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.     

Continuous fermentation to produce xanthan biopolymer: Effect of dilution rate

Single-stage continuous fermentations to produce xanthan gum have been run at dilution rates (D) from 0.023 to 0.196 hr^?1. Xanthan production rate (XPR) was a function of D. XPR increased from 0.34 g/hr/kg at D = 0.023 hr^?1 to the maximum 0.84 g/hr/kg at D = ca. 0.15 hr^?1. At D > 0.15 hr^?1 XPR decreased and at the highest D studied (0.196 hr^?1) was 0.69 g/hr/kg. Yield of xanthan from glucose consumed was 81–89%. Steady states ended between 6.5 and 8.7 turnovers when a variant strain occurred.     

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.     

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.     

Continuous enzymatic saccharification of cellulose with culture filtrates of trichoderma viride QM 6a.

Continuous saccharification of Solka Floc (cellulose pulp) in single and four-vessel stirred-tank reactor systems has been possible employing enzymes obtained directly from submerged fermentation of Trichoderma viride QM 6a. Studies on the effect of modification of the solid substrate, enzyme stability, substrate concentration, and the influence of reducing sugar concentration on the rate of hydrolysis are reported. While susceptibility of substrate to digestion is not affected by heating alone, it is strikingly increased by heating plus grinding, or by grinding following heating. Batch and steady state continuous saccharification experiments have yielded more than 5% reducing sugar in the effluent with a dilution rate of 0.025 hr^?1 at 50°C, at a substrate level of 10%. An average glucose concentration of 3.4% has been obtained in the effluent of a continuous saccharification using 5% substrate at the same dilution rate and temperature.     

Biological kinetic behaviors and operational performances in aerated and oxygenated systems

Biological kinetic behaviors of the oxygenated and aerated activated sludge process were studied and compared in both once-through and constant sludge recycle systems. The models derived by Herbert, Elsworth, and Telling [J. Gen. Microbiol., 14 , 601 (1956)] and Ramanathan and Gaudy [Biotechnol. Bioeng., 11 , 207 (1969)] were used for the studies of once-through and constant sludge recycle systems, respectively. Soft drink waste water was used for the growth limiting substrate. Temperature was controlled within 30 ± 2°C. The influent substrate concentration was maintained at 1,000 mg/liter. The experiments were conducted at various dilution rates (from \documentclass{article}\pagestyle{empty}\begin{document}$ \frac{1}{9} $\end{document} to 1/1.0 hr^?1), and recycle solids concentration values (from 5,000 to 10,000 mg/liter), with hydraulic recycle ratio, α, at 0.3. Biological kinetic constants were evaluated and compared. It was found that these constants were different for the aerated and oxygenated systems within a certain range of dilution rates studied. The critical dilution rates for diluting out effluent chemical oxygen demand (COD) occurred at 0.1 and 0.2 hr^?1in the once-through operation, and 0.2 and 0.4 hr^?1in the sludge recycle operation for aerated and oxygenated systems, respectively. Observed sludge yield values and specific growth rate were varied with the type of aeration and with and without constant sludge recycle concentration applied. Sludge carbohydrates and proteins content in the oxygenation system (cell recycle) were 10.1–21.6% and 35.6–52.2%. Sludge volume index in the air and oxygenation systems varied from 41.4 to 354 and 31.9 to 58.5, respectively.     

Continuous monitoring of cellulase action on microcrystalline cellulose

Summary Cellobiose oxidase from Phanerochaete chrysosporium was used for continuous monitoring of cellulase action on microcrystalline cellulose (Avicel). Two protocols are described, the parameter monitored being either the decline in electrode potential as ferricyanide is reduced or consumption of dioxygen. Most experiments used a commercial cellulase preparation from Trichoderma reesei and ferricyanide as acceptor. Within 1 min of an addition of cellulase, ferricyanide reduction reached a steady rate. This was converted into a rate of production of substrate for celobiose oxidase, in mol·min^–1. Experiments were conducted either with a constant concentration of cellulase and increasing Avicel, or with constant Avicel and increasing cellulase. Kinetic analysis of the experiments with constant cellulase indicated a K _mof 4.8 ± 1.0 (g cellulose)·1^–1, which was close to the value predicted from binding studies. The specific activity of the cellulase was measured as 375±25 mol·(g cellulase)^–1·min^–1 in experiments with a high cellulose concentration, but was less than half this value when the cellulose was saturated with cellulase. The maximal rate of cellulose degradation was 9.6±1.3 mol·(g cellulose)^–1·min^–1.     

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     

Isolation of a yeast-lyzing Arthrobacter species and the production of the lytic enzyme complex in batch and continuous-flow fermentors

A gram-negative bacterium strongly lytic toward living cells of the food yeast Saccharomyces fragilis was isolated by continuous-flow enrichment from compost. The organism was identified as a species of Arthrobacter. The extracellular lytic enzyme complex produced by this bacterium contained β-1,3-glucanase, mannan mannohydrolase, and proteolytic activities. The polysaccharases were inducible by whole yeast cells. In chemostat cultures on chemically defined media, synthesis of the polysaccharases was very slight and only detectable at dilution rates below 0.02 hr^?1. Enzyme production in defined media was not solely dependent on growth rate but also was influenced by the growth limiting substrate and the culture history. The production of individual depolymerases and of the lytic activity was studied in batch and chemostat cultures containing yeast as the limiting substrate. The maximum specific growth rate of the Arthrobacter under these conditions was 0.22 hr^?1. β-1,3-Glucanase and proteolytic activities were synthesized by exponentially growing bacteria but maximum lytic titers did not develop until the specific growth rate was declining, at which time mannan mannohydrolase syntheses was induced. In yeast limited chemostats polysaccharase syntheses were greatest at the lowest dilution rates examined, namely 0.02 hr^?1. Further optimization of enzyme production was achieved by feeding the Arthrobacter culture to a second-stage chemostat. A comparison of lytic enzyme productivities in batch and chemostat cultures has been made.     

Regulation of nitrogen levels for heterogeneous populations of sewage origin grown in completely mixed reactors

Heterogeneous populations of sewage origin were grown continuously at, dilution rates from 1/12 hr^?1 to dilute-out (1/1 hr^?1) using glucose (1000 mg/l) as carbon source and three concentrations of NH₃-N as the nitrogen source (COD:N = 70:1, 40:1, and 25:1). The effects of nitrogen level and growth rate (dilution rate) on substrate removal, biological solids production, cellular carbohydrate and protein, and NH-N in the effluent were examined. It was found that the optimum level of nitrogen supplementation for the synthetic nitrogen-deficient waste employed should not be based solely on the desired effluent quality with respect to COD removal but should include due consideration of reactor detention time (or dilution rate) and the allowable (or desirable) level of nitrogen leakage in the effluent.     

Yield and maintenance relations of yeast growth in the chemostat at superoptimal temperatures

A model is proposed that accounts for the decreases in yield which occur in chemostat cultures of mesophilic yeasts at superoptimal growth temperatures. Two yield depressing effects were identified, one due to increased maintenance requirements by the viable fraction of the population, the other due to energy substrate dissipation by the nonviable fraction. The two effects are functions of the dilution rate, as is the fraction of nonviable cells. Experimental results were obtained on the yield, maintenance, and dissipation of energy substrate in a glucose-limited chemostat culture of a respiration-deficient mutant of Saccharomyces cerevisiae at 39°C. The rates of glucose utilization for maintenance and for dissipation constituted, respectively, 33–28% and 15–9% of the total glucose utilization rate over the range of dilution rates tested (0.038–0.064 hr^?1), while the yield varied over this range from 0.066–0.085 g of biomass (dry wt) per gram of glucose.     

Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process

A mathematical model for enzymatic cellulose hydrolysis, based on experimental kinetics of the process catalysed by a cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] preparation from Trichoderma longibrachiatum has been developed. The model takes into account the composition of the cellulase complex, the structural complexity of cellulose, the inhibition by reaction products, the inactivation of enzymes in the course of the enzymatic hydrolysis and describes the kinetics of d-glucose and cellobiose formation from cellulose. The rate of d-glucose formation decelerated through the hydrolysis due to a change in cellulose reactivity and inhibition by the reaction product, d-glucose. The rate of cellobiose formation decelerated due to inhibition by the product, cellobiose, and inactivation of enzymes adsorbed on the cellulose surface. Inactivation of the cellobiose-producing enzymes as a result of their adsorption was found to be reversible. The model satisfactorily predicts the kinetics of d-glucose and cellobiose accumulation in a batch reactor up to 70–80% substrate conversion on changing substrate concentration from 5 to 100 g l^?1and the concentration of the enzymic preparation from 5 to 60 g l^?1.     

Kinetic model identification in mixed populations using continuous culture data

The kinetic behavior of heterogeneous microbial populations of sewage origin was studied in a single-stage, isothermal, continuous flow, completely mixed aeration tank. A series of experiments were carried out at various dilutions rates using glucose as the limiting substrate. The cell dry weight and substrate concentration in terms of chemical oxygen demand (COD) were continuously monitored. The results indicate that reproducible steady-state conditions can generally be obtained; however, multiple steady states were observed at dilution rates near washout. At low dilution rates (below about 0.1 hr^?1) the contribution of microorganism decay became appreciable. Using the multiresponse data of cell dry weight and COD, the parameter values in various existing growth models were estimated. The analyses of variance and residuals revealed that models proposed by Moser, Monod, and Contois, each with a decay term added, were significantly better than the other models which were tested.     

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.     

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.     

Ultrasound mediated enzymatic hydrolysis of cellulose and carboxymethyl cellulose

A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm^?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate V_max was enhanced by sonication relative to controls, but the value of the saturation constant K_m was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm^?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm^?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013     

Kinetics of biphasic growth of yeast in continuous and fed-batch cultures

The influence of dilution rate on the production of biomass, ethanol, and invertase in an aerobic culture of Saccharomyces carlsbergensis was studied in a glucose-limited chemostat culture. A kinetic model was developed to analyze the biphasic growth of yeast on both the glucose remaining and the ethanol produced in the culture. The model assumes a double effect where glucose regulates the flux of glucose catabolism (respiration and aerobic fermentation) and the ethanol utilization in yeast cells. The model could successfully demonstrate the experimental results of a chemostat culture featuring the monotonic decrease of biomass concentration with an increase of dilution rate higher than 0.2 hr^?1 as well as the maximum ethanol concentration at a particular dilution rate around 0.5 hr^?1. Some supplementary data were collected from an ethanol-limited aerobic chemostat culture and a glucose-limited anaerobic chemostat culture to use in the model calculation. Some parametric constants of cell growth, ethanol production, and invertase formation were determined in batch cultures under aerobic and anaerobic states as summarized in a table in comparison with the chemostat data. Using the constants, a prediction of the optimal control of a glucose fed-batch yeast culture was conducted in connection with an experiment for harvesting a high yield of yeast cells with high invertase activity.     

Effective production of biologically active water-soluble β-1,3-glucan by a coupled system of Agrobacterium sp. and Trichoderma harzianum

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10?g?L^?1 Tween-80 to the CFS-AT enhanced w-glucan production, presumably by loosening the curdlan ultrastructure and increasing the efficiency of curdlan hydrolysis. A two-stage glucose and DO control strategy was optimal for w-glucan production. At the T. harzianum cell growth stage, the optimal glucose flow rate and agitation speed were 2.0?g?L^?1 hr^?1 and 600?rpm, respectively, and at the w-glucan production stage, they were 0.5?g?L^?1 hr^?1 and 400?rpm, respectively. W-glucan production reached 17.31?g?L^?1, with a degree of polymerization of 19–25. Furthermore, w-glucan at high concentrations exhibited anti-tumor activity against MCF-7, HepG2, and Hela cancer cells in vitro. This study provides a novel, cost-effective, eco-friendly, and efficient microbial fermentation method for the direct production of biologically active w-glucan.