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14.9 Chemostat culture with protozoa Tetrahymena thermophila protozoa have a minimum doubling time of 6.5 hours when grown using bacteria as the limiting substrate. The yield of protozoal biomass is 0.33 g per g of bacteria and the substrate constant is 12 mg l -1. The protozoa are cultured at steady state in a chemostat using a feed stream containing 10 g l -1 of nonviable bacteria. (a) What is the maximum dilution rate for operation of the chemostat? (b) What is the concentration of T. thermophila when the operating dilution rate is one-half of the maximum? (c) What is the concentration of bacteria when the dilution rate is three-quarters of the maximum? (d) What is the biomass productivity when the dilution rate is one-third of the maximum?

14.10 Two-stage chemostat for secondary metabolite production A two-stage chemostat system is used for production of secondary metabolite. The volume of each reactor is 0.5 m 3; the flow rate of feed is 50 l h -1. Mycelial growth occurs in the first reactor; the second reactor is used for product synthesis. The concentration of substrate in the feed is 10 g l -1. Kinetic and yield parameters for the organism are: Yxs = 0.5 kg kg-1 Ks= 1.0 kg m-3 μmax = 0.12 h-1 ms= 0.025 kg kg-1 h-1 qp = 0.16 kg kg-1 h-1 Yps = 0.85 kg kg-1 Assume that product synthesis is negligible in the first reactor and growth is negligible in the second reactor. (a) Determine the cell and substrate concentrations entering the second reactor.

(b) What is the overall substrate conversion? (c) What is the final concentration of product?

14.11 Growth of algae in a continuous bioreactor with cell recycle Ammonium-limited growth of the bluegreen alga Oscillatoria agardhii is studied using a 1-litre chemostat. The temperature of the culture is controlled at 25°C and continuous illumination is applied at an intensity of 37 μE m -2 s-1. Sterile ammonia solution containing 0.1 mg l-1 N is fed to the cells. The maximum specific growth rate of the organism is 0.5 day -1 and the substrate constant for N is 0.5 μg l -1 . The yield of biomass on nitrogen is 18 mg of dry weight per mg of N. The reactor is operated to give a hydraulic residence time of 60 h. No extracellular products are formed. (a) What is the overall substrate conversion? (b) What is the biomass productivity? Intensification of the culture process is investigated using cell recycle. While keeping the feed rate of fresh medium the same as that used without recycle, a recycle stream containing three times the concentration of cells in the reactor outflow is fed back to the vessel at a volumetric flow rate that is one-quarter of that of the fresh feed. The substrate concentration s in a chemostat with cell recycle is given by the equation: s=

D K s (1+ α−αβ) μmax −D(1+ α−αβ)

where D is the dilution rate based on the fresh feed flow rate, μ max and Ks are the maximum specific growth rate and substrate constant for the organism, respectively, α is the volumetric recycle ratio, and β is the biomass concentration factor for the recycle stream. (c) By how much does using cell recycle reduce the size of the bioreactor required to achieve the same level of substrate conversion as that obtained without cell recycle?

14.12 Kinetic analysis of bioremediating bacteria using a chemostat A strain of Ancylobacter bacteria capable of growing on 1,2-dichloroethane is isolated from sediment in the river Rhine. The bacteria are to be used for onsite bioremediation of soil contaminated with chlorinated halogens. Kinetic parameters

for the organism are determined using data obtained from chemostat culture. A 1litre fermenter is used with a feed stream containing 100 μM of 1,2-dichloroethane. Steady-state substrate concentrations are measured as a function of chemostat flow rate.

(a) Determine μmax and KS for this organism. (b) Determine the maximum practical operating flow rate.

14.13Kinetic and yield parameters of an auxotrophic mutant An Enterobacter aerogenes auxotroph capable of overproducing threonine has been isolated. The kinetic and yield parameters for this organism are investigated using a 2-litre chemostat fed with medium containing 10 g l -1 glucose. Steady-state cell and substrate concentrations are measured at a range of reactor flow rates.

Determine the maximum specific growth rate, the substrate constant, the maintenance coefficient, and the true biomass yield from glucose for this culture.

14.14 Chemostat culture for metabolic engineering A chemostat of working volume 400 ml is used to obtain steady-state data for metabolic flux analysis. Lactobacillus rhamnosus is cultured at pH 6.0 using sterile medium containing 12 g l-1 glucose and no lactic acid. Lactic acid production is coupled with energy metabolism in this organism. Concentrations of biomass, glucose, and lactic acid are measured at five different operating flow rates.

(a) Determine μmax and Ks. (b) Determine ms and Yxs. (c) Determine mp and Ypx. (d) Determine Yps. (e) Determine the maximum practical operating flow rate for this system. (f) Determine the operating flow rate for maximum biomass productivity. (g) At the flow rate determined in (f), what are the steady-state rates of biomass and lactic acid production and substrate consumption?

14.15 Effect of axial dispersion on continuous sterilisation A 15-m3 chemostat is operated with dilution rate 0.1 h -1. A continuous steriliser with steam injection and flash cooling delivers sterilised medium to the fermenter. Medium in the holding section of the steriliser is maintained at 130°C. The concentration of contaminants in the raw medium is 10 5 ml-1; an acceptable contamination risk is one organism every 3 months. The Arrhenius constant and activation energy for thermal death are estimated as 7.5 3 10 39 h-1 and 288.5 kJ

gmol-1, respectively. The inner diameter of the steriliser pipe is 12 cm. At 130°C, the liquid density is 1000 kg m-3 and the viscosity is 4 kg m-1 h-1. (a) Assuming perfect plug flow, determine the length of the holding section. (b) What length is required if axial dispersion effects are taken into account? (c) If the steriliser is constructed with the length determined in (a) and operated at 130°C as planned, estimate the frequency of fermenter contamination

14.16 Contamination frequency after continuous sterilisation A continuous steriliser is constructed using a 21-metre length of pipe of internal diameter 8 cm. Liquid medium in the pipe is maintained at 128°C using saturated steam. At this temperature, the specific death constant of the contaminating organisms is 340 h-1 and the density and viscosity of the medium are 1000 kg m -3 and 0.9 cP, respectively. The concentration of contaminants in the raw medium is 6.5 3 105 ml-1. If sterile medium is required on the fermentation floor at a rate of 0.9 m3 h-1, what is the frequency of contamination in the fermentation factory?