metabolized dS/dt = rate of substrate metabolism, mg/L/hr Monod's data for E. coli and glucose showed a yield factor, Y, of 0.233 mg E. colilmg glucose metabolized at glucose concentrations between 25 mg/L and 200 mg/L. His data on Bacillus subtlis gave a yield factor, Y, of 0.218 mg B. subtilis/mg sucrose metabolized. These two different bacteria had similar yield factors on two related sugars. Later studies showed that these values were low, indicating that the substrate used by Monod may have been oxygen or nutrient deficient. CONTINUOUS FEED GROWTH PATTERNS Monod's next major contribution occurred in 1950, when he published his study on continuously fed bacteria growth systems. Monod developed a completely mixed bioreactor that could be maintained under aerobic conditions. By feeding a low concentration of substrate continuously, he found that the growth of bacteria was related to the fluid displacement time as long as it was greater than the generation time of the bacteria. Monod believed that when the fluid displacement was less than the generation time of the bacteria, the bacteria would be completely washed out of the system and there would be no bacterial growth. The effluent nutrient concentration would be the same as the influent nutrient concentration. Monod developed the following mathematical relationship between the rate of bacterial growth in the bioreactor and the displacement rate of bacteria from the bioreactor, Equation 3-5. dx/dt = (u D)x (3-5) where: x = microbial concentration in the bioreactor, mg/L. u = specific growth rate, 1/hr. D = displacement rate, Q/V or 1/t, 1/hr. t = time, hrs. Q = substrate flow rate, L/hr V = bioreactor volume, liters (L) The growth of bacteria will increase until the system comes to equilibrium. At equilibrium the rate of change in bacteria mass in the bioreactor is zero, dx/dt = 0. From the above equation, this means that either x or (\JL - D) must be zero. Since the microbial mass concentration, x, is not zero, (jj, - D) must be zero. This means that u = D at equilibrium. The rate of bacteria growth in a completely mixed, Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. continuously fed bioreactor is a function of the fluid retention time; i.e., p, = 1/t, until the retention period is so short that the bacteria can no longer divide before being washed from the system. Examination of the completely mixed bioreactor shows that the substrate will be removed by metabolism and by displacement. Metabolism results in part of the substrate being oxidized for energy and a corresponding part being converted to cell mass. Displacement results in loss of unmetabolized substrate in the effluent. Monod developed the following equation, Equation 3-6, to measure the rate of change in the substrate concentration in the bioreactor. dS/dt = D(S 0 -S)-(l/Y)(dx/dt) (3-6) where: dS/dt = rate of substrate metabolism, mg/L/hr dx/dt = rate of bacteria growth, mg/L/hr S = substrate concentration in bioreactor, mg/L S 0 = influent substrate concentration, mg/L Y = yield factor, mg/L x produced/mg/L S metabolized t = time, hrs. This equation can be solved for several variables. The concentration of bacteria in the bioreactor at equilibrium can be determined from Equation 3-7. x = Y(S 0 -S) (3-7) In effect, the bacteria concentration depends on the substrate removal and the efficiency of converting substrate into cell mass. When the residual substrate is quite small compared with the influent substrate, the cell mass concentration can be written as a function of the influent substrate, Equation 3-8. x = Y(S 0 ) (3-8) Determination of the residual substrate was made using Monod's original relationship for u at low substrate concentrations in Equation 3-9. S = KyOwt-l) (3-9) Equation 3-9 shows that the residual substrate in the treated effluent is related directly to the dilution rate D, the reciprocal of the fluid displacement time, 1/t. For a given set of bacteria and substrate, both Kj and (v^ are constants at a specific temperature. At long hydraulic retention times there will be very little unmetabolized substrate. As the product of (Vix and t approaches one, the Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. concentration of unmetabolized substrate will increase rapidly until the substrate concentration reaches the influent substrate concentration and growth stops. Both of Monod's studies have had a profound impact on the quantitative aspects of bacteriological growth at low organic substrate concentrations. The basic nature of these studies is such that many investigators are still using them today. It is important to recognize both the value and the limitations of Monod's equations if they are to be properly applied. Monod developed his equations from laboratory data and fundamental concepts. He demonstrated that the growth rate of bacteria becomes a function of the substrate concentration when substrate is limiting. He demonstrated that growth of bacteria was directly proportional to the substrate metabolism. His research also showed that the bacteria in continuous flow reactors always grew at log rates. On the negative side, there is the limitation of the specific growth rate factor, p,, at high and low substrate concentrations. Monod's failure to observe the effect of maintenance energy invalidates his mass calculations except at short detention periods. Monod's data on cell yield per unit substrate indicated that his substrate was limited in trace nutrients. Trace nutrients are essential for the maximum cell yield per unit substrate metabolized. Available oxygen may also have been limiting. The extended value of Monod's research was the stimulation of other investigators to carry out additional research on food limiting conditions. This is very important for environmental microbiologists working in the area of biological wastewater treatment. All of the important biological wastewater treatment systems operate under food limiting conditions. TYPICAL CALCULATIONS: An aerobic bioreactor, fed 1,000 mg/L glucose and containing E. coli, operated with a 6 hr retention period. 1. Determine the specific growth rate of the E. coli in the bioreactor. H = D=l/t=l/6 = 0.17/hr 2. Determine the maximum mass of E. coli. Information from Chapter 2 indicated Y = 0.49 mg VSS/mg glucose x = Y(S 0 ) = 0.49(1000) = 490 mg/1 VSS 3. Determine effluent glucose concentration. Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. K s = 4.0 mg/L glucose and (VK = 3.0/hr S = KS/CIVK t - 1) = 4.0/((3)(6) - 1) = 4.0/(1 8 - 1) = 0.24 mg/L glucose Monod's equations indicate that most of the glucose would have been metabolized with 6 hrs aeration. 4. Determine the effluent glucose concentration with one hr retention period. 5 = 4.0/((3)(1) - 1) = 4.0/2 = 2.0 mg/1 glucose Monod's equation indicates 99.8% glucose metabolism. With the same microbial mass the key would lie in the ability of the system to transfer sufficient oxygen for aerobic metabolism. 5. Determine the oxygen demand rate at both 6 hrs aeration and one hr aeration. Glucose has a COD of 1.07 mg COD/mg glucose hi Chapter 2 metabolism of glucose used 0.38 for energy 6 Hrs Aeration: Oxygen Used = 1.07(1000 - 0.24) (0.38)76 = 68 mg/L/hr for energy 1 Hr Aeration: Oxygen Used = 1 .07(1000 - 2) (0.38)71 = 406 mg/L/hr for energy The rate of oxygen demand increases rapidly as the detention time in the bioreactor is shortened. If the system is unable to transfer the oxygen, oxygen transfer will be the limiting factor controlling metabolism. Monod's equations were developed for aerobic systems with the substrate limiting. Several studies followed publication of Monod's research on continuous flow systems. One of the more detailed studies was made by Herbert, Elsworth and Telling in 1956. Their study attempted to determine the validity of Monod's theory and to show how easily continuously fed studies could be made. They used a 20- liter bioreactor fed 2,500 mg/L glycerol as the organic substrate and Aerobacter cloacae as the bacteria. Batch fed growth studies on this substrate and organism produced a u^ of 0.85/hr, a K s value of 12.3 mg/L and a Y value of 0.53 mg Copyright 2004 by Marcel Dekker, Inc. All Rights Reserved. [...]... Equation 3- 1 5 Carbonic CO2 + H2O -> - H2C 03 ( 3- 1 5) acid has two ionization constants, K! = 4 .30 x 10"7 and K2 = 5.61 x 10"11 The initial ionization causes the carbonic acid to break into hydrogen ions and bicarbonate ions, as shown in Equation 3- 1 6 The second ionization results in the bicarbonate H2CO3 -> H+ + HCCV ( 3- 1 6) ion breaking into hydrogen ions and carbonate ions, as shown in Equation 3- 1 7 -> H+... shown in Equation 3- 1 1 When a strong base, such as sodium HCl-J-I-r + Cr ( 3- 1 1) hydroxide is added to water, it ionizes completely with the formation of hydroxide ions, as shown in Equation 3- 1 2 On the other hand, weak acids such as organic NaOH - Na+ + OH" ( 3- 1 2) acids, do not ionize completely Acetic acid, C2H4O2, ionizes to form hydrogen ions and acetate ions, as shown in Equation 3- 1 3 Weak organic... solution, sodium bicarbonate is produced, as shown in Equation 3- 1 8 NaOH + H2CO3 -> NaHCO3 + HOH Copyright 2004 by Marcel Dekker, Inc All Rights Reserved ( 3- 1 8) Further addition of sodium hydroxide would push the bicarbonates to carbonates, as shown in Equation 3- 1 9 The pH is pushed higher as carbonates are formed NaOH + NaHCOj -> Na2CO3 + HOH ( 3- 1 9) The addition of carbon dioxide to the carbonates would... starts as ionized phosphoric acid as shown in Equation 3- 2 1 Phosphoric acid has three hydrogen atoms that ionize at different pH levels The three ionization constants for H3PO4 - H+ + H2PO4" -> H+ + HPO4= -> • H* + PO4 '3 Copyright 2004 by Marcel Dekker, Inc All Rights Reserved ( 3- 2 1) phosphoric acid are K, = 7.5 x 10 '3, K2 = 6.2 x 10"8, and K3 = 4.8 x 10' 13 At pH 7.0 the dihydrogen phosphate ions are 7,500... the pH close to the 7 - 8 range There are other weak bases and weak acids than the carbonate system Weak bases are primarily ammonia related compounds, such as the amines The amino group can absorb hydrogen ions, pushing the pH higher The addition of hydrogen ions causes the amine to become positively charged as shown in Equation 3- 2 0 for methyl amine CH3NH2 + H+ -> • CH3NH3+ ( 3- 2 0) Amino acids are important... unionized concentration remaining in solution, as shown in Equation 3- 1 4 The handbook shows that the ionization constant, K, Copyright 2004 by Marcel Dekker, Inc All Rights Reserved K = [H+] [CH3COO"]/[CH3COOH] ( 3- 1 4) for acetic acid is 1 75 x 1 0~5 at 25° C At a pH of 7.0 the hydrogen ion concentration is 1.0 x 10"7 From Equation 3- 1 3 the acetate ions are 175 times the acetic acid concentration For... bicarbonate, react with the organic acids, as shown in Equation 3- 2 2, to form acid salts with the release of carbon dioxide The pH will be depressed slightly as carbonic acid is formed When the organic acid salts are metabolized, sodium bicarbonate is reformed to NaHCO3 + CH3COOH -> CH3COONa + CO2 + HOH ( 3- 2 2) maintain the pH at the proper level In some batch-fed experiments, pH data collected at the start of... concentration as shown in Equation 3- 9 The pH scale provides an easy to use number for hydrogen ion ( 3- 9 ) concentration The pH scale operates from 0 to 14 with 0 to 7 being acidic and 7 to 14 being basic for water The pH scale was derived from the ionization of water as shown in Equation 3- 1 0 The hydrogen ion concentration decreases as the pH rises HOH . as follows. dN/dt=u,N ( 3- 1 ) where: N = number of bacteria t = time, hrs. u. = specific growth rate, 1/hr. Solving Equation 3- 1 for the number of bacteria results in Equation 3- 2 . N = N 0 (e^') . the following equation, Equation 3- 6 , to measure the rate of change in the substrate concentration in the bioreactor. dS/dt = D(S 0 -S )-( l/Y)(dx/dt) ( 3- 6 ) where: dS/dt = rate of . Equation 3- 8 . x = Y(S 0 ) ( 3- 8 ) Determination of the residual substrate was made using Monod's original relationship for u at low substrate concentrations in Equation 3- 9 . S