Chapter 3

Bacterial Growth

Raina M. Maier1.Draw a growth curve of substrate disappearance as a function of time. Label and define each stage of growth.The figure below shows an actual experiment of salicylate degradation (blue squares) and increase in cell mass (red circles). Three of the growth phases are shown, lag where essentially no substrate disappearance or cell growth occurs, exponential where the substrate rapidly disappears and the number of cells is doubled with each new generation, and stationary phase where the substrate has been used up and again there is essentially no cell growth. Note that there is no way to observe the death phase from a substrate disappearance curve.

2.Calculate the time it will take to increase the cell number from 104 CFU/ml to 108 CFU/ml assuming a generation time of 1.5 hr.The generation time is 1.5 hr, therefore determine the specific growth rate, u, from equation 3.6:

2 = eut

ln 2 = u(1.5 hr)

u = 0.46 hr-1 Plug this into equation 3.4:

ln X = ut + ln X0 ln 108 CFU/ml = (0.46 hr-1)(t) + ln 104 CFU/ml

t = 20 hr

3. You are given a microorganism that has a maximum growth rate (um) of 0.39 hr-1. Under ideal conditions (maximum growth rate is achieved), how long will it take to obtain 1 x 1010 CFU/ml if you begin with an inoculum of 2 x 107 CFU/ml?

ln X = ut + ln X0

ln 1 x 1010 CFU/ml = (0.39 hr-1)(t) + ln 2 x 107 CFU/ml

t = 15.9 hr4. Is there any way to increase the growth rate observed in question 3? There are two ways. Using the microbe you have been given, the only way to increase um would be to increase the temperature (as long as the microbe can tolerate that). Alternatively, one could find a different microbe that has a higher um.5.Write the Monod equation and define each of the constants.

u = um S

Ks + S

The constants in this equation are um and Ks.

um is the maximum growth rate that can be achieved by the microbe being studied at a defined temperature and for a particular substrate.

Ks is the half saturation constant and is defined as the substrate concentration at which growth (u) is occurring at um6.There are two special cases when the Monod equation can be simplified. Describe these cases and the simplified Monod equation that results.The first special case is when the substrate concentration (S) is much less than Ks. In this case the Monod equation simplifies to:

u = um S

KsThe second special case is when the substrate concentration (S) is much greater than Ks. In this case the Monod equation simplifies to:u = um7.List terminal electron acceptors used in anaerobic respiration in the order of preference (from an energy standpoint).See Table 3.3:

Nitrate

Manganese

Sulfate

Carbon dioxide

8.Define disproportionation.Under aerobic conditions, all of the carbon in a substrate is either incorporated into cell mass or complete oxidized to carbon dioxide to provide energy. Under anaerobic conditions, substrate carbon is either incorporated into cell mass or disproportionated into its most oxidized form (carbon dioxide) to provide energy and into its most reduced form (methane) when some of the carbon dioxide serves as a terminal electron acceptor.

9.Define the term critical dilution rate, Dc, and explain what happens in continuous culture when D is greater than Dc.Dc is the critical dilution rate in a continuous culture reactor. If the dilution rate D is greater than Dc, the cell growth rate cannot keep up with the dilution rate and cells will be washed out of the reactor with an accompanying decline in reactor operation efficiency.10.Compare the characteristics of each of the growth phases (lag, log, stationary, and death) for batch culture and environmental systems.Lag phase is generally short in a controlled batch culture system and is often simply a requirement for a physiological adjustment to a new substrate or set of growth conditions. In the environment, the lag phase is generally longer than in a controlled batch culture system. The reason for lag in the environment may be a low initial population number or even the absence of an essential gene.

Log phase in a controlled batch system is characterized by a period of rapid growth with a doubling occurring with each new generation. Scientists often optimize growth conditions to maximize the log phase to produce either cells or secondary metabolites that are economically important. In the environment, where cell numbers are relatively stable depending on the organic matter content in the system, the log phase occurs for only brief periods following addition of a new substrate or limiting nutrient.

Stationary phase in a controlled batch system is a state of no net growth although new cells are still being made while others are dying. Generally, the cell number is high and entrance into stationary phase indicates that either the substrate or an important nutrient has been used up and cells can no longer by synthesized optimally or the cell density is so high that further growth is inhibited. Often the stationary phase is an important period of growth for production of secondary metabolites. In the environment it is likely that a population (which may not even be very high in numbers) will exist in stationary phase for a very short period before entering dormancy and awaiting the next substrate pulse.

Death phase in a controlled batch system is characterized by a net loss of culturable cells due to a lack of required nutrients or a buildup of toxic by-products. In the environment, following cell growth response to an added substrate or nutrient, cell death will be observed (as a decline in the culturable numbers) until the system reaches its initial stable cell number.11. Compare and contrast the copiotrophic and oligotrophic style of life in the environment.

Oligotrophs are organisms that prefer low substrate concentrations and their growth is often characterized by low Ks values (achieve a normal growth rate a very low substrate concentrations). These are the organisms that live off slowly released residues from soil organic matter. Copiotrophs are organisms that prefer high substrate concentrations and are often characterized by higher Ks values (need higher substrate concentrations to achieve their normal growth rate). These are the organisms that take advantage of a newly added substrate, like a dead leaf or insect.