Development of high-molar-mass cellobiase complex by spontaneous protein-protein interaction in the culture filtrate ofTermitomyces clypeatus

The 450 kDa cellobiase fromTermitomyces clypeatus which migrates as a single band on IEF, PAGE and SDS-PAGE, was found to possess appreciable sucrase activity. The fungus produced sucrase and cellobiase constitutively in different media but with different activity ratios. The kinetics of secretion of the two enzymes was similar underin vivo andin vitro conditions. HPGPLC analysis of the culture filtrates indicated the presence of both sucrase and cellobiase in the same protein fractions of different molar mass, even in the 30-kDa protein fraction. No free sucrase or cellobiase could be detected in the culture filtrates. It was also observed that fractionation of cellobiase by (NH₄)₂SO₄ precipitation was different with different amounts of associated sucrase activity present in the culture filtrate. The (NH₄)₂SO₄-precipitated cellobiase fraction also contained cellobiases in proteins of widely varied molar mass ranges. However, none of the low-molar mass proteins other than the 450-kDa enzyme could be purified, as all low-molar-mass fractions spontaneously aggregated to the 450-kDa enzyme. Hydrophobic chromatography of the (NH₄)₂SO₄-precipitated fractions followed by HPGPLC of the eluted active fraction yielded both cellobiase-free sucrase and a very low sucrase-containing cellobiase fraction. The cellobiase fraction, homogeneous in PAGE, was also a high-molar-mass protein complex dissociating into a number of protein bands on SDS-PAGE. It was suggested that the 450-kDa cellobiase was not liberated by the fungus as a preformed enzyme complex but that the complex developed through interaction of cellobiase with sucrase underin vitro conditions and the possibility of the involvement of other proteins in the aggregation cannot be excluded.     

Development of a scale-up strategy for Chinese hamster ovary cell culture processes using the kLa ratio as a direct indicator of gas stripping conditions

Herein, we described a scale-up strategy focused on the dissolved carbon dioxide concentration (dCO₂) during fed-batch cultivation of Chinese hamster ovary cells. A fed-batch culture process for a 2000-L scale stainless steel (SS) bioreactor was scaled-up from similarly shaped 200-L scale bioreactors based on power input per unit volume (P/V). However, during the 2000-L fed-batch culture, the dCO₂ was higher compared with the 200-L scale bioreactor. Therefore, we developed an alternative approach by evaluating the k_La values of O₂ (k_La[O₂]) and CO₂ [k_La(CO₂)] in the SS bioreactors as a scale-up factor for dCO₂ reduction. The k_La ratios [k_La(CO₂)/k_La(O₂)] were different between the 200-L and 2000-L bioreactors under the same P/V condition. When the agitation conditions were changed, the k_La ratio of the 2000-L scale bioreactor became similar and the P/V value become smaller compared with those of the 200-L SS bioreactor. The dCO₂ trends in fed-batch cultures performed in 2000-L scale bioreactors under the modified agitation conditions were similar to the control. This k_La ratio method was used for process development in single-use bioreactors (SUBs) with shapes different from those of the SS bioreactor. The k_La ratios for the SUBs were evaluated and conditions that provided k_La ratios similar to the 200-L scale SS bioreactors were determined. The cell culture performance and product quality at the end of the cultivation process were comparable for all tested SUBs. Therefore, we concluded that the k_La ratio is a powerful scale-up factor useful to control dCO₂ during fed-batch cultures.     

Citric Acid Production from Hydrolysate of Pretreated Straw Cellulose by Yarrowia lipolytica SWJ-1b Using Batch and Fed-Batch Cultivation

In this study, crude cellulase produced by Trichoderma reesei Rut-30 was used to hydrolyze pretreated straw. After the compositions of the hydrolysate of pretreated straw were optimized, the study showed that natural components of pretreated straw without addition of any other components such as (NH₄)₂SO₄, KH₂PO₄, or Mg²⁺ were suitable for citric acid production by Yarrowia lipolytica SWJ-1b, and the optimal ventilatory capacity was 10.0 L/min/L medium. Batch and fed-batch production of citric acid from the hydrolysate of pretreated straw by Yarrowia lipolytica SWJ-1b has been investigated. In the batch cultivation, 25.4 g/L and 26.7 g/L citric acid were yields from glucose and hydrolysate of straw cellulose, respectively, while the cultivation time was 120 hr. In the three-cycle fed-batch cultivation, citric acid (CA) production was increased to 42.4 g/L and the cultivation time was extended to 240 hr. However, iso-citric acid (ICA) yield in fed-batch cultivation (4.0 g/L) was similar to that during the batch cultivation (3.9 g/L), and only 1.6 g/L of reducing sugar was left in the medium at the end of fed-batch cultivation, suggesting that most of the added carbon was used in the cultivation.     

Production of Cellulase by Clostridium Papyrosolvens CFR-703

Clostridium papyrosolvens producing filter paperase, carboxymethyl cellulase and cellobiase under anaerobic cultivation conditions at 35 °C is described. Higher activities of filter paperase and carboxymethylcellulases were assayed in 48 h culture filtrate, while maximum cellobiase accumulated in the culture broth at 72 h. Filter paperase, carboxymethylcellulase and cellobiase activities were optimum at 35 °C and pH values of 7.0, 6.5 and 7.5 respectively. Cultivation of the strain in 1000 ml Hungate bottles with 1% cellulose at pH 6.5 and 35 °C produced carboxymethyl cellulase, filter paperase and cellobiase activities of 45, 35 and 20 IU/ml respectively. This revised version was published online in July 2006 with corrections to the Cover Date.     

Influence of culture modes on the microbial production of hyaluronic acid by <Emphasis Type="Italic">Streptococcus zooepidemicus</Emphasis>

The principal objective of this study was to assess the effects of culture modes including batch culture, pulse fed-batch culture, constant feeding rate fed-batch culture, and exponential fed-batch culture on the production of hyaluronic acid (HA) by Streptococcus zooepidemicus. Batch cultures had the highest levels of HA productivity, whereas fed-batch cultures were more favorable with regard to cell growth, and exponential fed-batch cultures evidenced the highest cell concentrations. A two-step culture model was proposed to enhance HA production: an exponential fed-batch culture was conducted prior to 8 h and then sucrose supplementation was applied for 8 h to start the batch fermentation of S. zooepidemicus. HA production and productivity were increased by 36 and 37% in the proposed two-step culture process as compared with that observed in the batch culture, respectively. The proposed two-step culture model can be applied in the production of secondary metabolites, and particularly of the exopolysaccharides.     

Biotechnological aspects of the production of the anticancer drug podophyllotoxin

The natural lignan podophyllotoxin, a dimerized product of two phenylpropanoid moieties which occurs in a few plant species, is a pharmacologically important compound for its anticancer activities. It is used as a precursor for the chemical synthesis of the anticancer drugs etoposide, teniposide and etopophose. The availability of this lignan is becoming increasingly limited because of the scarce occurrence of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. Biotechnological production using cell culture may be considered as an alternative source. Selection of the best performing cell line, its maintenance and stabilization are necessary prerequisites for its production in bioreactors and subsequent scale-up of the cultivation process to the industrial level. Scale-up of growth and product yield depends on a multitude of factors, such as growth medium, physicochemical conditions, seed inoculum, type of reactor and processing conditions. The composition of the growth medium, elicitors and precursors, etc. can markedly influence the production. Optimum levels of parameters that facilitate high growth and product response in cell suspensions of Podophyllum hexandrum have already been determined by statistical design. P. hexandrum cells have successfully been cultivated in a 3-l stirred-tank bioreactor under low shear conditions in batch and fed-batch modes of operation. The batch kinetic data were used to identify the mathematical model which was then used to develop nutrient-feeding strategies for fed-batch cultivation to prolong the productive log phase of cultivation. An improvement in the production of podophyllotoxin to 48.8 mg l^–1 in a cell culture of P. hexandrum was achieved, with a corresponding volumetric productivity of 0.80 mg l^–1 day^–1, when the reactor was operated in continuous cell-retention mode. Efforts are being made to further enhance its production levels by the development of hairy root culture or by varying the channeling of precursors towards the desired biosynthetic pathway by molecular approaches.     

Biogas production and valorization by means of a two-step biological process

The scope of this research work was to investigate biogas production and purification by a two-step bench-scale biological system, consisting of fed-batch pulse-feeding anaerobic digestion of mixed sludge, followed by methane enrichment of biogas by the use of the cyanobacterium Arthrospira platensis. The composition of biogas was nearly constant, and methane and carbon dioxide percentages ranged between 70.5–76.0% and 13.2–19.5%, respectively. Biogas yield reached a maximum value (about 0.4 m³_biogas/kgCOD_i) at 50 days-retention time and then gradually decreased with a decrease in the retention time. Biogas CO₂ was then used as a carbon source for A. platensis cultivation either under batch or fed-batch conditions. The mean cell productivity of fed-batch cultivation was about 15% higher than that observed during the last batch phase (0.035 ± 0.006 g_DM/L/d), likely due to the occurrence of some shading effect under batch growth conditions. The data of carbon dioxide removal from biogas revealed the existence of a linear relationship between the rates of A. platensis growth and carbon dioxide removal from biogas and allowed calculating carbon utilization efficiency for biomass production of almost 95%.     

Production of alkaline serine protease subtilisin Carlsberg by Bacillus licheniformis on complex medium in a stirred tank reactor

Bacillus licheniformis (DSM 641) was cultivated on complex medium in batch and fed-batch operations in a 20-l working volume stirred tank reactor. The medium composition (maltose, glucose, sucrose, fructose, ammonia, phosphate) and O₂ and CO₂ in the off-gas were monitored on-line; pH, pO₂, turbidity, culture fluorescence were monitored in situ; optical density, concentrations of sugars, amino acids, phosphate, proteins, DNA, protease activity and total solids content were monitored off-line. Problems of on-line sampling, cell concentration monitoring, and culture fluorescence measurements and the influence of medium components on the enzyme productivity are discussed. Close relationships between variations of pH, pO₂, O₂ transfer rate and CO₂ production rate on the one hand and cell mass and fluorescence intensity on the other were demonstrated in batch and in fed-batch cultures. Using suitable cultivation conditions, alkaline protease with high volume activity [15300 units (U)/ml] and specific activity (510 U/mg) was produced. By replacing the complex medium with a semisynthetic one, the volumetric activity was reduced by a factor of ten (to 1650 U/ml), but the specific productivity by a factor of only two (to 210 U/ml). Correspondence to: K. Schügerl     

Production of biomass and recombinant human-like collagen in <Emphasis Type="Italic">Escherichia coli</Emphasis> processes with different CO<Subscript>2</Subscript> pulses

The evolution of CO₂ in a fed-batch culture of recombinant Escherichia coli containing human-like collagen (HLC) cDNA was determined with an O₂-enriched air supply (40%, v/v) in a 12.8 l fermentor; a maximum CO₂ concentration of 12.7% in the effluent gas was detected. The CO₂ pulse injection experiments showed that: (1) a 20% CO₂ pulse introduced in the batch cultivation phases inhibited cell growth but if introduced in the fed-batch cultivation phases slightly stimulated growth; and (2) CO₂ inhibited HLC expression only in the expression phase, where the final HLC concentration decreased by 34% under a 3 h 20% CO₂ pulse. The higher the CO₂ concentration and/or the longer the duration of the CO₂ pulse, the stronger the stimulatory or inhibitory effects. An erratum to this article can be found at     

Bioprocess optimization for pectinase production using <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a submerged cultivation system

Background

Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.

Results

The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH₄)₂SO₄, 4.33; K₂HPO₄, 1.36; MgSO₄.5H₂O, 0.05; KCl, 0.05; and FeSO₄.5H₂O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.

Conclusions

Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

     

Kinetic models for astaxanthin production by high cell density mixotrophic culture of the microalga Haematococcus pluvialis

High cell density cultivation of Haematococcus pluvialis for astaxanthin production was carried out in batch and fed-batch modes in 3.7-L bioreactors with stepwise increased light intensity control mode. A high cell density of 2.65 g L⁻¹ (batch culture) or 2.74 g L⁻¹ (fed-batch culture) was obtained, and total astaxanthin production in the fed-batch culture (64.36 mg L⁻¹) was about 20.5% higher than in the batch culture (53.43 mg L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, astaxanthin formation, as well as sodium acetate consumption was proposed. Good agreement was found between the model predictions and experimental data. The models demonstrated that the optimal light intensity for mixotrophic growth of H. pluvialis in batch or fed-batch cultures in a 3.7-L bioreactor was 90–360 μmol m⁻² s⁻¹, and that the stepwise increased light intensity mode could be replaced by a constant light intensity mode. Received 24 December 1998/ Accepted in revised form 23 April 1999     

Production of thuringiensin by fed-batch culture of Bacillus thuringiensis subsp. darmstadiensis 032 with an improved pH-control glucose feeding strategy

A improved pH-control fed-batch strategy for Bacillus thuringiensis subsp. darmstadiensis 032 producing thuringiensin was developed based on the analysis of the batch culture, constant rate fed-batch cultures and the original pH-control fed-batch. Having considered the pH variation and the glucose consumption status, the pH was adjusted from 6.5 to 7.0 by adding base in the late cultivation period of batch culture, and then the pH was kept at 7.0 by glucose feeding. The feeding was terminated when the pH could not be controlled by glucose feeding anymore. The proposed fed-batch strategy effectively avoided underfeeding or overfeeding, and it increased the thuringiensin yield and Y_P/X by 89.51% and 103.2% compared to that of the batch culture, respectively.     

Regulation of cellobiase secretion in Termitomyces clypeatus by co-aggregation with sucrase

Regulated secretory proteins are sorted via selective co-aggregation in eukaryotes. Cellobiase (C) of the filamentous fungus Termitomyces clypeatus remained co-aggregated with sucrase (S), and only one isoform of each of the enzymes was present in intra- and extracellular extracts. Kinetics of secretion of sucrase increased in vivo and in vitro in secreting (Sc) medium and decreased under non-secreting (NSc) conditions similar to those observed for cellobiase. In the Sc condition, total enzyme production and activity ratios of cellobiase and sucrase (C/S) in cell-bound, extra- and intracellular preparations increased with time and were significantly higher from those obtained in non-secretory media. It was concluded that secretion of sucrase in culture medium is under same cellular regulation as that of cellobiase, and sucrase is involved in regulating extracellular release of cellobiase through co-aggregation in the fungus. Received: 27 August 2001 / Accepted: 1 November 2001     

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The production yield of Coenzyme Q₁₀ (CoQ₁₀) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l⁻¹, the dry cell weight (DCW), specific CoQ₁₀ content, CoQ₁₀ production, and the production yield of CoQ₁₀ from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ₁₀ production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ₁₀ content, CoQ₁₀ production, and the product yield of CoQ₁₀ from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ₁₀ production results obtained (626.5 mg l⁻¹ of CoQ₁₀ and 9.25 mg g DCW⁻¹ of specific CoQ₁₀ content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ₁₀ production is available.     

Inferential control of the specific growth rate in fed-batch cultivation processes

Inferential control algorithms and systems for set-point control of the specific growth rate in fed-batch cultivation processes are presented. Realization of the proposed control systems does not require mathematical model and a priori knowledge of the culture of microorganisms under control. Feed-back signal in PID control loops of the investigated systems is based on measurements of the O₂ concentration in exhaust gas and the air supply rate, however, measurements of the other technological parameters related to the culture growth dynamics can also be applied.     

Effect of simple and complex carbon sources,low temperature culture and complex carbon feeding policies on poly-3-hydroxybutyric acid (PHB) content and molecular weight (Mw) from Azotobacter chroococcum 6B

The molecular weight (M _w) of poly-3-hydroxybutyrate (PHB), produced by shake-flask culture of Azotobacter chroococcum showed little variation with increasing glucose concentration as carbon source (being in the range of 400–500 kDa), while M _w increased from 300–400 to 640 kDa when grown with increasing concentration of sugar cane molasses. Molecular weight increased nearly 30% from 48 to 72 h culture time when 5% molasses as carbon source was used, while with glucose the highest M _w was reached at 48 h. Under fermentor cultivation A. chroococcum produced PHB with a relatively high M _w of 1590 kDa at 53 h culture time when grown in modified Burk's medium with glucose as carbon source at an initial C/N ratio (molar basis) of 69 under fermentor cultivation. A batch glucose-grown ammonium-limited fermentor culture was repeatedly fed with sugar cane molasses (initial C/N ratio 69) and it was observed that PHB content curve decreased at a slower rate than in the fed-batch culture in which glucose and sucrose were not consumed in the culture medium after the feed.     

Kinetics of α-amylase production in a batch and fed-batch culture of Bacillus subtilis with caseinate as nitrogen source and starch as carbon source

Summary Analysis of a large number of experimental data from the cultivation of Bacillus subtilis formed the basis for a kinetic model of the process explaining the effect of composition of the culture medium and of the growth rate on the rate of enzyme production. The resulting rate of formation of -amylase (EC 3.2.1.1) reflects the sum of the rate of enzyme production and the rate of its degradation as affected by the environment. The kinetic dependence confirms the previously described mechanism of regulation of enzyme biosynthesis. The mathematical model of the process served here to determine the optimal conditions for enzyme biosynthesis which were then verified in a fed-batch cultivation. The production of the enzyme in fed-batch culture was found to be twice that found in a batch cultivation.Symbols X biomass concentration, g·l^-1 - t time, h - S ₁ caseinate concentration, g·l^-1 - S ₂ starch concentration, g·l^-1 - P product concentration, U·ml^-1 - r _P specific rate of product formation, U·g^-1·h^-1 - R _P total rate of product formation, U·l^-1·h^-1 - Y yield coefficient - specific growth rate, h^-1     

Features of aspergillus japonicus cellobiase formation and optimization of its production

We studied some mechanisms of cellobiase formation in Asp. Japonicus VKM F-2092 aiming at the subsequent development of controlled cultivation to produce the enzyme. Formation of cellobiase was found to be of noninducible constitutive character. The enzyme was predominantly localized in the cytosole and the periplasm, its minor portion being cell-wall bound. The release of the enzyme into the medium was due to autolytic processes. The two-phaseness of cellobiase formation in Asp. Japonicus grown on a mineral medium with 0.5% glucose was shown to be determined by the following mechanisms:

• 1 Catabolic repression. We established glucose concentrations causing the repression and found it to be dependent on carbon sources in the growth medium.
• 2 Catabolic inhibition. Cellobiase activity was found to be inhibited by assumed glucose metabolites in the presence of nitrate nitrogen in the medium.

. Practical recommendations to optimize the process of cellobiase production by Asp. Japonicus are given.     

Kinetic models for phycocyanin production by high cell density mixotrophic culture of the microalga Spirulina platensis

Phycocyanin production by high cell density cultivation of Spirulina platensis in batch and fed-batch modes in 3.7-L bioreactors with a programmed stepwise increase in light intensity program was investigated. The results showed that the cell density in fed-batch culture (10.2 g L⁻¹) was 4.29-fold that in batch culture (2.38 g L⁻¹), and the total phycocyanin production in the fed-batch culture (0.795 g L⁻¹) was 3.05-fold that in the batch culture (0.261 g L⁻¹). An unstructured kinetic model to describe the microalga culture system including cell growth, phycocyanin formation, as well as glucose consumption was proposed. The data fitted the models well (r ² > 0.99). Furthermore, based on the kinetic models, the potential effects of light limitation and photoinhibition on cell growth and phycocyanin formation can be examined in depth. The models demonstrated that the optimal light intensity for mixotrophic growth of Spirulina platensis in batch or fed-batch cultures using a 3.7-L bioreactor was 80160 μE m⁻² s⁻¹, and the stepwise increase in light intensity can be replaced by a constant light intensity mode. Received 28 July 1998/ Accepted in revised form 8 October 1998     

Simple fed-batch cultivation strategy for the enhanced production of a single-sugar glucuronic acid-based oligosaccharides by a cellulose-producing <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> strain

The production of water-soluble single-sugar glucuronic acid-based oligosaccharides (WSOS) by a cellulose producing strain Gluconacetobacter hansenii PJK was studied in a periodically recycled and fed-batch cultivations using glucose/ethanol or glucose only. Fermentations were carried out in a 2 L jar fermenter equipped with a turbine impeller with 6 flat blades. WSOS were produced constantly but the bacterial cellulose (BC) production stopped at 48 h of cultivation in a periodically recycled culture using the exhausted medium supplemented with glucose and ethanol. Tremendous quantities of WSOS were obtained in fed-batch cultivations using glucose/ethanol (35.6 g/L at 132 h of cultivation) or glucose only (86 g/L after 240 h of cultivation) as the nutritional source. However, the BC production yield under these nutritional conditions decreased significantly in comparison to previous studies about the BC production by the same strain. The overall results revealed that G. hansenii is capable of producing enormous quantities of WSOS compared to those reported previously for compounds of a related chemical nature. Moreover, the WSOS production was found to be dependent on the pH of the culture broth.