Why do the microorganisms use oxygen to break down the organic matter - ie how is the oxygen used? 
The oxygen is used...

• as an electron acceptor in aerobic respiration
• in electron transfer reactions that build up a proton gradient for ATP synthesis 

Which of the following terms apply to a bacterium that is breaking down fats using oxygen?  

• Aerobic respiration
• Chemoorganotrophy
• Heterotrophy
• oxidative phosphorylation 

Fecal coliforms make good indicators because ... 

• They grow rapidly on selective and differential media.
• They are easy to identify by their patterns of growth on those media.
• They primarily exist in animal digestive tracts, so their presence is a reasonable indication of fecal contamination.
• They persist at least as long as fecal pathogens in water. 

One bacterial metabolism that is sometimes exploited to remove nitrogen from water is called "annamox". In annamox, _________ is used as an electron donor/energy source and _____________ is used as an electron acceptor. 

ammonia; nitrate

Annamox = anaerobic oxidation of ammonia 

Annamox bacteria grow using chemolithoautotrophy. Briefly, how do chemolithoautrophs make a living? That is, what characteristics do their energy sources have? Lithotrophy = "rock-eating"; these bacteria use reduced inorganic chemicals such as H2S or Fe2+ or NH4+ as energy sources. 

How do they use those energy sources to make ATP? High energy e- from the inorganic chemicals are passed through an electron transport chain to a terminal e- acceptor. As the electrons are passed from one carrier to another, protons are pumped across a membrane to form an electrochemical gradient; the proton motive force is relieved through an ATP synthase that phosphorylates ADP to ATP.

What are their carbon sources? Chemolithotrophs typically are autotrophs because they live in environments where little organic carbon is available. 

Water that has high amounts of nitrogen and phosphorous can support photoautotrophic growth of bacteria such as cyanobacteria. We discussed the 'Z scheme' of electron flow used in phototrophic plants and some phototrophic bacteria such as cyanobacteria. In the Z scheme, how does cyclic e- flow differ from noncyclic e- flow? 

Cyclic e- flow doesn't produce oxygen 

Bacteria besides the cyanobacteria are also capable of photoautotrophic growth. Electron flow in purple phototrophic bacteria is depicted below. 

[image]

P870 is the reaction center chlorophyll in a photosystem in this purple bacterium. It has an absorption peak (ie it absorbs light the most) of 870 nm. The reaction center chlorophyll absorbs light directly, or has energy transferred to it from antenna pigments in the photosystem. In it's excited state, it is a powerful reductant and it will pass it's electrons to a Bacteriochlorophyll or Bacteriopheophytin molecule as shown in the figure. 

 The excited electrons from P870 get passed through a series of electron carriers and then end back on P870. How do purple bacteria use these reactions? 

This cyclic electron flow is used to pump protons. As the electrons are passed from one carrier to another, protons are pumped across a membrane to form an electrochemical gradient; the proton motive force is relieved through an ATP synthase that phosphorylates ADP to ATP. 

Electrons sometimes get taken from Q (quinone) and used to reduce NAD+ to NADH in a process called reverse electron flow. These electrons can then be replaced by an electron donor such as hydrogen sulfide or ferrous iron. Is the purple bacterium using oxygenic photosynthesis or anoxygenic photosynthesis? 

In oxygenic photosynthesis, oxygen is produced when water is split and the resulting electrons are used to reduce a reaction center chlorophyll. In anoxygenic photosynthesis, as seen here, the source of electrons is something other than water, so no oxygen is released. 

Why do purple bacteria need to generate NADH? How do they use that NADH?  

These bacteria are autotrophs, that is, they rely on CO2 as a carbon source. To convert CO2 into organic molecules they need sources of energy but also sources of electrons and protons. The NADH is used to provide reducing power in autotrophic pathways (the Calvin cycle, for purple bacteria). 

NADH is being generated by 'reverse electron flow'. What is reverse electron flow, and why do organisms like purple phototrophic bacteria and some chemolithotrophs use reverse electron flow? 

Reverse e- flow uses rather than creates a proton gradient, moving electrons to a more reduced state. Organisms use this if their energy source doesn't release enough energy to directly reduce NAD+ to NADH, but does release enough energy to build up a proton gradient. 

You used several strategies and different culture media in order to isolate the predominant saltern organism in pure culture. Once you had a pure culture of the organism, you were able to grow it in the medium below:

Is this a defined medium or a complex medium? Explain. 

This is a defined medium, because known chemicals are added to it at known concentrations. 

You construct a growth curve for the organism growing in this medium. The organism is pigmented and sometimes slightly changes color as the culture gets more dense, so you decide to do plate counts rather than measure changes in absorbance. At one timepoint, you remove a 1 ml sample of the culture and add it to 99 ml of sterile medium. You mix that, and add 1 ml to another 99 ml of medium. Then you mix that, and add 1 ml to 9 ml of medium.

What is the total dilution in your final tube?

1/100 * 1/100 * 1/10 = 1/105 or can be expressed as 10-5  

You plate 0.1 ml of the final tube, and count 43 colonies. What was the cell concentration in the culture when you originally removed the 1 ml sample?

43 colonies/0.1 mlà430 cells/ml = Cf

Ci = Cf/D = (430 cells/ ml) / 10-5 = 4.3 * 107 cells/ml 

You inoculate cells from a slant culture into a 250 ml flask containing 50 ml of the medium on the previous page, and, using plate counts, you construct a growth curve. The results are depicted below. Next to the letters A, B, and C, write the name of that stage of the growth curve and a brief description of what the cells are doing in that stage. 

[image]

A= Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains temporarily unchanged. Although there is no apparent cell division occurring, the cells may be growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic activity.
The length of the lag phase is dependent on a wide variety of factors including the size of the inoculum; time necessary to recover from physical damage or

4

shock in the transfer; time required for synthesis of essential coenzymes or division factors; and time required for synthesis of new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.
2. B= Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth wherein all the cells are dividing regularly by binary fission, and are growing by geometric progression. The cells divide at a constant rate depending upon the composition of the growth medium and the conditions of incubation.
3. C= Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a closed system such as a test tube or flask). Population growth is limited by one of three factors: 1. exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3. exhaustion of space, in this case called a lack of "biological space". The stationary phase, like the lag phase, is not necessarily a period of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced after the active stage of growth). It is during the stationary phase that spore- forming bacteria have to induce the activity of dozens of genes that may be involved in sporulation process.
4. Death Phase. If incubation continues after the population reaches stationary phase, a death phase follows, in which the viable cell population declines. 

The first step in annotating your genome is to define open reading frames (ORFs). What is an ORF? What role do ORFs play in analyzing microbial genomes?

An ORF is an open reading frame. An ORF consists of a long string of nucleotides that doesn't have a stop codon in one of the reading frames. ORFs almost always = protein coding genes. By compiling a list of ORFs in a genome, and BLASTing them against annotated ORFs, you can determine what enzymes and other proteins an organism can make and hence what kind of metabolism it uses and what other functions it is capable of 

 In analyzing the genome sequence, you discover the genes for several rhodopsins. Rhodopsins can have different functions, one of which is a light- driven proton pump. What is the advantage to having a light-driven proton pump? How would cells likely use a light-driven proton pump? 

Organisms need to make ATP in order to drive energy-requiring reactions. Sometimes they need to rely on a chemical energy source but if this energy source is organic, then the total amount of biomass that can be synthesized is reduced if they are using the organic matter to generate a proton gradient. The advantages to having a light-driven proton pump are that they don't need to use up a chemical energy source and they can divert organic molecules to biomass synthesis. Cells would likely use a light-driven proton pump to create a PMF that can be used to make ATP, transport substances, turn flagella, etc.