Continuous fermentation to produce xanthan biopolymer: Laboratory investigation

Xanthan biopolymer has been produced by single-stage continuous fermentation with Xanthomonas campestris NRRL B-1459 in a medium of glucose, minerals, distillers' solubles, and urea for as long as 20 days. At the highest dilution rate studied (D = 0.0285 hr^?1), the steady state rate of xanthan production was 0.36 g/kg/hr and the steady state yield, basis glucose consumed, was 68%. Observations indicate that xanthan production rate is a function of pH and D.     

Continuous cultivation of Trichoderma viride on cellulose

Using ball milled cellulose as the only carbon source Trichoderma viride was grown in a continuous flow culture at pH = 5.0 and T = 30°C. Steady-state values for cell protein, cellulose, and cellulase for different substrate concentrations (4–11 g/liter) and dilution rates (0.033–0.080 hr^?1) were obtained. Under steady-state conditions, 50–75% of the cellulose was consumed indicating a critical dilution rate on 0.17 hr^?1. Cellulase activity (U/ml) in the fermentation broth increased slightly with increasing substrate concentration and decreased with increasing dilution rate, while the specific cellulase productivity (U/mg cell protein·hr) was fairly independent of the dilution rate, with a maximum around D = 0.05 hr^?1. Following step changes in substrate concentration and dilution rate, new steady-state values were reached after three to five residence times (cell protein and cellulose) and four to six residence times (celullase activity).     

Continuous fermentation to produce fungal protein. Effect of growth rate on the biomass yield and chemical composition of Fusarium moniliforme

Fusarium moniliforme was grown on a carob aqueous extract in a chemostat for fungal protein production. The substrate was adjusted to provide 0.5% carob sugars supplemented with inorganic salts. The dilution rate varied from 0.086 to 0.227 hr^?1 under constant conditions of temperature (30°C), pH (4.5), and oxygen saturation (60–80%). A yield of 0.709 g dry mycelium/g consumed carob sugar and a productivity value of 0.687 g dry mycelium/liter hr^?1 were obtained at μ = 0.205 hr^?1. The maintenance coefficient was 0.077 g carob sugar/g dry mycelium hr^?1. While the carbohydrate and purine content of dry mycelium increased at μ values from 0.114 to 0.205 hr^?1 both true (Lowry) and crude (N × 6.25) protein contents decreased at the same μ range. Maximum values of 36.3% true and 47.9% crude protein of dry mycelium were obtained at μ = 0.114 hr^?1, whereas a minimum purine content of 99.8 μmol/g corresponding to 6.42% nucleic acids was recorded at μ = 0.086 hr^?1. It was concluded that a continuous fermentation of carob aqueous extract using F. moniliforme should be operated at growth rates of approximately 0.205 hr^?1 in order to maximize protein production.     

Daughter cells as an important factor in determining the physiological state of yeast populations

Cells of Candida utilis grown in a single-stage chemostat at D = 0.05, 0.1, 0.25, and 0.35 hr^?l were separated into a fraction of scar-bearing mother cells and a fraction of scar-free daughter cells. The scar-free cells were transferred into small batch cultures where the length of the maturation phase, changes in length and width of cells, specific growth rate, and specific rate of RNA and protein synthesis were examined for 5 hr. The daughter cells grown at D = 0.05 hr^?1 were very small at the moment of separation from the mother cells (about one-third of the mother cell). Their maturation phase (in a batch culture), at the beginning of which they attain the specific growth rate approaching the μ_max of the strain used, lasts for 3 hr. On the other hand, daughter cells grown at D = 0.35 hr^?1 are almost the same size as the mother cells at the moment of separation. After transfer to a batch culture they begin to bud almost immediately. Similarly, in their other morphological and physiological parameters they differ strikingly from immature daughter cells which are formed at low specific growth rates. The importance of these differences from the point of view of mathematical modeling of growth processes is discussed.     

Continuous Decolorization of Molasses Waste Water with Mycelia of Coriolus versicolor Ps4a

Continuous decolorization of molasses waste water by mycelia of Coriolus versicolor Ps4a was studied using waste water from a baker’s yeast factory, treated by means of methane fermentation and with activated sludge. Optimum decolorization with bare pellet-type mycelia in shaking flasks needed the addition of glucose (0.5%) and peptone (0.05%) and aerobic conditions (1ppm of dissolved oxygen). Continuous decolorization in a bubbling column reactor showed a decolorization yield of approximately 75% in only 20 hr at a dilution rate (D) of 0.03 hr^?1 under the optimum conditions.

In order to continue the decolorization for a longer time, mycelia immobilized within Caalginate gel were tested in a bubbling column reactor under the optimum conditions. The immobilized mycelia showed an almost constant decolorization yield (65.7%) during continuous decolorization for 16 days at D = 0.22 hr^?1.     

Kinetics of the liquid-phase oxidation of acid ferrous sulfate by the bacterium Thiobacillus ferrooxidens

The kinetics of the batch-wise liquid-phase oxidation of ferrous sulfate by the organism Thiobacillus ferrooxidans has been studied over a range of temperatures from 20°C to 31°C and in the presence of an abundant supply of oxygen, carbon dioxide, and other nutrients. The rate of oxidation was found to be accurately described by the equation where t = time hr, S = concentration of ferrous ions g Fe⁺⁺/1., μ_m = maximum specific growth rate of bacteria, hr^?1. Y = mass of bacteria produced per gram of iron oxidized g/g, K = saturation constant, g Fe⁺⁺/l., and X = concentration of bacteria g/1. The value for the maximum specific growth rate, μ_m, was found to vary from 0.12 hr^?1 at 20°C to 0.20 hr^?1 at 31°C, while the value for the saturation constant K varied randomly between 1 and 2 g/1. A method has also been described which permitted evaluation of the relevant rate constants μ_m and K without direct knowledge of the bacterial population. This method was found to yield values of μ_m and K which agreed with values determined accurately by a statistical regression analysis of the experimental data.     

Production of the macrolide antibiotic tylosin in batch and chemostat cultures

The production of tylosin and related compounds by Streptomyces fradiae NRRL 2702 was studied in batch and chemostat cultures using a soluble synthetic medium. In batch culture, a trophophase–idiophase kinetic pattern was observed with tylosin, macrocin, and relomycin accumulating in the idiophase. When the organism was grown in chemostat culture, the specific rate of production of tylosin and related compounds (q_tylosin) was found to be a function of the growth rate. The maximum value of (q_tylosin) was observed when D = 0.017 hr^?1. At this growth rate only tylosin and relomycin accumulated in the medium. By varying the concentration of glucose in the ingoing medium it was possible to study the effects of glucose on tylosin synthesis in chemostat cultures. At a growth rate of 0.017 hr^?1, the maximum value of q_tylosin was 0.71 mg tylosin/g dry weight (DW)/hr when the glucose uptake rate was 7 mg glucose/g DW-hr. This value of q_tylosin was 40% greater than the maximum q_tylosin observed in batch culture. When glycerol was substituted for glucose in the medium, it was possible in chemostat culutures to get values of q_tylosin approximately 20% greater than those obtained with glucose at the same uptake rate. By varying the concentration of sodium glutamate in the ingoing medium it was possible to show that increasing the specific uptake rate of sodium glutamate increased the values of q_tylosin obtained. Similar chemostat experiments where the inorganic phosphate concentration in the ingoing medium was varied showed that increased the uptake of phosphate decreased the values of q_tylosin obtained. Also increasing the uptake rate of phosphate increased the relomycin-to-tylosin ratio. By taking into consideration the suppressing effects of glucose and the stimulating effects of sodium glutamate on tylosin synthesis, it was possible to formulate a medium that resulted in a value of q_tylosin of 1.1 mg/g/hr being obtained at a growth rate of 0.03 hr^?1. Batch fermentations with this medium did not follow a trophophase–idiophase kinetic pattern, but instead tylosin was actively synthesized during a period of rapid mycelial growth.     

Acute effects of Cu on oxygen consumption and 96 hr-LC50 values in the freshwater fish Tilapia sparrmani (Teleostei: Cichlidae) in Mooi River hard water,South Africa

The median lethal copper (Cu) concentration (96 hr-LC₅₀) values for acute Cu toxicity for Tilapia sparrmanii (live mass: 30 ± 8g) in Mooi River hard water of dolomitic origin at 20 °C, pH 7.9, was 68.1 μmol l^?1. At this 96 hr-LC₅₀ value the specific oxygen consumption rate (∈ O₂) decreased by 44.2 (± 2.1) % from a non-exposed value of 6.6 (±0.32) mmol O₂ kg^?1 hr^?1 to 3.63 (±0.23) mmol O₂ kg ^?1 hr^?1. At 46.4 μmol Cu l^?1, 100% of the exposed T. sparrmanii were still alive after 96 hours, but the ∈ O₂ decreased by a mean value of 1.65 (± 0.16) mmol O₂ kg^?1 fish hr^?1 or 25% (± 2.4). Contrary to Pb and Cd, Cu as CuCl₂ 2H₂O was not precipitated in hard water four days after it was dissolved. Thus T. sparrmanii and other cichlids are shown to be more than an order of magnitude more resistant to Cu as a toxicant than most salmonids.     

The application of constant recycle solids concentration in completely mixed oxygen activated sludge process

For better operational control of the completely mixed oxygen activated sludge process (CMOAS), a study concerning the kinetics, performance, and operational stability of the Ramanathan-Gaudy model was conducted. Short-term experiments were conducted at various dilution rates (1/9, 1/6, 1/3, 1/1.5, and 1/1.0 hr^?1) by using two recycle solids concentration values (5000 and 10,000 mg/liter). The influent substrate was an actual industrial organic wastewater (soft drink waste) and its concentration was maintained at 1000 mg/liter COD. The hydraulic recycle ratio, α, was maintained at 0.30. It was found that for CMOAS system with constant recycle cell concentration, a “steady state” with respect to reactor biological solids and effluent COD at different dilution rates could be attained. No appreciable dilute-out of reactor biological solids and substrate was observed up to the dilution rate of 1 hr^?1 for both systems of different X_R (5000 and 10,000 mg/liter). For the system of X_R = 5000 mg/liter, except the dilution rate of hr^?1, the effluent filtrate COD was lower than 100 mg/liter, the aerator biological solids concentration was about 1550 mg/liter, and the COD removal efficiency was higher than 90% for all dilution rates. For the system of X_R = 10,000 mg/liter, the effluent filtrate COD was lower than 71 mg/liter, the aerator biological solids concentration was about 2750 mg/liter, and the COD removal efficiency was higher than 90% throughout all the dilution rates selection in the present study. The value of the Sludge Volume Index (SVI) was the range of 37.0 to 58.5 and provided good settleability of sludge. The sludge yield was 0.53 for the system of X_R = 5000 mg/liter and 0.57 for the system of X_R = 10,000 mg/liter. The carbohydrate and the protein content of the cells were 10.1–21.6% and 35.6–50.6%, respectively. For predicting the reactor biological solid and effluent COD of the CMOAS system by using the Ramanathan-Gaudy model, two sets of values for the biological kinetic constants should be considered since it provided the best fit of predicted values of the observed values. In the present study, μ_m = 0.4 hr^?1, k_s = 92 mg/liter for 1/3 ? D ? 1, and μ_m = 0.05 hr^?1, k_s = 11.1 mg/liter for 1/9 ? D < 1/3 were used to calculate the predicted values of reactor biological solid and effluent filtrate COD.     

Non-carbon loss long-term continuous lactic acid production from mixed sugars using thermophilic Enterococcus faecium QU 50

In this study, a non-sterile (open) continuous fermentation (OCF) process with no-carbon loss was developed to improve lactic acid (LA) productivity and operational stability from the co-utilization of lignocellulose-derived sugars by thermophilic Enterococcus faecium QU 50. The effects of different sugar mixtures on LA production were firstly investigated in conventional OCF at 50°C, pH 6.5 and a dilution rate of 0.20 hr⁻¹. The xylose consumption ratio was greatly lower than that of glucose in fermentations with glucose/xylose mixtures, indicating apparent carbon catabolite repression (CCR). However, CCR could be efficiently eliminated by feeding solutions containing the cellobiose/xylose mixture. In OCF at a dilution rate ca. 0.10 hr⁻¹, strain QU 50 produced 42.6 g L⁻¹ of l -LA with a yield of 0.912 g g⁻¹-consumed sugars, LA yield of 0.655 g g⁻¹ based on mixed sugar-loaded, and a productivity of 4.31 g L⁻¹ hr⁻¹ from simulated energy cane hydrolyzate. In OCF with high cell density by cell recycling, simultaneous and complete co-utilization of sugars was achieved with stable LA production at 60.1 ± 3.25 g L⁻¹ with LA yield of 0.944 g g⁻¹-consumed sugar and LA productivity of 6.49 ± 0.357 g L⁻¹ hr⁻¹. Besides this, a dramatic increase in LA yield of 0.927 g g⁻¹ based on mixed sugar-loaded with prolonged operational stability for at least 500 hr (>20 days) was established. This robust system demonstrates an initial green step with a no-carbon loss under energy-saving toward the feasibility of sustainable LA production from lignocellulosic sugars.     

Translational and rotational diffusion of tobacco mosaic virus from polarized and depolarized light scattering

The translational and rotational dynamics of tobacco mosaic virus in sodium phosphate buffer (pH =7.5) solutions has been investigated by polarized and depolarized light scattering Rayleigh linewidth studies. For concentrations ranging from 1.75 × 10^?4 g ml^?1 to 0.25 × 10^?4 g ml^?1 the translational diffusion coefficient (D_T) has been found to be slightly concentration dependent and extrapolation to zero concentration gives D₀^20°C = 0.34 ± 0.01 × 10^?7 cm²S^?1. A full analysis of the polarized spectra obtained at high and low scattering angles and the depolarized spectra at near zero scattering angles has enabled these techniques to be compared and the rotational diffusion constant D_R to be determined. At a solution concentration of 1.75 × 10^?4 g ml^?1 a mean value is found to be D_R^20°C = 350 ± 30s^?1. These values of D_T and D_R are in approximate agreement with calculations based on models of the tobacco mosaic virus molecule as a cylindrical rod.     

Long term shear effects on a hybridoma cell line by dynamic perfusion devices

The long term shear effects on a hybridoma cell line were studied by the simulation of a hollow fiber perfusion system. Various mechanical/environmental stress conditions were applied and steady state concentrations of live, dead and lysed cells were measured or calculated in a continuous culture. From mathematical modeling, it is shown that inclusion of a lysed cell index (LCI) renders a better fit to the material balance equation at steady state. The specific cell death rate increased with increasing shear force as expected only when the LCI was included. Without the inclusion of the LCI, the calculated specific cell growth rates are about 25–60% of the value when included. The results reported may lend some insight to design improvements since most perfusion devices add shear stresses to the cells in the reactor.List of Symbols b ml/hr continuous culture flow rate - D hr^–1 dilution rate (b/V) - m g glucose/10⁹ cells/hr specific maintenance coefficient - S ₀ g/l feed substrate concentration - S g/l reactor substrate concentration - t hr time - V ml reactor volume - X ⁺ cells/ml live cell concentration - X ^– cells/ml dead cell concentration - X ⁰ cells/ml lysed cell concentration - Y _x/s 10⁹ cells/g glucose cell/substrate yield coefficient - hr^–1 specific growth rate - hr^–1 specific death rate - hr^–1 specific lysis rate - hr^–1 specific lysis rate for simultaneous death and lysis     

Production of Candida utilis protein from peat extracts

Sphagnum peat extracts or hydrolysates have been obtained and used as a culture medium for the production of Candida utilis biomass as single cell proteins. Acid hydrolysis of ground peat (4–60 mesh) in an autoclave operated under a set of conditions for acid strength (0.3-1.5 (v/v) H₂SO₄), holding time (1–4 hr), temperature (100–165°C), and weight ratio of dry peat to solution (3.3–16.7 g dry peat/100 g solution) yielded carbohydrate-rich extracts of different concentrations (1–34g/liter). The best yield (mg total carbohydrate/g dry peat) was obtained for a holding time of I hr and a temperature of 152°C. Low peat concentratio (4.1 g dry peat/100 g solution)resulted in high yield(280mg total carbohydrate/gdry peat) with a corresponding low carbohydrate content in hydrolysate (13 g/liter), while a lower yield with a higher carbohydrate content (34 g/liter)in hydrolysate were found when increasing peat concentration (16.7 g dry peat/100 g solution). Shake-fladk experiments using peat hydrolysates as the culture medium together with NH₄OH (～4.8 g/liter) and K₂HPO₄(5 g/liter) as nitrogen and phosphate supplement, respectively, gave a maximum biomass concentration of 7.5 g/liter after 60 hr at 30°C and 200rpm. Batch cultivation in a fermentor under controlled conditions for aeration (4.2 liter/min), agitation (500rpm), temperature (30°C), and pH (5.0) produced a maximum biomass of 10 g/liter after 20 hr with a specific growth rate of 0.13 hr^?1. For the continuous cultivation, a maximal biomass productivity of 1.24 g/gliter-he was obtained at a dilution rate of 0.125 hr ^?1. Monod's equation's equation has been used for the estimation of the coefficients μ_Max, K_s, and Y. It was found that the yield coefficient Y is not constant during the progress of batch cultivation.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

Tower fermentation using Zymomonas mobilis for ethanol production

Summary Flocculation was induced in a pure strain of the bacteria Zymomonas mobilis. When fermenting glucose to ethanol, cell densities of up to 40g/l were achieved and sustained in a 0.92 litre tower fermenter with dilution rates of up to 2.3 hr^-1. A maximum productivity of 100g EtOH/l/hr with 98% conversion of the 105g/l glucose feed was achieved. The limitation to performance with increase in throughput arose from incomplete fermentation of the feed glucose, rather than washout of the flocculated bacteria.     

Protection from illegal fishing and shark recovery restructures mesopredatory fish communities on a coral reef

The recovery of communities of predatory fishes within a no‐take marine reserve after the eradication of illegal fishing provides an opportunity to examine the role of sharks and other large‐bodied mesopredatory fishes in structuring reef fish communities. We used baited remote underwater video stations to investigate whether an increase in sharks was associated with a change in structure of the mesopredatory fish community at Ashmore Reef, Western Australia. We found an almost fourfold increase in shark abundance in reef habitat from 0.64 hr^?1 ± 0.15 SE in 2004, when Ashmore Reef was being fished illegally, to 2.45 hr^?1 ± 0.37 in 2016, after eight years of full‐time enforcement of the reserve. Shark recovery in reef habitat was accompanied by a two and a half‐fold decline in the abundance of small mesopredatory fishes (≤50 cm TL) (14.00 hr^?1 ± 3.79 to 5.6 hr^?1 ± 1.20) and a concomitant increase in large mesopredatory fishes (≥100 cm TL) from 1.82 hr^?1 ± 0.48 to 4.27 hr^?1 ± 0.93. In contrast, near‐reef habitats showed an increase in abundance of large mesopredatory fishes between years (2.00 hr^?1 ± 0.65 to 4.56 hr^?1 ± 1.11), although only smaller increases in sharks (0.67 hr^?1 ± 0.25 to 1.22 hr^?1 ± 0.34) and smaller mesopredatory fishes. Although the abundance of most mesopredatory groups increased with recovery from fishing, we suggest that the large decline of small mesopredatory fish in reef habitat was mostly due to higher predation pressure following the increase in sharks and large mesopredatory fishes. At the regional scale, the structure of fished communities at Ashmore Reef in 2004 resembled those of present day Scott Reefs, where fishing still continues today. In 2016, Ashmore fish communities resembled those of the Rowley Shoals, which have been protected from fishing for decades.     

Production of xanthan by in-flow cultures of Xanthomonas campestris

The kinetics of xanthan formation in Xanthomonas campestris continuous and fed-batch fermentations was studied along with metabolic changes due to growth rate variation. A maximum growth rate within the range 0.11–0.12 h^–1 was obtained from the continuous culture data in defined medium, producing xanthan at rates up to 0.36 g l^–1 h^–1 corresponding to a maximum 67% glucose conversion at a dilution rate (D) of 0.05 h^–1. Comparatively, fed-batch cultivation was more efficient, producing maximum xanthan at 0.75 g l^–1 h^–1 and 63% glucose conversion at 0.1 h^–1. When reaching D=0.062 h^–1 in continuous cultures, a change was observed and the values of the specific rate of substrate consumption shifted, initiating an uncoupled growth region expressing a lack of balance of the catabolic and anabolic reactions. The deviation was not accompanied by a change in specific xanthan production indicating that xanthan metabolism was not affected by D. For fed-batch-grown X. campestris cells within the range D=0.03–0.1 h^–1, both metabolic parameters changed linearly with the growth rate showing a wide region coupled to growth. Outside that range, glucose accumulated and the specific xanthan production dropped, suggesting substrate inhibition. Correspondence to: J. C. Roseiro     

Comparative Fluxome and Metabolome Analysis of Formate as an Auxiliary Substrate for Penicillin Production in Glucose‐Limited Cultivation of Penicillium chrysogenum

During glucose‐limited growth, a substantial input of adenosine triphosphate (ATP) is required for the production of β‐lactams by the filamentous fungus Penicillium chrysogenum. Formate dehydrogenase has been confirmed in P. chrysogenum for formate oxidation allowing an extra supply of ATP, and coassimilation of glucose and formate has the potential to increase penicillin production and biomass yield. In this study, the steady‐state metabolite levels and fluxes in response to cofeeding of formate as an auxiliary substrate in glucose‐limited chemostat cultures at the dilution rates (D) of both 0.03 h^?1 and 0.05 h^?1 are determined to evaluate the quantitative impact on the physiology of a high‐yielding P. chrysogenum strain. It is observed that an equimolar addition of formate is conducive to an increase in both biomass yield and penicillin production at D = 0.03 h^?1, while this is not the case at D = 0.05 h^?1. In addition, a higher cytosolic redox status (NADH/NAD⁺), a higher intracellular glucose level, and lower penicillin productivity are only observed upon formate addition at D = 0.05 h^?1, which are virtually absent at D = 0.03 h^?1. In conclusion, the results demonstrate that the effect of formate as an auxiliary substrate on penicillin productivity in the glucose‐limited chemostat cultivations of P. chrysogenum is not only dependent on the formate/glucose ratio as published before but also on the specific growth rate. The results also imply that the overall process productivity and quality regarding the use of formate should be further explored in an actual industrial‐scale scenario.     

Use of continuous-culture techniques for determining the growth kinetics of a celluloytic Thermoactinomyces sp.

In order to better understand the kinetics of cellulose degradation by Thermoactinomyces sp., continuous-culture experiments were performed utilizing the various intermediates of cellulose degradation as the feed substrates. Steady-state data from the glucose runs suggest that this organism has a growth yield of 0.42 g cell/g glucose, and a specific maintenance of 0.24 g glucose/g cell/hr. The Monod equation did not seen to model the growth well, since a plot of 1/D vs. 1/S gave a maximum specific growth rate that was even lower than one of the steady-state dilution rates. A dynamic washout experiment suggested a maximum specific specific growth rate of 0.36 hr^?1 and indicated that glucose is only slightly growth inhibitory as the inhibition constant, K_i, is 19 g glucose/liter. An equation for substrate concentration for washout conditions was derived. This equation predicted the transient glucose concentration relatively well. A fill-and-draw technique was investigated for determination of the growth parameters. It was not successful because of difficulties in contamination and accurately monitoring the dissolved oxygen in the small highly agitated vessel. However, the technique could be useful in studying the growth characteristics of sludge in a waste treatment system where contamination is not a worry. One could cover the medium surface and use a nonsterilizable dissolved oxygen probe of high sensitivity membrane to overcome these difficulties.