Enhanced production of cellulase usingAcidothermus cellulyticus in fed-batch culture

Summary Fed-batch fermentations of Acidothermus cellulolyticus utilizing mixtures of cellulose and sugars were investigated for potential improvements in cellulase enzyme production. In these fermentations, we combined cellulose from several sources with various simple sugars at selected concentrations. The best source of cellulose for cellulase production was found to be ball-milled Solka Floc at 15 g/l. Fed-batch fermentations with cellobiose and Solka Floc increased cell mass only slightly, but succeeded in significantly enhancing cellulase synthesis compared to batch conditions. Maximum cellulase activities obtained from fermentations initiated with 2.5 g cellobiose/l and 15 g Solka Floc/l were 0.187 units (U)/ml, achieved by continuous feeding to maintain <0.1 g cellobiose/l, and 0.215 U/ml using the same initial medium when 2.5 g cellobiose/l was step-fed after the sugar was nearly consumed. In batch, dual-substrate systems consisting of simple sugars with Solka Floc, substrate inhibition was evident in terms of specific growth rates, specific productivity values, and maximum enzyme yields. Limiting concentrations of glucose or sucrose at 5 g/l, and cellobiose at 2.5 g/l, in the presence of Solka Floc, yielded cellulase activities of 0.134, 0.159, and 0.164 U/ml, respectively. Offprint requests to: M. E. Himmel     

Enhanced cellulase production in fed-batch culture of Trichoderma reesei C30

The use of a fed-batch cultivation of the fungus Trichoderma reesei (C30) allows cellulase [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] production to occur under optimum conditions, and results in extremely high enzyme titres and productivities. Enzyme levels of 26 U ml^?1 at productivities >130 U l^?1 h^?1 have been achieved. These results are compared with the values obtained in two-stage continuous cultivation of the organism at optimum pH and temperature.     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Enhanced phenylpyruvic acid production with Proteus vulgaris in fed-batch and continuous fermentation

Phenylpyruvic acid is a deaminated form of phenylalanine and is used in various areas such as development of cheese and wine flavors, diagnosis of phenylketonuria, and to decrease excessive nitrogen accumulation in the manure of farm animals. However, reported phenylpyruvic acid fermentation studies in the literature have been usually performed at shake-flask scale with low production. In this study, phenylpyruvic acid production was evaluated in bench-top bioreactors by conducting fed-batch and continuous fermentation for the first time. As a result, maximum phenylpyruvic acid concentrations increased from 1350 mg/L (batch fermentation) to 2958 mg/L utilizing fed-batch fermentation. Furthermore, phenylpyruvic acid productivity was increased from 48 mg/L/hr (batch fermentation) to 104 and 259 mg/L/hr by conducting fed-batch and continuous fermentation, respectively. Overall, this study demonstrated that fed-batch and continuous fermentation significantly improved phenylpyruvic acid production in bench-scale bioreactor production.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

Efficient acetic acid production by repeated fed-batch fermentation using two fermentors

Summary In acetic acid fermentation, the number of viable cells decrease as the acetic acid concentration increases to more than about 40 g/l, which means that the productivity attainable by conventional fed-batch and repeated fed-batch operations using one fermentor is limited. In this paper, based on a fed-batch experiment using Acetobacter aceti 2096, a mathematical model was developed. The optimization carried out showed the superiority of repeated fed-batch operation using two fermentors. The performance evaluation was made with respect to productivity and product concentration. It was shown to be attractive in practice to use multiple fermentors, in particular for high product concentrations. Experiments were then conducted to ascertain the simulation results. Offprint requests to: T. Kobayashi     

Ethanol production from paper material using a simultaneous saccharification and fermentation system in a fed-batch basis

In this work, a recycled paper-derived feedstock was used to produce ethanol by the simultaneous saccharification and fermentation (SSF) process using the thermotolerant yeast Kluyveromyces marxianus CECT 10875. At standard SSF conditions, the highest yield (about 80% of theoretical) was obtained at low substrate concentration and high enzyme loading. With increasing substrate concentration, mixing difficulties appeared which prevented an adequate SSF process performance and limited ethanol production. An SSF fed-batch procedure was then used which permitted an increase in substrate concentrations while maintaining SSF yields similar to that obtained at standard SSF, thus allowing an increased final ethanol production (about 18 g/l).     

Efficient production of gene products by repeated fed-batch fermentation using multiple fermentors

In the cultivation of genetically engineered microorganisms, cell growth decreases after induction, and it becomes eminent when a product such as β-galactosidase is harvested, indicating that conventional repeated fed-batch operation cannot be used to increase productivity. In this study, the use of multiple fermentors to overcome such difficulties was attempted. A mathematical model was developed based on fed-batch experiments using Escherichia coli JM103 harboring the pUR2921 plasmid encoding the β-galactosidase structural gene. Computer simulation demonstrated that improved performance can be attained by repeated fed-batch operation using multiple fermentors. Experiments were then conducted to ascertain the operating conditions.     

Enhanced sophorolipid production by feeding-rate-controlled fed-batch culture

To develop the easier control method for fed-batch culture of sophorolipid production, we chose rapeseed oil as the most productive oil and compared their productivities in relation to different concentrations of glucose. The optimal concentration of glucose was 30 g/L for sophorolipid production. A fed-batch method was conducted using Candida bombicola ATCC 22214 with rapeseed oil as a secondary substrate. The feeding rate of rapeseed oil was dependent on pH and was calculated by the consumption rate of NaOH and rapeseed oil. The glucose concentration was constantly maintained between 30 and 40 g/L. As a result, we have produced a crude sophorolipid up to 365 g/L for 8 days through a feeding-rate-controlled fed-batch process.     

Enhanced production of recombinant streptokinase in Escherichia coli using fed-batch culture

Fed-batch culture strategy is often used for increasing production of heterologous recombinant proteins in Escherichia coli. This study was initiated to investigate the effects of dissolved oxygen concentration (DOC), complex nitrogen sources and pH control agents on cell growth and intracellular expression of streptokinase (SK) in recombinant E. coli BL21(DE3). Increase in DOC set point from 30% to 50% did not affect SK expression in batch culture where as similar increase in fed-batch cultivation led to a significant improvement in SK expression (from 188 to 720 mg l⁻¹). This increase in SK could be correlated with increase in plasmid segregational stability. Supplementation of production medium with yeast extract and tryptone and replacement of liquid ammonia with NaOH as pH control agent further enhanced SK expression without affecting cell growth. Overall, SK concentration of 1120 mg l⁻¹ representing 14-fold increase in SK production on process scale-up from flask to bioreactor scale fed-batch culture is the highest reported concentration of SK to date.     

Bacterial cellulose production by fed-batch fermentation in molasses medium

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.     

Optimized nikkomycin production by fed-batch and continuous fermentation

We performed fed-batch and continuous fermentations to extend the time of maximal nikkomycin production by Streptomyces tendae Tü 901/S 2566. This was achieved by the fed-batch culture technique. Furthermore, high productivity was obtained at slow growth rates in a continuous fermentation process. Different dilution rates with and without carbon limitation were done and the results were compared. Correspondence to : T. Schüz     

Enhanced poly(L-malic acid) production from pretreated cane molasses by Aureobasidium pullulans in fed-batch fermentation

Poly(L-malic acid) (PMA) is a natural polyester with many attractive properties for biomedical application. However, the cost of PMA production is high when glucose is used as a carbon source. To solve this problem, cane molasses as a low-cost feedstock was applied for the production of PMA. Six pretreatment methods were applied to cane molasses before fermentation. Pretreatment with combined tricalcium phosphate, potassium ferrocyanide, and sulfuric acid (TPFSA) removed significant amounts of metal ions from cane molasses. The PMA concentration increased from 5.4?g/L (untreated molasses) to 36.9?g/L (TPFSA-pretreated molasses) after fermentation in shake flasks. A fed-batch fermentation strategy was then developed. In this method, TPFSA-pretreated cane molasses solution was continuously fed into the fermentor to maintain the total sugar concentration at 20?g/L. This technique generated approximately 95.4?g/L PMA with a productivity of 0.57?g/L/hr. The present study indicated that fed-batch fermentation using pretreated cane molasses is a feasible technique for producing high amounts of PMA.     

Enhanced cellulase production in mutants of Thermomonospora curvata

Thermomonospora curvata, a thermophilic actinomycete, secretes multiple forms of endo-beta 1-4-glucanase (EG)when grown on cellulose-mineral salts liquid medium. The EG activity(measured as carboxymethyl cellulose hydrolysis) was separated by ion exchange chromatography into three distinct components which differ in their kinetic properties. Exposure of Thm. curvata to ultraviolet light, N-nitrosoguanidine, or ethane methyl sulfonate produced mutants with enhanced EG production. Selection of colonies which cleared cellulose agar plant containing 2-deoxtglucose of glycerol yielded mutants having 1.5 to 2.6 times the extracellular EG and saccharifying activity (measured by filter-paper and cotton-fiber hydrolysis). The secretion of extracellular protein was increased proportionally in mutant cultures.     

An Algorithm for operating a fed-batch fermentation using quasi-steady state

The determination of an optimum feeding profile of a fed-batch fermentation requires the solution of a singular optimum control problem, which is often complicated by changes in the process kinetics during the fermentation. The procedure of optimization may be sufficiently simple, if the feeding part of fermentation is carried out in the quasi-steady state. In this work an algorithm for operating a fed-batch fermentation using mentioned regime is offered. The algorithm supposes a periodical correction of the feeding strategy. Applying to fed-batch lysine fermentation demonstrate efficacy of this algorithm over frequently used strategies.     

Enhanced spore production ofBacillus thuringiensis by fed-batch culture

Summary Fed-batch culture was carried out to increase cell mass followed by batch culture for spore production ofbacillus thuringiensis. High cell mass obtained by increasing the feeding glucose concentration in constant fed-batch culture which supported fast cell growth resulted in good sporulation during subsequent batch culture, and the maximum cell mass of 72.6 g/L and spore concentration of 1.25×10¹⁰ spores/mL could be obtained.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.