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.     

A novel design of solid state fermenter and its evaluation for cellulase production by Trichoderma viride SL-1

Summary A novel design of a large scale solid state fermenter, designated as PPSSF--Periodic Pressure Solid State Fermenter--was constructed. Due to the respiration created by periodical variations of air pressure, air was uptaken / expelled from the fermenting mass and heat moved from the fermenter easily. It is possible to operate the unit at different capacities simply by adding or removing the bins. The results of its evaluation for enzyme formation by Trichoderma viride SL-1 indicated that a 2–3 fold increased cellulase production can be obtained in this system.     

Shikonin production and secretion by hairy root cultures of Lithospermum erythrorhizon

Summary Hairy root cultures of Lithospermum erythrorhizon were established by transformation of in vitro grown shoots with Agrobacterium rhizogenes 15834. Hairy roots cultured on Murashige and Skoog solid medium did not produce any red pigments. However, the hairy roots cultured in Root Culture solid or liquid media produced a large amount of red pigments, which were released to the medium. The addition of adsorbents to the culture medium stimulated shikonin production by ca. 3-fold. Using this method an air-lift fermenter system was established, equipped with a XAD-2 column, which continuously produced ca. 5 mg/day of shikonin during a period of more than 220 days.     

Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread

Aims: Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air‐lift fermenters. Among surfactants tested, Tweens (Tween‐20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X‐100 inhibited the enzyme production. The mould produced phytase optimally at a_w 0·95, and it declined sharply below this a_w value. The enzyme production was comparable in air‐lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca‐alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween‐80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca‐alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high‐soluble phosphate. Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air‐lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.     

A potential economical substrate for large-scale production of Bacillus thuringiensis var. kurstaki for caterpillar control

Bacillus thuringiensis var. kurstaki has been widely used in caterpillar control programs. Large-scale production of this bacterium is expensive because of the high cost of the raw materials used in the medium. In this study, we attempted to develop an economical medium, based on inexpensive, locally available raw materials using a 3-L fermenter. Parthenium hysterophorus L. extract based culture medium resulted in highest toxicity (LC₅₀ 14.628 µg mL–¹) against 7-day-old Spodoptera litura (Fab) larvae, spore count (4.1 × 10⁹ spores mL^–1) and biomass (4.9 g L^–1) within a short fermentation time of 36 h. It was 512 times cheaper than the nutrient broth (standard medium) used for B. thuringiensis production. Hence, this parthenium extract based culture medium was considered most economical with potential for the large-scale industrial production of B. thuringiensis.     

Cultivation characteristics ofIsaria japonica

Cultivation characteristics of fruit-body (synnema) formation ofIsaria japonica were examined using liquid and solid media in order to produce fruit-bodies on a large scale. Mycelia grew well at 18–28°C on PDA medium with an initial pH of 7.0. The formation of fruit-bodies ofI. japonica was induced by lowering temperature to below 20°C in PD liquid medium. In sawdust-rice bran basal medium mixed with pupal powder prepared from silkworms (Bombyx mori), the fresh weight of fruit-bodies increased with increasing content of pupal powder. The highest yields of fruit-bodies were obtained in carbon-rich barley grain medium supplemented with pupal powder. The fruit-bodies grown under CO₂ concentrations of 1,000 μl/L had coral-like, many-branched synnemata with numerous conidiospores, whereas those formed under high concentrations (9,000 μl/L) of CO₂ had unbranched and longer synnemata. High concentrations of CO₂ remarkably inhibited conidiospore formation on synnemata. Continuous high-intensity illumination at 2.93 W·m⁻² inhibited the elongation of synnemata, and low-intensity illumination at 0.088 W·m⁻² slightly inhibited the branching of synnemata. Fruit-bodies were produced on the pupa metamorphosed from living larvae ofAgrotis fucosa placed on the surface of a culture ofI. japonica incubated in sawdust-rice bran medium.     

Morphogenetic effect of glycerol on tissue cultures of the red seaweed Grateloupia doryphora

Explants of Grateloupia doryphora were cultivated in Provasoli Enriched Seawater culture medium (PES) supplemented with glycerol (0.1, 0.3, 0.5 or 0.8 mol 1^–1) or carbohydrates (0.1 or 0.3 mol 1^–1 mannose, glucose and galactose) and agar (3, 8, 15 g 1^–1 ). The osmolality of the medium was adjusted by dilution of the seawater (70 or 100%, v/v). The increase in fresh weight of explants cultivated in liquid medium with glycerol (0.3 mol 1^–1) and without glycerol was compared. All experiments were carried out in the light, except for one assay in which the explants were cultivated in the dark. Glycerol was an effective carbon source for the vegetative propagation of G. doryphora in solid and liquid media. Mannose, glucose and galactose all had no effect on growth or morphogenesis of the explants. In solid media the main effect of glycerol was as a morphogenetic inductor, with PES70 (70% seawater) + 0.1 or 0.3 mol 1^–1 glycerol + 3 or 8 g 1^–1 agar the best formulation. An increase in the concentration of agar in glycerol-containing medium reduced the morphogenetic capacity of the explants, which developed into compact cell masses. The effects of glycerol were observed only in explants cultivated under light.     

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 g L^–1 day^–1 after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 g L^–1 day^–1, a productivity of 5 g L^–1 day^–1 was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.     

Mixture design as first step for improved glutaminase production in solid-state fermentation by isolated Bacillus

Aim: Investigation of mixture‐design impact on glutaminase production by isolated Bacillus sp. Methods and Results: An augmented simplex centroid design was used to optimize a three (wheat bran, Bengal gram husk and palm seed fibre) component mixture for glutaminase production. Selected substrate materials showed impact on glutaminase production values at individual level by Bengal gram husk [2789 U gds^?1 (gram dry substrate] and in two‐level combination with wheat bran and Bengal gram husk (maximum of 3300 U gds^?1). Conclusion: Bengal gram husk is the most suitable substrate medium for glutaminase production by Bacillus sp. Maximum glutaminase production is achieved using solid‐substrate mixture at two‐level combinations in the ratio of 66 : 34 for Bengal gram husk and wheat bran, respectively. Significance and Impact of the Study: The present study has significance in large‐scale production of glutaminase at commercial level with the use of multi‐substrate rather than single‐substrate/support material.     

Studies on the growth of Thiobacillus ferrooxidans

Summary A method for enumeration of viable numbers of Thiobacillus ferrooxidans using membrane filters on ferrous-iron agar is presented. Factors affecting colony production were the concentration and brand of agar, pH of the medium, and type of membrane filter. The results suggest that inhibition of T. ferrooxidans by agar is a result of the acid hydrolysis of agar, the main product of which is d-galactose. Colony development was suppressed by aged medium, by acid-hydrolysed agar and by 0.1% galactose. Sartorius and Millipore membrane filters were suitable for the experiments, whereas Oxoid MF-50 membranes virtually suppressed the production of colonies. The method was employed to follow growth of T. ferrooxidans in pH 1.3 medium. The viable cell numbers were correlated with ¹⁴CO₂-fixation and ferrous iron oxidation. Generation time was 6 h 22 min with a yield of 2.2×10¹² organisms/g atom Fe²⁺ oxidized. Growth of T. neapolitanus on thiosulphate medium was not affected by agar-type or membrane filters and yield of the organism was 1.5×10¹³ organisms/g molecule Na₂S₂O₃ oxidized.     

Morphology engineering of Aspergillus oryzae for l-malate production

Aspergillus oryzae is a competitive natural producer for organic acids, but its production capacity is closely correlated with a specific morphological type. Here, morphology engineering was used for tailoring A. oryzae morphology to enhance l -malate production. Specifically, correlation between A. oryzae morphology and l -malate fermentation was first conducted, and the optimal range of the total volume of pellets in a unit volume of fermentation broth (V value) for l -malate production was 120–130 mm³/ml. To achieve this range, A. oryzae morphology was improved by controlling the variation of operational parameters, such as agitation speed and aeration rate, and engineered by optimizing the expression of cell division cycle proteins such as tyrosine-protein phosphatase (CDC14), anaphase promoting complex/cyclosome activator protein (CDC20), and cell division control protein 45 (CDC45). By controlling the strength of CDC14 at a medium level, V value fell into the optimal range of V value and the final engineered strain A. oryzae CDC14(3) produced up to 142.5 g/L l -malate in a 30-L fermenter. This strategy described here lays a good foundation for industrial production of l -malate in the future, and opens a window to develop filamentous fungi as cell factories for production of other chemicals.     

Pilot production of Clonostachys rosea conidia in a solid‐state fermentor optimized using response surface methodology

The nonpathogenic, saprophytic fungus Clonostachys rosea is one of the most powerful fungal biological control agents (BCAs). However, the production of fungal BCAs is still a major constraint for their large‐scale use and commercialization. Here, we developed a novel solid‐fermentation reactor that is light transparent and ventilated both at the top and the bottom, and optimized C. rosea cultivation conditions in solid‐state fermentation using response surface methodology. The growth area of spores provided by the novel fermentor was two times that of the traditional one. A quadratic polynomial model was developed, which indicated the effects of variables on the conidia yield. The greatest spore production of 3.50 × 10¹⁰ spores/g‐dry‐matter was obtained after 11 days at the initial moisture content of 69.2% w/w, the medium thickness of 3.84 cm, and the porosity of 0.37%. The optimized spore yield was increased by one order of magnitude. The fermentation time was shortened from 15 to 11 days. With the novel solid‐fermentation reactor, increase in C. rosea spores production and decrease in fermentation time were achieved. Current data imply that both the novel solid‐fermentation reactor designed and the optimized fermentation conditions are suitable for industrial‐scale C. rosea spore production.     

Improved performance in γ-polyglutamic acid production by Bacillus subtilis LX on industrial scale by impeller retrofitting and its unstructured microbial growth kinetics model

Abstract

We conducted industrial scale γ-polyglutamic acid (γ-PGA) production by Bacillus subtilis (B. subtilis) LX and modeled its microbial growth kinetics based on a logistic regression. We found that the use of a three-layer impeller including a lower semicircular disc impeller and two-layers of six-wide-leaf impellers were able to both increase γ-PGA yields and decrease fermentation time as compared with two-layer Rushton impellers. Indeed, our results revealed that the optimal γ-PGA yield (20.67?±?2.19?g/L) was obtained after 40?hr in the impeller retrofitted fermenter, and this yield was 29.7% higher than that in Rushton impellers fixed fermenter. The microbial growth kinetics of B. subtilis LX in this system were established, and the model was consistent with the experimental data (R² = 0.924) suggesting that it was suitable for describing the microbial growth kinetics underlying γ-PGA production on an industrial scale. In addition, biomass yield (Y_x/s-glucose), γ-PGA yield (Y_p/s-glucose), γ-PGA yield (Y_{p/s-glutamate}), and the correlation between γ-PGA production and B. subtilis LX (Y_p/x) were found to be 0.043, 0.133, 0.743, and 3.090?g/g, respectively, in the impeller retrofitted fermenter, as compared with 0.036, 0.103, 0.629, and 2.819?g/g, respectively, in the two-layer Rushton impeller fermenter.     

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.     

Cellulose degradation and carboxymethylcellulase production by Sporocytophaga myxococcoides grown in an air-lift fermenter

Summary Sporocytophaga myxcoccoides was grown in a 31 air-lift fermenter using a medium containing 2% w/v insoluble cellulose. The cellulose content of the medium reduced the k_La of the fermenter but during growth the dissolved oxygen concentration did not fall below 75% saturation. Rates of cellulose degradation and extracellular enzyme production were similar to those reported for a stirred-tank fermenter.     

Effect of culture conditions on growth and sporulation ofBacillus thuringiensis subsp.israelensis and development of media for production of the protein crystal endotoxin

Summary The influence of medium composition on the inoculum and production stages of theBacillus thuringiensis subsp.israelensis bioinsecticide fermentation was investigated. Media which inhibited sporulation were selected for inoculum development stages. Bioinsecticide production media were designed to produce high cell counts and >90% sporulation in a 48h fermentation. Maximum insecticidal activity occurred at the point of maximum bacterial cell lysis/spore release. A process involving two inoculum stages and a 48h production stage in a 40 l fermenter yielded a viable cell count of 6.5 x 10⁹/ml with greater than 95% sporulation. Good correlation existed between spore counts and bioinsecticide activity.     

Fungi on the hair of large mammals in Egypt

Several isolates of Candida albicans were tested for production of chlamydoconidia and metabolic changes when grown on several different solid and liquid media. A liquid medium, consisting solely of sterilized skimmed milk and a solid medium containing processed cheese stimulated more rapid and greater production of chlamydoconidia than the corn meal agar and the other media tested.     

Establishment of hairy root lines and analysis of gentiopicroside in the medicinal plant <Emphasis Type="Italic">Gentiana macrophylla</Emphasis>

The leaf explants from the eight-week-old Gentiana macrophylla Pall sterile seedlings were precultured for three days on Murashige and Skoog (MS) agar medium with 1 mg/l of 6-benzyladenine, and then incubated in MS liquid medium containing Agrobacterium rhizogenes R1000 for 2 h with shaking. After 10 days of culture on hormone-free MS agar medium with 500 mg/l cefotaxime under illumination of 300 μmol/(m² s) with a 16-h photoperiod at 24°C, up to 18.3% of the infected explants produced typical hairy roots (HRs). Finally, three stable HR lines with no morphologically visible differences were obtained. The HR lines could vigorously grow and propagate on/in hormone-free half-strength MS solid/liquid medium. Moreover, they changed from white to green when moved from darkness to the light and spontaneously produced calli, which were able later to produce adventitious buds. PCR analysis revealed that three HR lines, which were undergone to continuous subculturing over two years, still contained the rolC gene from the A. rhizogenes Ri plasmid. HPLC revealed the HR line possessing a certain capacity of synthesizing gentiopicroside (0.11 mg/g dry wt).     

Antimicrobial solid media for screening non-sterile Arabidopsis thaliana seeds

Stable genetic transformation of plants is a low-efficiency process, and identification of positive transformants usually relies on screening for expression of a co-transformed marker gene. Often this involves germinating seeds on solid media containing a selection reagent. Germination on solid media requires surface sterilization of seeds and careful aseptic technique to prevent microbial contamination, but surface sterilization techniques are time consuming and can cause seed mortality if not performed carefully. We developed an antimicrobial cocktail that can be added to solid media to inhibit bacterial and fungal growth without impairing germination, allowing us to bypass the surface sterilization step. Adding a combination of terbinafine (1 μM) and timentin (200 mg l⁻¹) to Murashige and Skoog agar delayed the onset of observable microbial growth and did not affect germination of non-sterile seeds from 10 different wild-type and mutant Arabidopsis thaliana accessions. We named this antimicrobial solid medium “MSTT agar”. Seedlings sown in non-sterile conditions could be maintained on MSTT agar for up to a week without observable contamination. This medium was compatible with rapid screening methods for hygromycin B, phosphinothricin (BASTA) and nourseothricin resistance genes, meaning that positive transformants can be identified from non-sterile seeds in as little as 4 days after stratification, and transferred to soil before the onset of visible microbial contamination. By using MSTT agar we were able to select genetic transformants on solid media without seed surface sterilization, eliminating a tedious and time-consuming step.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.