Section one Answer 5 of the following 6 problems (3 marks each) 1.1) Explain the major reactions of the Sulfur cycle by pointing out: a) the environmental conditions needed (e.g. electron donors or acceptors, the type of microbes involved) c) the significance of the process to industry and environment d) speculate on the likely mechanisms of ATP generation e) Show the stoichiometrically correct equation of two important reactions involved

Exam format: Detailed questions (e.g. 6 out of 8) 50% Short answer questions (e.g. 10 out of 12) 25% Calculations (e.g. growth contants, stoichiometry, chemostat) 25% Calculatios can be integrated within short answer questions 1.1) Explain the major reactions of the Sulfur cycle by pointing out: a) the environmental conditions needed (e.g. electron donors or acceptors, the type of microbes involved) c) the significance of the process to industry and environment d) speculate on the likely mechanisms of ATP generation e) Show the stoichiometrically correct equation of two important reactions involved

1.2) Explain what the 4 growth constants are that describe microbial growth. Explain how you can determine the growth constants from experimental data.

1.3) Explain the fermentation pathway of either butyric, propionic or the acetone butanol fermentation under the following headings: a) Overall “strategy” of fermentation. b) Points and mechanism of ATP generation c) Internal electron acceptors used to regenerate NADH d) Describe in words to individual reactions of the pathway e) Show by using an electron and carbon flow diagram that the electron and carbon balance is correct

Nitrogen cycle, nutrient removal 1.4) Nitrification and Denitrification are important in nitrogen removal from wastewater. Elaborate on the principle of biological nitrogen removal in waste water treatment plants. In your answer explain: a) The need for nitrogen removal from wastewater b) The respective role of nitrification and denitrification in nitrogen removal c) Chemical equations for both reactions d) process conditions needed for nitrification and denitrification for waste water treatment e) The traditional way of making use of sequential nitrification and denitrification in waste water treatment f) The principle of simultaneous nitrification and denitrification (SND) g) How SND can be accomplished

1.5) Explain the phenomenon of interspecies hydrogen transfer (e.g. Methanobacillus omelianskii) as part of the anaerobic digestion bioprocess. Explain in detail how ΔG calculations (Gibbs Free Energy Change) can be used to determine under at which hydrogen concentrations the hydrogen producing or hydrogen consuming bacterium can operate under energetically favourable conditions (spontaneous reaction). If possible, use a sketch that relates the Gibbs Free Energy Change to the hydrogen concentration

1.6) Glutamate is the most widely produced amino acid. Explain the biochemistry of the process and point out the requirements to the organisms to allow overproduction and excretion of this amino acid.

Section 2 short answer questions: Answer 10 out of the following 12 questions (1.5 marks each) 2.1) Explain how citric acid is produced during a) growth (idiophase) b) product formation (trophophase) Explain why the citric acid cycle (TCA cycle) is not interrupted when citric acid is excreted.

2.2) During the microbial reduction of nitrate (NO3-) to nitrite (NO2-) bacteria use nitrate as electron acceptor. Explain why it is not quite correct to say that nitrate reducing bacteria make use of the oxygen atoms in the nitrate molecule as an electron acceptor.

2.3) How would you predict the expected growth yield (Y) of an organism (g of biomass formed per g of substrate degraded) that aerobically oxidises ethanol to acetic acid (vinegar production)? Show your calculation and explain all your assumptions.

2.4) An unknown organic substrate is degraded in a biogas digester to 4.9 L of methane gas. If this substance was degraded by aerobic bacteria, how much oxygen would be used?

2.5) How much methane will be produced from the anaerobic digestion of hydroxy- butyrate (CH3-CHOH-CH2-COOH)? What is the expected ratio of CH4/CO2 from this conversion?

2.6) A reactor with a kLa of 25h-1 aerobically converts propanol (CH3-CH2- CH2OH) to CO2. What is the maximum propanol oxidation rate that can be obtained by the limited oxygen supply?

2.7) What is the advantage of biomass feedback in bioprocesses. Give an example and show the effect on productivity (R).

2.8) Sketch the effect on oxygen concentration and oxygen uptake rate that will be the result of a substrate spike added to a starved, aerobic bacterial culture. Show by using units how you can quantify the amount of organic substrate that was degraded. Use acetate (CH3-COOH) as an example.

2.6) A reactor with a kLa of 25h-1 aerobically converts propanol (CH3-CH2- CH2OH) to CO2. What is the maximum propanol oxidation rate that can be obtained by the limited oxygen supply?

2.9) Explain the advantages and limitations of using chemostats for industrial processes compared to batch cultures. Productivity, contamination, revertants (back-mutation to wild strain = “contamination from inside)

2.10) Establish the stoichiometrically correct equation for the conversion of ethanol (CH3-CH2OH) to CO2 (to be more precise to bicarbonate) for sulfate- reducing bacteria using sulfate (SO42-) as electron acceptor to produce HS-.

2.11) In continuous culture (e.g. chemostat) the productivity is related to the flowrate into the reactor and the biomass concentration. What would be the productivity (assuming the biomass, X is the product) of a 10 L chemostat with a flow rate of 2.5 L/h and a biomass concentration of 15 g/L ?

2.12) Explain the effect of oxygen use by heterolactic bacteria. What is their advantage in using oxygen when they have not electron transport chain?

2.10) Establish the stoichiometrically correct equation for the conversion of ethanol (CH3-CH2OH) to CO2 (to be more precise to bicarbonate) for sulfate- reducing bacteria using sulfate (SO42-) as electron acceptor to produce HS-.

Process control of high cell density cultures. Recombinant strains of E.coli have been shown to excrete acetate as an undesired and disruptive endproduct if glucose as the principal stubstrate is present in excess. This fermentation behaviour in spite of the excesss of oxygen is called Crabtree effect. Explain how a process control program could be designed that provides the glucose to a growing E.coli cultures such that it does not become saturating (and hence avoids the production of acetate). Give possible reasons why acetate is excreted in spite of the presence of oxygen.

Batch (productivity (max total) Chemostat Fed batch (HCDC) SBR

Anaerobic respirations are different to anaerobic fermentation in that external electron acceptors other than oxygen are used to build up a proton gradient that allows ATP generation via the ATP synthase. In contrast to oxygen respiration, anaerobic respirations form reduced endproducts, which can in turn serve as the electron donor for aerobic bacteria. Explain with an example the endproduct that is formed and the re- oxidation of the reduced endproduct (we had examples of the deep ocean environment). Show exactly how many electrons are being added / removed for both the anaerobic respiration and the re-oxidation.

Explain the purpose of SND via NO2-

Explain the reasoning that allows nitrification and denitrification to occur at the same time. Point out the significance of N removal to protect the environment

Explain PID control

Competition of organisms for the same substrate. Explain how the growth constants and the Gibbs Free energy change of the reaction can determine the outcome of competition for a common substrate (such as hydrogen).

2.10) Establish the stoichiometrically correct equation for the conversion of ethanol (CH3-CH2OH) to CO2 (to be more precise to bicarbonate) for sulfate- reducing bacteria using sulfate (SO42-) as electron acceptor to produce HS-.