Chapter 3 Bacterial Growth Raina M. Maier 1. 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 CF !ml to 10" CF !ml assuming a generation time of 1.# hr. The generation time is 1. hr, therefore determine the specific growth rate, u, from equation !."# $ % eut ln $ % u(1. hr) u % &.'" hr(1 )lug this into equation !.'# ln * % ut + ln *& ln 1&, -./0ml % (&.'" hr(1)(t) + ln 1&' -./0ml

t % $& hr $. %ou are given a microorganism that has a ma&imum growth rate 'um( of 0.$) hr*1. nder ideal conditions 'ma&imum growth rate is achieved(+ how long will it take to obtain 1 & 1010 CF !ml if ,ou begin with an inoculum of 2 & 10- CF !ml. ln * % ut + ln *& ln 1 x 1&1& -./0ml % (&.!1 hr(1)(t) + ln $ x 1&2 -./0ml t % 1 .1 hr 4. /s there an, wa, to increase the growth rate observed in 0uestion $. There are two ways. /sing 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). 3lternatively, one could find a different microbe that has a higher um. #. 1rite the 2onod e0uation and define each of the constants. u % um 4 5s + 4

The constants in this equation are um and 5s. um is the maximum growth rate that can be achieved by the microbe being studied at a defined temperature and for a particular substrate. 5s is the half saturation constant and is defined as the substrate concentration at which growth (u) is occurring at 6 um 3. 4here are two special cases when the 2onod e0uation can be simplified. Describe these cases and the simplified 2onod e0uation that results. The first special case is when the substrate concentration (4) is much less than 5s. 7n this case the 8onod equation simplifies to# u % um 4 5s

The second special case is when the substrate concentration (4) is much greater than 5s. 7n this case the 8onod equation simplifies to# u % um -. List terminal electron acceptors used in anaerobic respiration in the order of preference 'from an energ, standpoint(.

4ee Table !.!# Nitrate 8anganese 4ulfate -arbon dioxide ". Define disproportionation. /nder aerobic conditions, all of the carbon in a substrate is either incorporated into cell mass or complete oxidi9ed to carbon dioxide to provide energy. /nder anaerobic conditions, substrate carbon is either incorporated into cell mass or disproportionated into its most oxidi9ed 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. ). Define the term critical dilution rate+ Dc+ and e&plain what happens in continuous culture when D is greater than Dc. :c is the critical dilution rate in a continuous culture reactor. 7f the dilution rate : is greater than :c, the cell growth rate cannot ;eep 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+ stationar,+ and death( for batch culture and environmental s,stems. <ag phase is generally short in a controlled batch culture system and is often simply a requirement for a physiological ad=ustment to a new substrate or set of growth conditions. 7n 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. <og phase in a controlled batch system is characteri9ed by a period of rapid growth with a doubling occurring with each new generation. 4cientists often optimi9e growth conditions to maximi9e the log phase to produce either cells or secondary metabolites that are economically important. 7n 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. 4tationary phase in a controlled batch system is a state of no net growth although new cells are still being made while others are dying. >enerally, 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 synthesi9ed optimally or the cell density is so high that further growth is inhibited. ?ften the stationary phase is an important period of growth for production of secondary metabolites. 7n the environment it is li;ely 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.

:eath phase in a controlled batch system is characteri9ed by a net loss of culturable cells due to a lac; of required nutrients or a buildup of toxic by(products. 7n 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 st,le of life in the environment. ?ligotrophs are organisms that prefer low substrate concentrations and their growth is often characteri9ed by low 5s 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. -opiotrophs are organisms that prefer high substrate concentrations and are often characteri9ed by higher 5s values (need higher substrate concentrations to achieve their normal growth rate). These are the organisms that ta;e advantage of a newly added substrate, li;e a dead leaf or insect.