Production of beta-glucan and related glucan-hydrolases by Botryosphaeria rhodina

AIMS: Characterization of beta-glucan production from Botryosphaeria rhodina DABAC-P82 by detecting simultaneously glucan-hydrolytic enzymes and their localization, culture medium rheology and oxygen transfer. METHODS AND RESULTS: Mycelium growth, beta-glucan production, substrate consumption and glucan-hydrolytic enzymes were monitored both in shaken flasks and in a 3-l stirred-tank bioreactor. Glucan production (19.7 and 15.2 g l(-1), in flask and bioreactor, respectively) was accompanied by extra-cellular and cell-bound beta-glucanase and beta-glucosidase activities. In the bioreactor scale, in the time interval of 0-78 h the apparent viscosity of the culture broth exhibited a general increase; thereafter, it began to reduce, probably because of the above glucan-hydrolytic activities. Moreover, the culture media collected after 45 h behaved as solid-like materials at shear rates smaller than 0.001 s(-1), as pseudo-plastic liquids in the middle shear rate range and as Newtonian ones at shear rates greater than 1000 s(-1). CONCLUSION: The greatest beta-glucan accumulation in the bioreactor was found to be associated with nitrogen and dissolved oxygen concentrations smaller than 0.15 g l(-1) and 25%, respectively, and with the peak points of the glucan-degrading enzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A careful analysis of the critical factors (such as, culture broth rheology, oxygen mass transfer and glucan-hydrolytic enzymes) limiting the beta-glucan production by B. rhodina is a prerequisite to maximize beta-glucan yield and production, as well as to define the process flow sheet capable of maximizing biopolymer recovery, solvent re-utilization and glucose consumption.     

Exopolysaccharide production from Sclerotium glucanicum NRRL 3006 and Botryosphaeria rhodina DABAC-P82 on raw and hydrolysed starchy materials

AIMS: Evaluation of fermentative usage of raw starchy materials for exopolysaccharide (EPS) production by Sclerotium glucanicum NRRL 3006 and Botryosphaeria rhodina DABAC-P82. METHODS AND RESULTS: Non-hydrolysed corn starch, soft wheat flour, potato flour, cassava flour, sweet and industrial potato flours, and corn starch hydrolysed to different dextrose equivalent (DE) were tested in shaken culture for EPS production. Both fungal strains produced EPS on all tested materials but the production was maximum on hydrolysed corn starch (30.5 and 19.8 g l(-1) by B. rhodina and S. glucanicum on corn starch at 100 and 62 DE, respectively). CONCLUSIONS: Raw starchy materials as such and, in particular, partially or totally hydrolysed corn starch could be used profitably for EPS production by S. glucanicum and B. rhodina. SIGNIFICANCE AND IMPACT OF THE STUDY: The excellent EPS production, productivity and yield of B. rhodina DABAC-P82 when grown on 60 g l(-1) of totally hydrolysed corn starch.     

Production of swainsonine from Metarhizium anisopliaein stirred-tank and air-lift reactors

Growth of Metarhizium anisopliaein modified starch-casein medium produced 61, 50, and 58 mg swainsonine/l when cultured in shaken flasks, stirred-tank and air-lift reactors respectively. Over approximately 45 h, the maximum swain-sonine specific productivity was 0.47 mg/g.h in a stirred tank reactor and 0.32 mg/g.h in an air-lift reactor. After 120 h, increasing broth viscosity was encountered in the latter fermenter.     

β-Glucan production by Botryosphaeria rhodina on undiluted olive-mill wastewaters

Botryosphaeria rhodina produced beta-glucan when grown on undiluted olive-mill wastewaters (OMW). The production of exopolysaccharide increased with the COD up to 17.2 g l(-1) on the most loaded OMW (151 and 66 g l(-1) of COD and total sugar, respectively). The total phenol content of OMW was reduced from 8 to 4.1 g l(-1).     

Production of bacterial cellulose by Acetobacter xylinumwith an air-lift reactor

Bacterial cellulose was produced by Acetobacter xylinum subsp. surcrofermentans BPR2001 in a 50 liter air-lift reactor using fructose as the main carbon source. When air was supplied, the production of the cellulose was only 2.3 g/l in 80 h but when O -fortified air was supplied, the cellulose concentration increased to 5.63 g/l in 28 h and the productivity of the cellulose in an air-lift reactor with O -fortified air supply was comparable to that in a mechanically agitated jar fermenter.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Air-bubbling, hollow-fiber reactor with cell bleeding and cross-flow filtration

Continuous asymmetric reduction of dyhydrooxoisophorone (DOIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) was achieved by a thermophilic bacterium Bacillus stearothermophilus NK86-0151. Three reactors were used: an air-bubbling hollow-fiber reactor with cell bleeding and cross-flow filtration, an air-lift reactor, and a CSTR with PAA immobilized cells. The maximum cell concentration of 11.1 g dry wt L(-1) was obtained in an air-bubbling hollow-fiber reactor, while in the other reactors the cell densities were between 3.5 and 4.1 g dry wt L(-1) The optimum bleed ratio was 0.1 at the dilution rate 0.3 h(-1) in the hollow-fiber reactor. The highest viable cell concentration was maintained in the dilution range of 0.4-0.7 h(-1) by a combination of proper cell bleeding and cross-flow filtration. The maximum volumetric productivity of 4-HTMCH reached 826 mg L(-1) h(-1) at the dilution rate 0.54 h(-1). This value was 4 and 2 times higher than those in the air-lift reactor and CSTR, respectively. The increasing viable cell concentration increased the volumetric productivity of 4-HTMCH. A cell free product solution was continuously obtained by cross-flow filtration.     

Evaluation of plastic-composite supports in repeated fed-batch biofilm lactic acid fermentation by Lactobacillus casei

A customized stirred-tank biofilm reactor was designed for plastic-composite supports (PCS). In repeated-batch studies, the PCS-biofilm reactors outperformed the suspended-cell reactors by demonstrating higher lactic acid productivities (2.45 g l(-1) h(-1) vs 1.75 g l(-1) h(-1)) and greater glucose consumption rates (3.27 g l(-1) h(-1) vs 2.09 g l(-1) h(-1)). In the repeated fed-batch studies, reactors were spiked periodically with concentrated glucose (75%) to maintain a concentration of approximately 80 g of glucose l(-1) in the bioreactor. In suspended-cell fermentations with 10 g of yeast extract (YE) l(-1) and zero, one, two, and three glucose spikes, the lactic acid productivities were 2.64, 1.58, 0.80, and 0.62 g l(-1) h(-1), respectively. In comparison, biofilm reactors with 7 g of YE l(-1) and zero, one, two, and three glucose spikes achieved lactic acid productivities of 4.20, 2.78, 0.66, and 0.94 g l(-1) h(-1), respectively. The use of nystatin (30 U ml(-1)) subdued the contaminating yeast population with no effect on the lactic acid productivity of the biofilm reactors, but it did affect productivity in the suspended-cell bioreactor. Overall, in repeated fed-batch fermentations, the biofilm reactors consistently outperformed the suspended-cell bioreactors, required less YE, and produced up to 146 g of lactic acid l(-1) with 7 g of YE l(-1), whereas the suspended-cell reactor produced 132 g l(-1) with 10 g of YE l(-1).     

Studies on citric acid production with immobilized Yarrowia lipolytica in repeated batch and continuous air-lift bioreactors

Citric acid was produced from glucose in repeated-batch shake-flask and continuous air-lift cultivations by calcium-alginate-immobilized Yarrowia lipolytica A-101 yeast. The medium composition was systematically studied in a batch system by using experimental design and empiric modelling. The highest citric acid product concentration of 39 g/l was reached with a medium containing 150 g/l of glucose, 0.105 g/l of potassium dihydrogen phosphate, 0.84 g/l of magnesium sulphate and 21 mg/l of copper sulphate (5.2 mg/l of copper). The results were further improved by hardening the alginate carrier beads with glutaraldehyde, and by activation of the immobilized biocatalyst in a nutrient solution. In continuous air-lift bioreactors with varying height-to-diameter ratio the highest productivity of 350 mg/l per hour with a dilution rate of 0.023 l/h and a citric acid product concentration of 12 g/l was reached with a ratio of 3. Correspondence to: H. Kautola     

Shear stress enhances microcin B17 production in a rotating wall bioreactor, but ethanol stress does not

Stress, including that caused by ethanol, has been shown to induce or promote secondary metabolism in a number of microbial systems. Rotating-wall bioreactors provide a low stress and simulated microgravity environment which, however, supports only poor production of microcin B17 by Escherichia coli ZK650, as compared to production in agitated flasks. We wondered whether the poor production is due to the low level of stress and whether increasing stress in the bioreactors would raise the amount of microcin B17 formed. We found that applying shear stress by addition of a single Teflon bead to a rotating wall bioreactor improved microcin B17 production. By contrast, addition of various concentrations of ethanol to such bioreactors (or to shaken flasks) failed to increase microcin B17 production. Ethanol stress merely decreased production and, at higher concentrations, inhibited growth. Interestingly, cells growing in the bioreactor were much more resistant to the growth-inhibitory and production-inhibitory effects of ethanol than cells growing in shaken flasks.     

Light/dark cycles in the growth of the red microalga porphyridium sp

The effect of light/dark cycles on the growth of the red microalga Porphyridium sp. was studied in a tubular loop bioreactor with light/dark cycles of different frequencies and in two 35-L reactors: a bubble column reactor and an air-lift reactor. Photon flux densities were in the range of 50 to 300 μE m-2 s-1, and flow rates were 1 to 10 L min-1. Under conditions of low illumination and high flow rates, similar results were obtained for the bubble column and air-lift reactors. However, higher productivities-in terms of biomass and polysaccharide-were recorded in the air-lift reactor under high light intensity and low gas flow rates. The interactions of both photosynthesis and photoinhibition with the fluid dynamics in the bioreactors was taken as the main element that allowed us to interpret the differences in performance of the bubble column and the air-lift reactor. It is suggested that the cyclic distribution of dark periods in the air-lift reactor facilitates better recovery from the photoinhibition damage suffered by the cells. Copyright 1998 John Wiley & Sons, Inc.     

Production of cellulase by Aspergillus niger biofilms developed on polyester cloth

AIMS: To compare cellulase production by Aspergillus niger ATCC 10864 biofilms on polyester cloth and freely suspended cultures in shaken flasks and microbioreactors of bubble column type. METHODS AND RESULTS: Both shaken flasks and oxygenated microbioreactors containing 40 ml of production medium were used to compare cellulase secretion by free mycelium and biofilm cultures. Free mycelium cultures grew better in flasks than in microbioreactors producing compact and fluffy pellets, respectively, while the opposite was found for biofilm cultures without any visible change in biofilm morphology. Cellulase activities and volumetric productivities attained by biofilms in flask cultures were 70% higher than that produced by free mycelium cultures and threefold higher when biofilms were grown in microbioreactors. CONCLUSIONS: Fungal biofilms developed on polyester cloth in both flasks and microbioreactors produce higher cellulase yields and volumetric productivities than free mycelium cultures at lower biomass levels. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present study are of commercial and biological interest. All productivity parameters revealed that fungal biofilms may be used for the production of cellulase and other proteins in various types of bioreactors. Moreover, they may be used as model systems to study differential gene expression related to cell adhesion.     

Itaconic acid production from xylose in repeated-batch and continuous bioreactors

Summary Itaconic acid production from xylose by immobilized Aspergillus terreus TKK 200-5-2 mycelia was optimized both in repeated shake-flask fermentations and in continuous column bioreactors using statistical experimental design and empirical modelling. Using continuous 9-1 scale air-lift bioreactors, a pH of 2.5, aeration rate of 0.6 v/v per minute and residence time of 160 h gave the highest itaconic acid concentration. In air-lift bioreactors a cubic carrier size of 0.5 cm gave a 3.3-fold higher product concentration than 1-cm cubes. Packed-bed column reactors had a higher production rate than air-lift reactors. Offprint requests to: H. Kautola     

Enhancement of chrysogenin production in cultures of Penicillium chrysogenum by uronic acid oligosaccharides

Additions of 50 to 100 g of acid-hydrolysed alginate oligosaccharides ml^–1 and enzyme-hydrolysed pectin oligosaccharides to 24- to 48-h cultures of Penicillium chrysogenum, ATCC 9480, led to enhanced production of chrysogenin by over 30 to 40% in shaken flasks and bioreactors. Some of the oligosaccharides also promoted biomass formation but were not used as a carbon source.     

Factors influencing the production of a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid, by Pseudomonas aeruginosa PR3 (NRRL B-18602) in batch cultures

Pseudomonas aeruginosa PR3 (NRRL B-18602) converts oleic acid to a novel compound, 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). Parameters that included medium volume, cell growth time, gyration speed, pH, substrate concentration, and dissolved oxygen concentration were evaluated for a scale-up production of DOD in batch cultures using Fernbach flasks and a bench-top bioreactor. Maximum production of about 2 g DOD (38% yield) was attained in Fernbach flasks containing 500 ml medium when cells were grown at 28 degrees C and 300 rpm for 16-20 h and the culture was adjusted to pH 7 prior to substrate addition. Increases of medium volume and substrate concentration failed to enhance yield. When batch cultures were initially conducted in a reactor, excessive foaming occurred that made the bioconversion process inoperable. This was overcome by a new aeration mechanism that provided adequate dissolved oxygen to the fermentation culture. Under the optimal conditions of 650 rpm, 28 degrees C, and 40-60% dissolved oxygen concentration, DOD production reached about 40 g (40% yield) in 4.5 L culture medium using a 7-L reactor vessel. This is the first report on a successful scale-up production of DOD.     

Effect of phosphate buffer concentration on the batch xylitol production by Candida guilliermondii

AIMS: To evaluate the effect of phosphate buffer concentration on growth and xylitol production by Candida guilliermondii FTI 20037. METHODS AND RESULTS: Fermentations runs were carried out in batch mode employing semisynthetic medium supplemented with phosphate buffer at different concentrations (from 200 to 600 mmol l(-1)). The xylitol yield (Y(P/S)) and volumetric productivity (Q(P)) were improved when the fermentation medium was supplemented with phosphate buffer at concentration of 600 mmol l(-1). Under this condition (Y(P/S)) and (Q(P)) values were 0.75 g g(-1) and 0.66 g l(-1) h(-1), respectively, whereas in the absence of the phosphate buffer these values decreased to 0.52 g g(-1) and 0.44 g l(-1)h(-1) respectively. CONCLUSIONS: The use of phosphate buffer at 600 mmol l(-1) promoted an easier pH control during shake flasks fermentation of C. guilliermondii. In addition the xylitol yield and productivity were significantly improved in response to the supplementation of potassium phosphate in the medium. The increase in these parameters could be related to both osmotic effect and pH control. SIGNIFICANCE AND IMPACT OF THE STUDY: This approach provided a method for improving the xylitol production from semisynthetic medium by C. guilliermondii, being possible their use as a simple strategy to achieve efficient fermentation processes employing complex medium such as lignocellulosic hydrolysates.     

Scale-up study on suspension cultures of Taxus chinensis cells for production of taxane diterpene

Suspension cells of Taxus chinensis were cultivated in both shake flasks and bioreactors. The production of taxuyunnanine C (TC) was greatly reduced when the cell cultures were transferred from shake flasks to bioreactors. Oxygen supply, shear stress and stripping-off of gaseous metabolites were considered as potential factors affecting the taxane accumulation in bioreactors. The effects of oxygen supply on the cell growth and metabolism were investigated in a stirred tank bioreactor by altering its oxygen transfer rate (OTR). It was found that both the pattern and amount of TC accumulation were not much changed within the range of OTR as investigated. Comparative studies on the cell cultivation in low shear and high shear generating bioreactors suggest that the decrease of TC formation in bioreactors was not due to the different shear environments in different cultivation vessels. An incorporation of 2% CO(2) in the inlet air was beneficial for the cell growth, but did not improve the TC production in bioreactors. Furthermore, the effects of different levels of ethylene addition into the inlet air on the cell growth and TC production were investigated in a bubble column reactor. The average cell growth rate increased from 0.146 to 0.204 d(-1) as the ethylene concentration was raised from 0 to 50 ppm, and both the content and production of TC were also greatly improved by ethylene addition. At an ethylene concentration of 18 ppm, the highest TC content and volumetric production in the reactor reached 13.28 mg/(g DW) and 163.7 mg/L, respectively, which were almost the same as those in shake flasks. Compared with the control reactor (bubble column without ethylene supplementation), the maximum TC content was increased by 82% and the total production of TC was doubled. The results indicate that ethylene is a key factor in scaling up the process of the suspension cultures of T. chinensis from a shake flask to a bioreactor.     

Oxidative stress induces alkaloid production in Uncaria tomentosa root and cell cultures in bioreactors

The effect of oxidative stress on indole alkaloids accumulation by cell suspensions and root cultures of Uncaria tomentosa in bioreactors was investigated. Hydrogen peroxide (H₂O₂, 200 μM) added to U. tomentosa cell suspension cultures in shaken flasks induced the production of monoterpenoid oxindole alkaloids (MOA) up to 40.0 μg/L. In a stirred tank bioreactor, MOA were enhanced by exogenous H₂O₂ (200 μM) from no detection up to 59.3 μg/L. Root cultures grew linearly in shaken flasks with a μ=0.045 days^?1 and maximum biomass of 12.08±1.24 g DW/L (at day 30). Roots accumulated 3α‐dihydrocadambine (DHC) 2354.3±244.8 μg/g DW (at day 40) and MOA 348.2±32.1 μg/g DW (at day 18). Exogenous addition of H₂O₂ had a differential effect on DHC and MOA production in shaken flasks. At 200 μM H₂O₂, MOA were enhanced by 56% and DHC by 30%; while addition of 800 and 1000 μM H₂O₂, reduced by 30–40% DHC accumulation without change in MOA. Root cultures in the airlift reactor produced extracellular H₂O₂ with a characteristic biphasic profile after changing aeration. Maximum MOA was 9.06 mg/L at day 60 while at this time roots reached ca. 1 mg/L of DHC. Intracellular H₂O₂ in root cultures growing in the bioreactor was 0.87 μmol/g DW compared to 0.26 μmol/g DW of shaken flasks cultures. These results were in agreement with a higher activity of the antioxidant enzymes superoxide dismutase and peroxidase by 6‐ and 2‐times, respectively. U. tomentosa roots growing in the airlift bioreactor were exposed to an oxidative stress and their antioxidant system was active allowing them to produce oxindole alkaloids.