Improved simultaneous production of mycelial biomass and polysaccharides by submerged culture of <Emphasis Type="Italic">Hericium erinaceum</Emphasis>: optimization using a central composite rotatable design (CCRD)

The aim of this study was to optimize the culture medium used for the mycelial growth and production of intracellular polysaccharides (IPS) and exopolysaccharides (EPS) in a submerged culture of Hericium erinaceum. Of the various factors examined, including carbon and nitrogen sources, vitamins, mineral elements, and initial pH, those that proved to have a significant effect were then tested using a 2⁴ central composite rotatable design (CCRD). Under the optimal culture conditions, the maximal yield of biomass reached 14.24 ± 0.45 g l⁻¹ and was 1.85-fold higher than in the basal medium. The kinetics of EPS biosynthesis in a bioreactor showed that although the highest yield of EPS (2.75 ± 0.27 g l⁻¹) could be obtained on day 8, the process of biosynthesizing high molecular weight polysaccharides proceeded until the depletion of the carbon source in the medium (after 14 days of cultivation). Our results could be very helpful in the large-scale production of bioactive polysaccharides from H. erinaceum.     

pH control strategy in a shaken minibioreactor for polysaccharide production by medicinal mushroom Phellinus linteus and its anti-hyperlipemia activity

A process at various pH values ranging from 4.5 to 7.5 for production of mycelia and extracellular polysaccharide (EPS) by P. linteus fermentation in pH-controlled shaken bioreactor was investigated. A two-stage pH control strategy in which pH value was kept at 6.5 for the first 24 h, and then switched to 4.5 was developed successfully to enhance simultaneously the cell growth and EPS production. The maximum cell density and EPS production reached 15.13 ± 0.1 g/l on day 6 and 6.74 ± 0.1 g/l on day 4, respectively. The anti- hyperlipemia effect of EPS and intracellular polysaccharide (IPS) extracted from mycelia were observed that both EPS and IPS can obviously reduce the serum triglyceride (TG), the blood cholesterol (TC) and serum low density lipoprotein (LDL) level, and increase the high density lipoprotein (HDL) level of the hyperlipemia mice. Polysaccharides from submerged cultivation of medicinal fungus P. linteus have favorable potency to develop anti-hyperlipermia drugs.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Effects of conditions ofPenicillium funiculosum G-15 cultivation on production of extracellular glucose oxidase

Effects of conditions ofPenicillium funiculosum G-15 cultivation on the production of extracellular glucose oxidase were studied. The data showed that surface and submerged methods of cultivation can be used for obtaining a glucose oxidizing enzyme. The optimum conditions for submerged cultivation (25°C., initial pH 5.0, and aeration of 3 1/1 per min) and surface cultivation (temperature 25°C and initial pH 4.0) providing the maximum levels of glucose oxidase synthesis were determined.     

Enhancement of exopolysaccharide production from Ganoderma lucidum using a novel submerged volatile co-culture system

Based on the impact of volatile organic compounds (VOCs) on secondary metabolite pathways, a novel submerged volatile co-culture system was constructed, and the effects of thirteen fungal and bacterial VOCs were investigated on Ganoderma lucidum exopolysaccharides production. The results demonstrated at least a 2.2-fold increase in exopolysaccharide (EPS) specific production yield in 6 days submerged volatile co-culture of G. lucidum with Pleurotus ostreatus. Therefore, P. ostreatus was selected as a variable culture, and the effects of agitation speed, inoculum size, initial pH, and co-culture volume on EPSs production were investigated using a Taguchi L₉ orthogonal array. Finally, the highest concentration of EPSs (3.35 ± 0.22 g L^?1) was obtained under optimized conditions; initial pH 5.0, inoculum size 10%, 150 rpm, and 3:1 volume ratio of variable culture to main culture.     

Production of a potentially novel antimicrobial compound by a biofilm-forming marine Streptomyces sp. in a niche-mimic rotating disk bioreactor

After initial small-scale experiments, a 25.0 l rotating disk bioreactor (RDBR) was investigated for the cultivation of a biofilm-forming salt-tolerant Streptomyces sp. MS1/7, producing an antimicrobial compound. Peak activity attainment rate, PAAR (ratio of the peak antimicrobial activity, PAMA and the time taken to attain PAMA) was determined. Of the three pH values examined (8.0, 9.0 and 10.0) maximum PAAR (1.82 mm/h) was attained at pH 9.0. Three aeration rates (9.0, 6.0 and 3.0 l/min) were considered at three levels (25, 50 and 75%) of disk submergence. At the highest aeration rate and 50% submergence level, PAMA (41 mm), PAAR (1.86 mm/h) and biofilm density (BD, 0.91 g/ml) attained their highest values. At any given aeration rate, PAMA was always higher at 50% submergence level. This supports our earlier premise that ideal intertidal conditions, 12 h periods of immersion and emersion, promote maximum BD and antimicrobial production in the niche-mimic RDBR.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Optimization of production of C-phycocyanin and extracellular polymeric substances by Arthrospira sp.

The key factors influencing the production of C-phycocyanin (C-PC) and extracellular polymeric substances (EPS) by photoautotrophic culture of Arthrospira sp. were optimized using Taguchi method. Six factors were varied at either three or two levels as follows: light intensity at three levels; three initial culture pHs; two species of Arthrospira; three concentrations of Zarrouk’s medium; three rates of aeration of the culture with air mixed with 2% v/v carbon dioxide; and two incubation temperatures. All cultures ran for 14 days. The optimal conditions for the production of C-PC and EPS were different. For both products, the best cyanobacterium proved to be Arthrospira maxima IFRPD1183. The production of C-PC was maximized with the following conditions: a light intensity of 68 µmol photons m⁻² s⁻¹ (a diurnal cycle of 16-h photoperiod and 8-h dark period), an initial pH of 10, the full strength (100%) Zarrouk’s culture medium, an aeration rate of 0.6 vvm (air mixed with 2% v/v CO₂) and a culture temperature of 30 °C. The concentration of Zarrouk’s medium was the most important factor influencing the final concentration of C-PC. The optimal conditions for maximal production of EPS were as follows: a light intensity of 203 µmol photons m⁻² s⁻¹ with the earlier specified light–dark cycle; an initial pH of 9.5; a 50% strength of Zarrouk’s medium; an aeration rate of 0.2 vvm (air mixed with 2% v/v CO₂); and a temperature of 35 °C. Production of C-PC and EPS in raceway ponds is discussed.

     

Optimization of probiotic and lactic acid production by Lactobacillus plantarum in submerged bioreactor systems

Biomass and lactic acid production by a Lactobacillus plantarum strain isolated from Serrano cheese, a microorganism traditionally used in foods and recognized as a potent probiotic, was optimized. Optimization procedures were carried out in submerged batch bioreactors using cheese whey as the main carbon source. Sequential experimental Plackett–Burman designs followed by central composite design (CCD) were used to assess the influence of temperature, pH, stirring, aeration rate, and concentrations of lactose, peptone, and yeast extract on biomass and lactic acid production. Results showed that temperature, pH, aeration rate, lactose, and peptone were the most influential variables for biomass formation. Under optimized conditions, the CCD for temperature and aeration rate showed that the model predicted maximal biomass production of 14.30 g l⁻¹ (dw) of L. plantarum. At the central point of the CCD, a biomass of 10.2 g l⁻¹ (dw), with conversion rates of 0.10 g of cell g⁻¹ lactose and 1.08 g lactic acid g⁻¹ lactose (w/w), was obtained. These results provide useful information about the optimal cultivation conditions for growing L. plantarum in batch bioreactors in order to boost biomass to be used as industrial probiotic and to obtain high yields of conversion of lactose to lactic acid.     

Influence of some environmental factors on Rhizopus nigricans submerged growth in the form of pellets

Morphological changes of a steroid transforming filamentous fungus Rhizopus nigricans were studied by altering submerged growth conditions at inoculum sizes previously determined to favor pelleted growth. Beside the main classification between pelleted and clumpy growth forms, the size, concentration and structure of pellets were characterized at different cultivating temperature, initial pH value, medium composition, additives, and aeration conditions. Initial pH below 4 and above 7, the presence of Ca²⁺ and Tween-80 gave rise to the clumpy growth, otherwise pelleted growth prevailed. Among tested factors the pellet size was mainly influenced by the inoculum size and the presence of baffles and Ca²⁺ in cultivation medium. The formation of smooth pellets, prerequisite for further application in the process of steroid biotransformation, resulted in cultivations at lower temperature, high agitation rates in shaken cultures without baffles and at high nitrogen concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of Cultivation Parameters on the Morphology of Rhizopus arrhizus and the Lactic Acid Production in a Bubble Column Reactor

The use of filamentous Rhizopus for lactic acid production is facing a challenge due to its low yield mainly caused by the difficulty to control its morphology in submerged fermentation processes. This study was aimed at investigating the impacts of cultivation parameters on the morphology of Rhizopus arrhizus DAR 36017 and lactic acid production using waste potato starch in a laboratory scale bubble column reactor (BCR). The fungal morphology was significantly influenced by carbon sources, process pH, starch concentrations, sparger designs and aeration rates. The favorable morphology for lactic acid production was a freely dispersed small pellet, which was achieved under operation conditions at pH 5.0–6.0, starch concentrations of 60–120 g/L and aeration rates of 0.2–0.8 vvm using a sintered stainless steel disc sparger. Optimal cultivation conditions at pH 6.0 and an aeration rate of 0.4 vvm resulted in the formation of freely dispersed small pellets and 103.8 g/L lactic acid with a yield of 87 % from 120 g/L liquefied potato starch in 48 h. The overall results in terms of lactic acid yield and productivity are comparable to those reported in previous studies using immobilized Rhizopus cells in batch fermentations.     

Optimization for xylanase and cellulase production from Aspergillus niger ATTC 6275 in palm oil mill wastes and its application

Optimization of enzyme production from Aspergillus niger ATCC 6275 under both submerged and solid-substrate cultivation was investigated. Results from submerged cultivation using palm oil mill effluent revealed that pretreatment of ground palm cake did not improve enzyme production. Addition of 0.60g NH4NO3/l generated maximum activity of xylanase and cellulase (CMCase). The optimum aeration rate was 1.2 v/v min. Under solid-substrate cultivation, the results indicated that heating and alkali treatment of the ground palm cake gave no further improvement in enzyme production. The optimal N-source was 2% urea. Optimal initial moisture contents for xylanase and CMCase activities were 60% and 50% respectively, with temperature optima of 30°C and 35°C, respectively. The optimal inoculum size was 1× 108 spores/g palm cake with an initial pH of 4.5–5.0. The maximum activities of xylanase (282.9U/g) and CMCase (23.8U/g) were obtained under the optimum conditions. Solid-substrate cultivation was a better method for the production of enzyme, particularly xylanase, from A. niger ATCC 6275. The application of these enzymes to decanter effluent showed the separation of oil and grease and suspended solids from the effluent. This is comparable to the result achieved from using the commercial xylase preparation Meicelase and superior to the effect of Sumyzyme.     

Effect of conditions of culturing Penicillium funiculosum G-15 on production of extracellular glucose oxidase

Effects of conditions of Penicillium funiculosum G-15 cultivation on the production of extracellular glucose oxidase were studied. The data showed that surface and submerged methods of cultivation can be used for obtaining a glucose oxidizing enzyme. The optimum conditions for submerged cultivation (25 degrees C, initial pH 5.0, and aeration of 3 l/l per min) and surface cultivation (temperature 25 degrees C and initial pH 4.0) providing the maximum levels of glucose oxidase synthesis were determined.     

From field sampling to pneumatic bioreactor mycelia production of the ectomycorrhizal mushroom Laccaria trichodermophora

In order to increase survival rates of greenhouse seedlings destined for restoration and conservation programs, successful mycorrhization of the seedlings is necessary. To reforest forest ecosystems, host trees must be inoculated with ectomycorrhizal fungi and, in order to guarantee a sufficient supply of ectomycorrhizal inoculum, it is necessary to develop technologies for the mass production of ectomycorrhizal fungi mycelia. We selected the ectomycorrhizal fungus Laccaria trichodermophora, due to its ecological traits and feasible mycelia production in asymbiotic conditions. Here, we report the field sampling of genetic resources, as well as the highly productive nutritional media and cultivation parameters in solid cultures. Furthermore, in order to achieve high mycelial production, we used strain screening and evaluated pH, carbon source concentration, and culture conditions of submerged cultures in normal and baffled shake flasks. The higher productivity culture conditions in shake flasks were selected for evaluation in a pneumatic bioreactor, using modified BAF media with a 10 g/L glucose, pH 5.5, 25 °C, and a volumetric oxygen transfer coefficient (K_La) of 36 h⁻¹. Under those conditions less biomass (12–37 %) was produced in the pneumatic bioreactor compared with the baffled shake flasks. This approach shows that L. trichodermophora can generate a large biomass concentration and constitute the biotechnological foundation of its mycelia mass production.     

Studies of a pelleted growth form of Rhizopus nigricans as a biocatalyst for progesterone 11α-hydroxylation

The noncoagulative type of pellet formation can be induced in submerged cultivation of the filamentous fungus Rhizopus nigricans. The size and constitution of the hyphal agglomerates obtained varied with changes in inoculum size and agitation speed for given media composition and cultivation conditions. The physiological state of mycelium, used for a further process of biotransformation, was estimated by following the growth kinetics, pH value and substrate utilization during submerged cultivation. Namely, differences in pellet morphology and physiology affect the ability of R. nigricans to hydroxylate progesterone at the 11α position. A repeated batch procedure revealed the best maintenance of biotransformation capacity for pellets, obtained from the growth phase of cultivation at high agitation speed and with low inoculum size.     

Optimization of production of caffeine demethylase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. in a bioreactor

The effect of pH, aeration rate, and agitation rate on specific productivity of caffeine demethylase from Pseudomonas sp. was studied in a bioreactor. Maximum specific productivity of caffeine demethylase of 2,214 U g cell dry weight⁻¹ h⁻¹ was obtained at 0.27 vvm, 700 rpm, and pH 7.0. Under these conditions, volumetric oxygen transfer coefficient was 74.2 h⁻¹, indicating that caffeine demethylase production by Pseudomonas sp. was highly oxygen-dependent. Different metabolite formation at different agitation and aeration rates can be used as a strategy for recovery of pharmaceutically important metabolites from caffeine by manipulation of conditions in a bacterial culture. This is the first report on production of high levels of caffeine demethylase in bioreactors.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.     

Effect of aeration and agitation on the production of mycelial biomass and exopolysaccharides in an enthomopathogenic fungus Paecilomyces sinclairii

AIMS: The objective of the present study was to investigate the influence of aeration rate and agitation intensity on the production of mycelial biomass and exopolysaccharide (EPS) in Paecilomyces sinclairii. METHODS AND RESULTS: The P. sinclairii was cultivated under various aeration and agitation conditions in a 5 l stirred-tank bioreactor. The highest mycelial biomass (30.5 g l-1) and EPS production (11.5 g l-1) were obtained at a high aeration rate (3.5 v.v.m.) and at a high agitation speed (250 rev min-1). The apparent viscosities (6000-8000 cP) of fermentation broth increased rapidly towards the end of fermentations at high aeration and agitation conditions. CONCLUSIONS: The high level of dissolved oxygen achieved at a high aeration rate (3.5 v.v.m.) associated with higher hyphal density eventually resulted in enhanced EPS production. Agitation intensity was also proved to be a critical factor influencing on both the mycelial biomass and EPS production: high agitation speeds up to 250 rev min-1 were preferred to the yields of biomass and EPS production. SIGNIFICANCE AND IMPACT OF THE STUDY: The critical effects of aeration and agitation in the culture process of P. sinclairii were found, which is widely applicable to other kinds of basidiomycetes or ascomycetes in their submerged culture processes.     

Production and partial characterization of the exopolysaccharide from Pleurotus sajor caju

Microbial exopolysaccharides (EPSs) are very important because they are used in biotechnological applications in different industrial areas. The aim of the study was to determine the best EPS producer Pleurotus sp., to optimize EPS production and to perform partial purification and characterization of the produced EPS. After the production conditions were optimized, the EPS was isolated and partially purified. EPS was characterized by HP-TLC, 1H-NMR, FT-IR, and TGA. Hydroxyl, superoxide, and DPPH radical scavenging activities of the EPS were also investigated spectrophotometrically. The best EPS producer and its incubation period in submerged fermentation were determined as Pleurotus sajor caju and on 5 days, respectively. Culture conditions to increase EPS production were optimized as follows (in per liter): 90 g of glucose, 10 g of yeast extract, 10 g of peptone, and 100 mM of Mg2+. The optimal initial pH, temperature, and an agitation rate of culture were determined as 5.0, 25 °C, and 150 rate min−1, respectively. The highest EPS production was determined as 33.32 ± 1.6 g L−1. After isolation of EPS, one active fraction was obtained by gel filtration chromatography. EPS is composed mainly of glucose according to HP-TLC analysis. To the results, EPS had a complex structure by having carbohydrate and protein contents. The produced EPS had high degradation temperature as well as high antioxidant activity.     

Comparative evaluation of modified bioreactors for enhancement of growth and secondary metabolite biosynthesis usingPanax ginseng hairy roots

Hairy root cultures have demonstrated great promise in terms of their biosynthetic capability toward the production of secondary metabolites, but continue to constitute a major challenge with regard to large-scale cultures. In order to assess the possibility of conducting mass production of biomass, and the extraction of useful metabolites fromPanax ginseng. P. ginseng hairy roots, transformed byRhizobium rhizogenes KCTC 2744, were used in bioreactors of different types and sizes. The most effective mass production of hairy roots was achieved in several differently sized air bubble bioreactors compared to all other bioreactor types. Hairy root growth was enhanced by aeration, and the production increased with increasing aeration rate in a 1 L bioreactor culture. It was determined that the hairy root growth rate could be substantially enhanced by increases in the aeration rate upto 0.5 wm, but at aeration rates above 0.5 wm, only slight promotions in growth rates were observed. In 20 L air bubble bioreactors, with a variety of inoculum sizes, the hairy roots exhibited the most robust growth rates with an inoculum size of 0.1% (w/v), within the range 0.1 to 0.7% (w/v). The specific growth rates of the hairy roots decreased with increases in the inoculum size.