Modelling of the process yields of a whey fermentation

Summary The biomass yields (y) and COD reduction efficiencies (η) of a whey fermentation by Kluyveromyces fragilis were studied in a 100-1 fermenter at various stirrer speeds and lactose concentrations, and compared to those obtained in 10-1 and 15-1 fermenters at constant values of the oxygen transfer coefficient (k_La) and air velocity. The empirical models previously constructed by using the 15-1 fermenter data could be used to predict the yields on the other scales by calculating for each run the 15-1 fermenter which would provide the same oxygen transfer coefficient measured by the sulphite method on each fermenter under study. To make this model independent of stirrer speeds used in each generic fermenter, the effect of aeration and mixing was incorporated into an overall parameter (k_La) and the values of y and η were correlated only with temperature, lactose level and k_L a, since these variables were approximately orthogonal. The validity of this model was finally checked against the yields reported by Wasserman et al. (1961) in a 6-m³ fermenter, thus confirming the capability of the model to provide a reliable basis for further scale-up on the production scale.     

An effective and simplified scale-up strategy for acarbose fermentation based on the carbon source control

The scale-up strategy for acarbose fermentation by Actinoplanes sp. A56 was explored in this paper. The results obtained in shake-flask cultivation demonstrated that the ratio of maltose and glucose had significant effects on the biosynthesis of acarbose, and the feeding medium containing 3:1 (mass ratio) of maltose and glucose was favorable for acarbose production. Then the correlation of the carbon source concentration with acarbose production was further investigated in 100-l fermenter, and the results showed that 7.5–8.0 g of total sugar/100 ml and 4.0–4.5 g of reducing sugar/100 ml were optimal for acarbose production. Based on the results in 100-l fermenter, an effective and simplified scale-up strategy was successfully established for acarbose fermentation in a 30-m³ fermenter, by using total sugar and reducing sugar as the scale-up parameter. As a result, 4,327 mg of acarbose/l was obtained at 168 h of fermentation.     

Real-time fluid dynamics investigation and physiological response for erythromycin fermentation scale-up from 50 L to 132 m<Superscript>3</Superscript> fermenter

The physiological response of erythromycin fermentation scale-up from 50 L to 132 m³ scale was investigated. A relatively high oxygen uptake rate (OUR) in early phase of fermentation was beneficial for erythromycin biosynthesis. Correspondingly, the maximal consistency coefficient (K) reflecting non-Newtonian fluid characteristics in 50 L and 132 m³ fermenter also appeared in same phase. Fluid dynamics in different scale bioreactor was further investigated by real-time computational fluid dynamics modeling. The results of simulation showed that the impeller combination in 50 L fermenter could provide more modest flow field environment compared with that in 132 m³ fermenter. The decrease of oxygen transfer rate (OTR) in 132 m³ fermenter was the main cause for impairing cell physiological metabolism and erythromycin biosynthesis. These results were helpful for understanding the relationship between hydrodynamic environment and physiological response of cells in bioreactor during the scale-up of fermentation process.     

Dissolved oxygen as principal parameter for conidia production of biocontrol fungi Trichoderma viride in non-Newtonian wastewater

Dissolved oxygen (DO) concentration was selected as a principal parameter for translating results of shake flask fermentation of Trichoderma viride (biocontrol fungi) to a fermenter scale. All fermentations were carried out in a 7.5 l automated fermenter with a working volume of 4 l. Fermentation performance parameters such as volumetric oxygen transfer coefficient (k _L a), oxygen uptake rate (OUR), rheology, conidia concentration, glucose consumption, soluble chemical oxygen demand, entomotoxicity and inhibition index were measured. The conidia concentration, entomotoxicity and inhibition index were either stable or improved at lower DO concentration (30%). Variation of OUR aided in assessing the oxygen supply capacity of the fermenter and biomass growth. Meanwhile, rheological profiles demonstrated the variability of wastewater during fermentation due to mycelial growth and conidiation. In order to estimate power consumption, the agitation and the aeration requirements were quantified in terms of area under the curves, agitation vs. time (rpm h), and aeration vs. time (lpm h). This simple and novel strategy of fermenter operation proved to be highly successful which can be adopted to other biocontrol fungi.     

Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli.

In dialysis fermentations inhibiting metabolites can be removed from cell suspensions resulting in a prolonged exponential growth phase and higher production yields. Because of successful high cell density cultivations of Escherichia coli in a laboratory dialysis reactor, a scale-up of the process was investigated. To provide sufficient membrane area for dialysis in a technical scale fermenter, an external membrane module was used, that was also applied for oxygen supply to the culture in the external loop. Cultivations with recombinant E. coli K12, with and without induction, in 2- and 300-l reactors were carried out using external modules. Cell densities exceeding 190 g l(-1), previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-l reactor was increased to the 3.8-fold of industrial fed-batch-fermentations.     

Improved production of <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. ECU1011 acetyl esterase by medium design and fed-batch fermentation

We optimized culture medium and batch-fed fermentation conditions to enhance production of an acetyl esterase from Pseudomonas sp. ECU1011 (PSAE). This enzyme enantioselectively deacetylates α-acetoxyphenylacetic acid. The medium was redesigned by single-factor and statistical optimization. The addition of ZnSO₄ enhanced enzyme production by 37%. Yeast extract concentration was directly associated with the enzyme production. The fermentation was scaled up in a 5-l fermenter with the optimized medium, and the correlations between enzyme production and dissolved oxygen, pH, and feeding strategy were investigated. The fermentation process was highly oxygen-demanding, pH sensitive and mandelic acid-inducible. The fermentation pH was controlled at 7.5 by a pH and dissolved oxygen feedback strategy. Feeding mandelic acid as both a pH regulator and an enzyme inducer increased the enzyme production by 23%. The results of the medium redesign experiments were confirmed and explained in fed-batch culture experiments. Mathematical models describing the fermentation processes indicated that the enzyme production was strongly associated with cell growth. The optimized pH and dissolved oxygen stat fed-batch process resulted high volumetric production of PSAE (4166 U/l, 7.2-fold higher than the initial) without enantioselectivity decline. This process has potential applications for industrial production of chiral mandelic acid or its derivatives.     

Optimization of whey fermentation in a jar fermenter

Summary The fermentation of whey by Kluyveromyces fragilis IMAT 1872 was studied in a 15-l jar fermenter to assess the influence of temperature, lactose concentration, aeration and agitation on the biomass yield.Optimization of the operating parameters resulted in a 57.2% yield.The observations were analyzed in a composite design. Canonical analysis and an F-test showed that only the first two principal axes (i.e., oxygen transfer coefficient and lactose inhibition factors) of the system under study were significant. Therefore, the observations were fitted to a quadratic expression, by using only these factors: 86% of the data fall within a 10% deviation band.This empirical model allowed the formulation of an operating strategy to select the set of conditions yielding the maximum value of biomass.     

The effect of low oxygen uptake rate on the fatty acid profile of the oleaginous yeast Apiotrichum curvatum

Summary The effect of limiting the available oxygen on the fatty acid profile of Apiotrichum curvatum ATCC 20509 during growth on sulphuric acid casein whey was studied. At oxygen uptake rates (OUR) lower than 7 mmol O₂/l per hour, applied during the oil accumulating phase of the fermentation, a decrease in total unsaturated fatty acids was observed. It was possible to decrease the unsaturated fatty acids (oleate from 55% to 41% and linoleate from 9% to 3%) by limiting the OUR of the culture to <3 mmol O₂/l per hour. However at this low OUR, a lower oil coefficient (a measure of the efficiency of lactose substrate conversion to oil) was recorded. Furthermore the fermentation time was increased. An OUR of 5 mmol O₂/l per hour appeared to be the limit below which adverse effects on oil yields and increased fermentation times occurred. At this OUR, the accumulated oil contained 45% oleate and 5% linoleate. These effects were demonstrated in a 20-l air-lift fermentor and confirmed in a scaled down 500-l industrial type bubble column fermentor. Offprint requests to: R. J. Davies     

Effect of cycling dissolved oxygen concentrations on product formation in penicillin fermentations

Summary Limitations in mass and momentum transfer coupled with high hydrostatic pressures create significant spatial variations in dissolved gas concentrations in large fermenters. Microorganisms are subjected to fluctuating environmental conditions as they pass through the zones in a stirred vessel or along a closed loop fermenter.A 7-litre fermenter was modified to simulate the dissolved gas and hydrostatic pressure gradients in large vessels.The effect of cycling dissolved oxygen tension (DOT) on penicillin production by Penicillium chrysogenum P1 was investigated. The fermentation was affected by evironmental conditions such as medium composition, pH, size of inoculum, stirrer speed and DOT. Inoculum size below 10% (v/v) and stirrer speeds above 850 rpm caused significant reductions in specific prenicillin production rates (q_pen). q_pen values were measured at different constant DOT levels. Below 30% air saturation q_pen decreased sharply and no production was observed at 10%. Penicillin synthesis was impaired irreversibly below 10% DOT. The same profile was observed at higher stirrer speeds and air flow rates indicating that the effect was a physiological one. Oxygen uptake of the culture was affected significantly below 7% DOT, demonstrating that the critical DOT values for penicillin production and oxygen uptake are two distinct parameters. Carrying out the fermentation at one atmosphere over pressure was found to have no effect. When the dissolved oxygen concentration of the culture medium was cycled around the critical DOT for penicillin production, a considerable decrease in the specific penicillin production rate was observed. The effect was reversible but not transient, indicating a shift in cell metabolism.These results demonstrate the unfavourable effect of fluctuating environmental conditions on culture performance in stirred tanks. They suggest that these effects should be accounted for during strain selection, process development and scale up stages of an industrial process if the productivities in small scale vessels are to be obtained.     

Production of cellulase-free xylanase by a thermotolerant Streptomyces sp. grown on agricultural waste and media optimization using mixture design and Plackett–Burman experimental design methods

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 on finely ground cane bagasse at 55 °C. The optimal medium composition was developed by applying the mixture design and linear mathematical program, and evaluated using the Plackett–Burman experimental design. The best composition of basal medium was found by using the mixture design method. The highest xylanase activity, 10.6 IU, was obtained after 6 days of fermentation in shaked flask at 100 rpm, 55 °C, pH 7. Both experimental designs showed that trace elements induced xylanase production. With fermentation in a 5-l fermenter, xylanase activity of 12.5 IU was achieved.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Cultivation of anaerobic fungi in a 10-l fermenter system for the production of (hemi-)cellulolytic enzymes

 Two species of anaerobic fungi, i.e. Piromyces strain E2 and Neocallimastix patriciarum strain N2, were cultivated in a 10-l batch fermenter with filter- paper cellulose as the carbon source. The accumulation of fermentation products, production of extracellular protein and (hemi-)cellulolytic enzymes were monitored during growth. Growth of Piromyces E2 in the fermenter resulted in a shift in the fermentation pattern to more acetate and formate and less ethanol, lactate, succinate and malate, possibly because of removal of hydrogen. The specific activities of Avicelase, endoglucanase, β-glucosidase and xylanase were up to threefold higher compared to small batch cultures. Enzyme activities produced per gram of cellulose were up to four times the values reported for Piromyces E2 grown in a semi-continuous coculture with the methanogen Methanobacterium formicicum. The performance of fermenter enzyme preparations from the anaerobic fungi with respect to hydrolysis of Avicel compared well to that of preparations of Trichoderma reesei. However, addition of exogenous β-glucosidase was indispensible with the latter preparation for the complete conversion to glucose. Received: 14 December 1995/Received revision: 19 March 1996/Accepted: 25 March 1996     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

Optimization of whey fermentation in a shaken-flask fermenter

Summary A composite design technique was used to optimize the fermentation of whey by Kluyveromyces fragilis IMAT 1872. The experimental variables (temperature T, salts NP, yeast-extract YE and lactose level L), but not pH, were found to be significant at a level of 5%. The optimal operating conditions were determined (T=36.4°C, pH=5.1, NP=0.47% w/v, YE=0.114% w/v, and L=24.75 g/l). Temperature and lactose level exhibited a great influence on the biomass yield about the stationary point. Canonical analysis showed that these variables are not mutually independent. The optimal conditions will be used as starting point for a factorial design on a jar fermenter.     

Construction of a novel recombinant Escherichia coli strain capable of producing 1,3–propanediol and optimization of fermentation parameters by statistical design

Summary The structural gene yqhD from a wild-type Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme and the structural gene dhaB from Citrobacter freundii encoding glycerol dehydratase were amplified by using the PCR method. The temperature control expression vector pHsh harboring the yqhD and dhaB genes was transformed into E. coli JM109 to yield the recombinant strain E. coli JM109 (pHsh-dhaB-yqhD). The response surface method (RSM) was then applied to further optimize the fermentation condition of the recombinant strain. A mathematical model was then developed to show the effect of each medium composition and their interactions on the production of 1,3-propanediol by recombinant strain E. coli JM109. The model estimated that a maximal yield of 1,3-propanediol (43.86 g/l) could be obtained when the concentrations of glycerol, yeast extract and vitamin B₁₂ were set at 61.8 g/l, 6.2 g/l and 49 mg/l, respectively; and the fermentation time was 30 h. These predicted values were also verified by validation experiments. Compared with the values obtained by other runs in the experimental design, the optimized medium resulted in a significant increase in the yield of 1,3-propanediol. The yield and productivity under the optimal parameters and process can reach 43.1 g/l and 1.54 g/l/h. Maximum 1,3-propanediol yield of 41.1 g/l was achieved in a 5-l fermenter using the optimized medium. This makes the engineered strain have potential application in the conversion of glycerol to 1,3-propanediol on an industrial scale.     

Production of lactobionic acid from whey by Pseudomonas sp. LS13-1

Pseudomonas sp. LS13-1 was isolated as a producer of lactobionic acid from whey and when grown with 207 g whey l^-1 (150 g lactose l^-1 equivalent) and three intermittent additions of 69 g whey l^-1 (50 g lactose l^-1 equivalent) in a fed-batch culture at pH 5.5 in a 2-l jar fermenter, it produced 175 g lactobionic acid l^-1 after 180 h. In a lactose medium it produced 240 lactobionic acid l^-1 from a total of 300 g lactose l^-1 after 155 h. With the addition of 20 CaCO₃ l^-1 instead of pH control, 290 g lactobionic acid l^-1 was produced in the lactose medium after 155 h with a yield of higher than 90% (mon mol^-1).     

Optimization of gluconic acid synthesis by removing limitations and inhibitions

The optimization task was performed using the gluconic acid synthesis by the Acetobacter methanolicusMB 58 strain. The microorganisms were grown continuously on methanol as the growth substrate. After finishing the growth process by the deficiency of N and P, the gluconic acid synthesis was started by adding glucose. The synthesis process was performed continuously. The oxygen transfer rate depended on the gluconic acid concentration. During the growth process, the oxygen transfer rate reached a value of about 13 g O₂ · kg^?1 · h^?1using a 30-l glass fermenter equipped with a 6 blade stirrer and fully baffled. This rate declined to a value of between 2 and 5 g O₂ · kg^?1 · h^?1 in the presence of gluconic acid concentrations above 150 g gluconic acid · kg^?1medium. The yield (g gluconic acid · g^?1glucose) depended on the gluconic acid concentration and amounted to y = 0.7 in relation to 150 g gluconic acid · kg^?1medium and y = 0.8 in relation to 200 g · kg^?1medium, respectively. The fermenters were coupled with ultrafiltration moduls (Fa. ROMICON and Fa. SARTORIUS). The biomass concentrations amounted from 5 to 40 g dry mass kg^?1medium. The ultrafiltration modules retained the biomass within the fermentation system. A glucose solution (30 to 50 weight percent glucose) was continuously dosed into the fermenter. The retention time was chosen between 2 and 30 h. The gluconic acid synthesis rate reached values of up to 32 g gluconic acid · kg^?1 · h^?1. Within a range of up to 250 g gluconic acid · kg^?1medium, the acid concentration had no influence on the enzyme activity.     

Development of Media and Automated Liquid Fermentation Methods to Produce Desiccation-Tolerant Propagules ofTrichoderma harzianum

A suitable medium was developed from modified Richard's medium plus V8 juice (RM8) to produce high levels of desiccation-tolerant conidia ofTrichoderma harzianumstrain 1295-22. The addition of 9% (v/v) glycerol to RM8 improved both biomass production and desiccation tolerance of the conidia ofT. harzianum.This medium was then used in a laboratory scale fermenter (1.5 liter) to determine optimal operating conditions. The optimal temperature for conidial production and desiccation tolerance improvement in the fermenter was 32°C when dissolved oxygen was maintained at 50% saturation of air, and the stirring rate was 1000 revolutions per minute. The initial water potential of the medium (with 9% glycerol) was −3.7 MPa, the pH was 6, and neither was controlled during fermentation. Changes in medium pH and dissolved oxygen were associated with the stages of morphological development and conidiation. The pH of the medium decreased concurrently with germ-tube elongation and mycelium development and then increased to 6.0–6.2 at phialide formation. Intensive conidiation occurred at pH 6.3–6.5 and reached its maximal level at 6.9–7.1. Changes in pH values could be used as indicators to monitor the morphological development and conidiation ofT. harzianumduring fermentation. The use of a 48-h-old culture inoculum, rather than conidial inoculum, to start fermentation reduced the time required to complete the shift from vegetative growth to phialide formation. Intensive conidiation occurred immediately after the addition of culture inoculum and reached maximum levels within 68 h of fermentation. Dry weight of biomass increased with the duration of fermentation and was greatest at 96 h. However, no improvements in conidia/gram and CFU/gram were achieved after 72 h of fermentation. The desiccation tolerance of conidia harvested at 72 or 96 h was significantly (P = 0.05) greater than that of conidia harvested at 48 h of fermentation. Results obtained from this study could be used for further scale-up of the fermentation process.     

基因重组大肠杆菌表达HrpNEcc蛋白的发酵条件及诱导条件优化

对已构建好的表达HrpNEcc蛋白的工程菌BL21(DE3)/pET30a(+)hrpN Ecc的摇瓶发酵条件及乳糖诱导进行优化, 通过在7L发酵罐中放大发酵实验,以期提高蛋白产量并降低生产成本。在摇瓶中优化的发酵及诱导条件是：5% 的接种量,TB培养基,菌体培养至对数生长前期,添加3g/L外源诱导剂乳糖时,HrpNEcc蛋白产量可达417.60mg/L,比不添加乳糖时提高了36.73%,比用IPTG诱导时提高了16.85%。7L发酵罐中发酵,获得菌体湿重达到57.24g/L（WCW）,可溶性HrpNEcc蛋白产量占细胞总蛋白的50.2%,为3.29 g/L。     

Biotechnological lycopene production by mated fermentation of <Emphasis Type="Italic">Blakeslea trispora</Emphasis>

A semi-industrial process (800-l fermentor) for lycopene production by mated fermentation of Blakeslea trispora plus (+) and minus (–) strains has been developed. The culture medium was designed at the flask scale, using a program based on a genetic algorithm; and a fermentation process by means of this medium was developed. Fermentation involves separate vegetative phases for (+) and (–) strains and inoculation of the production medium with a mix of both together. Feeding with imidazole or pyridine, molecules known to inhibit lycopene cyclase enzymatic activity, enhanced lycopene accumulation. Different raw materials and physical parameters, including dissolved oxygen, stirring speed, air flow rate, temperature, and pH, were checked in the fermentor to get maximum lycopene production. Typical data for the fermentation process are presented and discussed. This technology can be easily scaled-up to an industrial application for the production of this carotenoid nowadays widely in demand.