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.     

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.     

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.     

Rapid ethanol fermentation of cellulose hydrolysate. II. Product and substrate inhibition and optimization of fermentor design

High concentrations of both ethanol and sugar in the fermentation broth inhibit the growth of yeast cells and the rate of product formation. Inhibitory effects of ethanol on the yeast strain Saccharomyces cerevisiae NRRL-Y-132 were studied in batch and continuous chemostat cultures. Growth was limited by either glucose or ethanol. Feed medium was supplemented with different ethanol concentrations. Ethanol was found to inhibit growth and the activity of yeast to produce ethanol in a noncompetitive manner. A linear kinetic pattern for growth and product formation was observed according to μ = μ_m (1 – P/P_m) and v = v_m (1 – P/P_m′), where μ_m is the maximum specific growth rate at P = 0 (hr^?1); P_m is the maximum specific product formation rate at P = 0 (hr^?1); P_m is the maximum ethanol concentration above which cells do not grow (g/liter); P_m′ is the maximum ethanol concentration above which cells do not produce ethanol (g/liter). Substrate inhibition studies were carried out using short-time experimental techniques under aerobic and anaerobic condition. The degree of substrate inhibition was found to be higher than that has been reported for ethanol fermentation of pure sugar. The kinetic relationships thus obtained were used to compute growth, substrate utilization, and alcohol production patterns and have been discussed with reference to batch and continuous fermentation of enzymatically produced bagasse hydrolysate.     

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 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.     

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.     

Studies of fungal growth and intermediary carbon metabolism under steady and non-steady state conditions

The physiology of Aspergillus nidulans strain 224 has been studied under conditions of batch- and glucose-limited chemostat-culture and the effect of different steady state growth rates and dissolved oxygen tensions (DOT) examined. Measurements of the specific activities of selected glucose enzymes, the extent of oxygen uptake inhibition by glycolytic inhibitors, and radiorespirometric analyses were made in order to follow the variations in glucose catabolism, which occurred under these conditions. Greatly increased activity of the hexosemonophosphate (HMP) pathway was found during: (i) exponential growth of batch cultures; (ii) at near maximum specific growth rates (μ = 0.072 hr^?1) (DOT = 156 mm Hg); and (iii) at low DOT levels (<30 mm Hg) (μ = 0.050 hr^?1) in chemostat cultures. These changes in glucose eatabolism have been discussed in terms of the biosynthetic demands of the fungus under the influence of changing growth pressures. Preliminary studies also have been made of transition state behavior following stepwise alteration of the DOT. A new steady state was established after 4–5 culture doublings during which period an “overshoot” in HMP pathway activity occurred; these kinetics are indicative of a derepression of certain glucose enzymes. Low molecular weight phenols are synthesized during the exponential phase in batch cultures and these are further metabliized to a major secondary metabolite, melanin, at the onset of stationary phase conditions. The kinetics of tyrosinase production in steady state chemostats differs from those that might be predicted for an enzyme associated solely with secondary metabolism. A primary physiological role for this oxidase in Aspergillus nidulans has been postulated.     

THE EFFECT OF TEMPERATURE ON L-LACTIC ACID PRODUCTION AND METABOLITE DISTRIBUTION OF Lactobacillus casei

The effect of temperature on the growth and L-lactic acid production of Lactobacillus casei G-03 was investigated in a 7-L bioreactor. It was found that the maximum specific growth rate (0.27 hr^?1) and L-lactic acid concentration (160.2 g L^?1) were obtained at a temperature of 41°C. Meanwhile, the maximum L-lactic acid yield, productivity, and dry cell weight were up to 94.1%, 4.44 g L^?1 hr^?1, and 4.30 g L^?1, respectively. At lower or higher temperature, the Lactobacillus casei G-03 showed lower acid production and biomass. Moreover, the main metabolite distribution of strain G-03 response to variations in temperatures was studied. The results suggested that temperature has a remarkable effect on metabolite distribution, and the maximum carbon flux toward lactic acid at the pyruvate node was obtained at 41°C, which had the minimum carbon flux toward acetic acid.     

Pulp and paper mill solid wastes as substrates for single-cell protein production

We have investigated the upgrading of some typical pulp and paper mill solid wastes into protein-enriched animal feed using the cellulolytic fungus Chaetomium cellulolyticum. The waste residues used were six different primary clarifier sludges and a sample of tertiary centricleaner rejects. These were obtained from mills whose modes of operation spanned the range typically in present-day usage: groundwood, sulfite, semichemical, Kraft, and thermomechanical pulping, with and without bleaching. Crude protein production from the solid waste residues is compared to that obtainable from fermentation of untreated or caustic-pretreated sawdusts. Some of these waste residues, especially the Kraft pulp mill rejects, appear to be promising sources of substrate for single-cell protein production. In these preliminary findings, up to 28% dry weight crude protein content of the product has been obtained at specific growth rates of up to 0.12hr^?1 on direct utilization of the wastes.     

Relationship between maintenance energy requirement,mineral salts and efficiency of glucose to ethanol conversion by Zymomonas mobilis

Summary Minimizing the usage of glucose carbon for growth and cell maintenance energy requirement, specific glucose uptake rates, specific ethanol production rates were increased 5-fold. At 0.2 hr^–1 and Y_g = 0.007–0.009, ethanol production rates of 7.99–8.46 g/ltr/hr, Q_p values of 14.85 g/g/hr were obtained. This relationship is discussed in regard to glucose fermentation efficiency.     

Partitioning of river metabolism identifies phytoplankton as a major contributor in the regulated Murray River (Australia)

1. River metabolism was measured over an annual cycle at three sites distributed along a 1000 km length of the lowland Murray River, Australia. 2. Whole system metabolism was measured using water column changes in dissolved oxygen concentrations while planktonic and benthic metabolism were partitioned using light‐dark bottles and benthic chambers. 3. Annual gross primary production (GPP) ranged from 775 to 1126 g O₂ m^?2 year^?1 which in comparison with rivers of similar physical characteristics is moderately productive. 4. Community respiration (CR) ranged from 872 to 1284 g O₂ m^?2 year^?1 so that annual net ecosystem production (NEP) was near zero, suggesting photosynthesis and respiration were balanced and that allochthonous organic carbon played a minor role in fuelling metabolism. 5. Planktonic rates of gross photosynthesis and respiration were similar to those of the total channel, indicating that plankton were responsible for much of the observed metabolism. 6. Respiration rates correlated with phytoplankton standing crop (estimated as the sum of GPP plus the chlorophyll concentration in carbon units), yielding a specific respiration rate of ?1.1 g O₂ g C^?1 day^?1. The respiration rate was equivalent to 19% of the maximum rate of phytoplankton photosynthesis, which is typical of diatoms. 7. The daily GPP per unit phytoplankton biomass correlated with the mean irradiance of the water column giving a constant carbon specific photon fixation rate of 0.35 gO₂ g Chl a^?1 day^?1 per μmole photons m^?2 s^?1 (ca. 0.08 per mole photons m^?2 on a carbon basis) indicating that light availability determined daily primary production. 8. Annual phytoplankton net production (NP) estimates at two sites indicated 25 and 36 g C m^?2 year^?1 were available to support riverine food webs, equivalent to 6% and 11% of annual GPP. 9. Metabolised organic carbon was predominantly derived from phytoplankton and was fully utilised, suggesting that food‐web production was restricted by the energy supply.     

Optimization of recombinantEscherichia coli fed-batch fermentation for bovine somatotropin

Summary Optimum specific growth rate for the production of recombinant bovine somatotropin (bST) was investigated in fed-batchE. coli fermentation with a controlled specific growth rate. Maximum specific bST concentration based on cell mass decreases linearly with the controlled specific growth rate. Productivity of bST is expressed by a quadratic equation as a function of the specific growth rate and has a maximum value at =0.23 hr^–1.     

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.     

Modeling of growth kinetics for an isolated marine bacterium,Oceanimonas sp. BPMS22 during the production of a trypsin inhibitor

Abstract

Protease inhibitors significantly control physiologically relevant protease activities. Protease inhibitors from marine microbial sources are unique due to their rough living environmental conditions. In the present study, a protein protease inhibitor (PI) was produced from marine Oceanimonas sp. BPMS22. Seven different media were screened for the growth of the bacterium and production of PI. Different carbon and nitrogen sources were screened and optimized for the specific protease inhibitor activity. Three different growth models were checked for the best fit of the bacterial growth. A modified Gompertz model was selected as the best model for the growth of Oceanimonas sp. BPMS22 with the maximum specific growth rate of 0.165?hr^?1 and doubling time of 4.2?hr. The production of PI takes place during the non-growing phase of the bacterial growth. A kinetic model for the production of PI during non-growing phase was used for studying various process parameters. From the model, the maximum trypsin inhibitor formation rate of 0.3802?IU per mg of biomass per hour was observed at 49.91?hr.     

Citric acid production from glucose. II. Growth and excretion kinetics in a trickleflow fermentor

The growth and citric acid production kinetics of Saccharomycopsis lipolytica on glucose is investigated in a trickle-flow fermentor. Liquid hold-up and oxygen-transfer coefficient in the reactor column filled with cylindrical wood chips have been determined and found in agreement with chemical engineering correlations. Citric acid production starts at the end of the growth phase and proceeds at a constant specific rate of 0.025 hr^?1for about 80 hr. The fermentor can then be regenerated by addition of ammonia, which induces new growth and excretion phases. Comparing the metabolic behavior of free and immobilized cells, two main kinetic differences are observed. First, the growth phase is linear with the bound cells instead of exponential in the stirred fermentor. Second, in the trickle-bed fermentor acid productivity and oxygen acid yield are reduced by 30%. Oxygen diffusional limitations, mainly in the biomass film, and alterations in bound cell metabolism are shown to be responsible of the kinetic modifications. Simple modelizations of oxygen diffusion effects are also presented to support the interpretation of the experimental data.     

Production and partial characterization of β-1,3-glucanase obtained from Rhodotorula oryzicola

The current study aims to assess the kinetics of population growth of Rhodotorula oryzicola and the production of β-1,3-glucanase (EC 3.2.1.39) enzyme by this yeast. It also aims to obtain the optimum conditions of β-1,3-glucanase enzymatic activity by varying the pH as well as to study the enzyme thermostability. R. oryzicola population doubled within 12?hr. During this period, 9.26 generations were obtained, with 1?hr and 29?min of interval from one generation to the other, with specific growth rate (µ) of 0.15 (hr^?1). The entire microorganism growth process was monitored during β-1,3-glucanases production, and the maximum value was obtained in the stationary phase in the 48-hr fermentation period. pH and temperature optimum values were 4.7 and 96°C, respectively. The enzyme maintained 88% of its activity when submitted to the temperature of 90°C for an incubation period of 1?hr. The results show that the enzyme can be used in industrial processes that require high temperatures and acidic pH.     

Continuous culture of Thiobacillus ferrooxidans on a zinc sulfide concentrate

A zinc sulfide concentrate was leached microbiologically by Thiobacillus ferrooxidans in a continuous stirred tank reactor. A model was developed to predict, the leaching kinetics when the bacterial growth rate was not limited by any substrate other than the zinc concentrate, and it was modified to explain the observed results. Stable steady sates were obtained over a range of dilution rates from 0.0171 to 0.1038 hr^?1. Because a solid substrate was used, the specific growth rate of the bacaeria was not a unique function of the subastrate concentration, and conventional contnuous culture theory based on the Monod equation did not apply to this system. The leaching rates and bacterial growth rates were first order in mineral surface area cocentration.     

Dialysis fermentation. I. Enhanced production of salicylic acid from naphthalene by Pseudomonas fluorescens

The conversion of naphthalene to salicylic acid by Pseudomonas fluorescens was studied as a model for dialysis fermentation. In a demonstration experiment, the continual removal of the product by dialysis and by intermittent replenishment of the dialysate reservoir caused cyclical changes in the concentrations of viable cells and product. The cumulative total amount of both cell mass and salicylate, however, continued to increase steadily until the experiment was terminated after 15 days. At this time the rate of product formation was highest and still increasing, although less than 10% of the cells were viable. The terminal amount of salicylate was about 20-fold greater than the maximum reached in the control fermentation, and was calculated to be 2.6-fold more productive even if the control were optimally recycled. Methods were projected to achieve still further improvements.     

DISTRIBUTION AND PHYSIOLOGICAL DETERMINANTS OF BLUE-GREEN ALGAL NITROGEN FIXATION ALONG A THERMOGRADIENT1

The capacity of thermal algal-bacterial mats to fix nitrogen (N₂) was examined in an alkaline thermal stream, Rabbit Creek, of Yellowstone National Park. Nitrogenase activity and nitrogen-fixation rates of mat cores placed in serum bottles and incubated in situ were estimated by the acetylene-reduction technique. Active nitrogenase was not detected at 60 or 65 C in either the blue-green algal or bacterial undermat components of the mats. Acetylene was reduced by all mats ≤55 C along the thermogradient; mean fixation estimates for the mats ranged from 7 to 5,028 nmoles N₂ fixed · mg Chl a^?1· hr^?1. Maximum fixation occurred at 35 C in the stream; statistical comparison of mean rates ordered the thermogradient mats according to estimated activities: 35 > 40 > 30 > 50 ≥ 55 ≥ 45 C. Mats (≤40 C) dominated by species of Calothrix accounted for ca. 97% of the total nitrogen fixation observed in the stream; the remaining activity was associated with mats containing Mastigocladus laminosus Cohn. Light intensity significantly affected fixation rates of the Calothrix mats which responded in a linear fashion from 9–100% full sunlight (ca. 1,900 μEin · m^?2· sec^?1). Calothrix mats from 30 and 40 C had maximum nitrogenase activity at their growth temperature suggesting that nitrogen fixation along the thermogradient was optimally adapted to in situ temperatures.