Cellulolytic enzymes on lignocellulosic substrates in solid state fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The production of cellulolytic enzymes by Aspergillus niger on lignocellulosic substrates groundnut fodder, wheat bran, rice bran and sawdust in solid state fermentation in a laboratory scale was compared. Czapek Dox liquid broth amended with cellulose (0.5%) was used to moisten lignocellulosic solid supports for cultivation of Aspergillus niger. The production of filter paperase, carboxymethyl cellulase and -glucosidase were monitored at daily intervals for 5 days. The peak production of the enzymes occurred within 3 days of incubation. Among solid supports used in the study, wheat bran was the best solid matrix followed by groundnut fodder in production of cellulolytic enzymes in solid state fermentation. Groundnut fodder supported significant production of FPase (2.09 FPU/g), CMCase (1.36 U/g) and -glucosidase activity (0.0117 U/g) in solid state fermentation. Considerable secretion of protein (5.10 mg/g) on groundnut fodder at peak time interval 1st day of incubation was recorded.     

High-yield spore production from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> by solid state fermentation

Bacillus licheniformis was grown for 48 h at 37°C in solid state fermentation; a maximum of 1.7 × 10¹¹ spores/g dry substrate were obtained using rice straw powder (300 g/kg) and wheat bran (700 g/kg) supplemented with glucose (40 g/kg), peptone (20 g/kg), yeast extract (20 g/kg), KH₂PO₄ (10 g/kg) and CaO (5 g/kg) with an initial moisture content of 65%.     

L-glutaminase production by <Emphasis Type="Italic">Trichoderma koningii</Emphasis> under solid-state fermentation

Solid state fermentation was conducted for the production of L-glutaminase by Trichoderma koningii Oud.aggr. using different agro-industrial byproducts inlcuding wheat bran, groundnut residues, rice hulls, soya bean meal, corn steep, sesamum oil cake, cotton seed residues and lentil industrial residues as solid substrates. Wheat bran was the best substrate for induction of L-glutaminase (12.1 U/mg protein) by T. koningii. The maximum productivity (23.2 U/mg protein) and yield (45.0 U/gds) of L-glutaminase by T. koningii occurred using wheat bran of 70% initial moisture content, initial pH 7.0, supplemented with D-glucose (1.0%) and L-glutamine (2.0% w/v), inoculated with 3 ml of 6 day old fungal culture and incubated at 30°C for 7 days. After optimization, the productivity of L-glutaminase by the solid cultures of T. koningii was increased by 2.2 fold regarding to the submerged culture.     

Evaluation of agro-food byproducts for gluconic acid production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> ORS-4.410

Certain cost-effective carbohydrate sources in crude as well as after purification were utilized as the sole sources of carbon for gluconic acid production using Aspergillus niger ORS-4.410 under submerged fermentation. Crude grape must (GM) and banana-must (BM) resulted into significant levels of gluconic acid production i.e. 62.6 and 54.6 g/l, respectively. The purification of grape and banana-must led to a 20–21% increase in gluconic acid yield. Molasses as such did not favour gluconate production (12.0 g/l) but a significant increase in production (60.3 g/l) was observed following hexacyanoferrate (HCF) treatment of the molasses. Rectified grape must (RGM) appeared to be best suitable substrate which after 144 h resulted in 73.2 g of gluconic acid/l with 80.6% yield followed by the yield obtained from the rectified banana must (RBM) (72.4%) and treated cane molasses (TM) (61.3%). Abundant growth of mould A. niger ORS-4.410 was observed with crude grape (0.131 g/l/h) and banana must (0.132 g/l/h).     

Physiological characterisation of <Emphasis Type="Italic">acuB</Emphasis> deletion in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The acuB gene of Aspergillus niger is an ortholog of facB in Aspergillus nidulans. Under carbon-repression conditions, facB is repressed, thereby preventing acetate metabolism when the repressing carbon source is present. Even though facB is reported to be repressed directly by CreA, it is believed that a basal level of FacB activity exists under glucose-repressive conditions. In the present study, the effect of deletion of acuB on the physiology of A. niger was assessed. Differences in organic acid and acetate production, enzyme activities and extracellular amino and non-amino organic acid production were determined under glucose-repressing and -derepressing conditions. Furthermore, consumption of alternative carbon sources (e.g. xylose, citrate, lactate and succinate) was investigated. It was shown that AcuB has pleiotropic effects on the physiology of A. niger. The results indicate that metabolic pathways that are not directly involved in acetate metabolism are influenced by acuB deletion. Clear differences in organic acid consumption and production were detected between the ∆acuB and reference strain. However, the hypothesis that AcuB is responsible for basal AcuA activity necessary for activation of acetate metabolic pathways, even during growth on glucose, could not be confirmed. The experiments demonstrated that also when acuB was deleted, no acetate was formed. Therefore, AcuB cannot be the only activator of AcuA, and another control mechanism has to be available for activating AcuA.     

Cost-effective production of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> using simple netting bag solid bioreactor

A novel simple solid state fermentation method, netting bag bioreactor (Φ 120 × 800 mm), was developed and used to cultivate Bacillus licheniformis as probiotics. High spore yield (1.2 × 10¹¹ CFU/g dry substrate) has been obtained by using this method. Comparing to the tray bioreactor and the packed bed bioreactor for Bacillus fermentation, the netting bag method was more cost-effective, time- and space-saving and the material cost is also as low as ca. US $293 per 1,000 kg spores. Thus, netting bag SSF can be widely applied to produce probiotic bacteria in developing areas.     

Biosorption of manganese by <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Summary Biosorption of manganese from its aqueous solution using yeast biomass Saccharomyces cerevisiae and fungal biomass Aspergillus niger was carried out. Manganese biosorption equilibration time for A. niger and S. cerevisiae were found to be 60 and 20 min, with uptakes of 19.34 and 18.95 mg/g, respectively. Biosorption increased with rise in pH, biomass, and manganese concentration. The biosorption equilibrium data fitted with the Freundlich isotherm model revealed that A. niger was a better biosorbent of manganese than S. cerevisiae.     

Statistical optimization for tannase production from <Emphasis Type="Italic">Aspergillus niger</Emphasis> under submerged fermentation

Statistically based experimental design was employed for the optimization of fermentation conditions for maximum production of enzyme tannase from Aspergillus niger. Central composite rotatable design (CCRD) falling under response surface methodology (RSM) was used. Based on the results of ‘one-at-a-time’ approach in submerged fermentation, the most influencing factors for tannase production from A. niger were concentrations of tannic acid and sodium nitrate, agitation rate and incubation period. Hence, to achieve the maximum yield of tannase, interaction of these factors was studied at optimum production pH of 5.0 by RSM. The optimum values of parameters obtained through RSM were 5% tannic acid, 0.8% sodium nitrate, 5.0 pH, 5 × 10⁷ spores/50mL inoculum density, 150 rpm agitation and incubation period of 48 h which resulted in production of 19.7 UmL⁻¹ of the enzyme. This activity was almost double as compared to the amount obtained by ‘one-at-a-time’ approach (9.8 UmL⁻¹).     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

<Emphasis Type="Italic">In vitro</Emphasis> colonization of hydrophilic contact lenses by <Emphasis Type="Italic">Aspergillus niger</Emphasis>

In vitro colonization of hydrophilic contact lenses by Aspergillus niger was investigated. Five strains of the fungus, four polymers, two culture media and four incubation periods were considered for analysis. Only the 2700 strain colonized the lenses. The degrees of adhesion and invasion varied significantly according to the characteristics of the culture under investigation. Journal of Industrial Microbiology & Biotechnology (2002) 29, 6–9 doi:10.1038/sj.jim.7000255 Received 06 August 2001/ Accepted in revised form 23 March 2002     

Response surface optimization of fermentation conditions for producing xylanase by <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">niger</Emphasis> SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of the dwarf pomegranate (<Emphasis Type="Italic">Punica granatum</Emphasis> L. var. <Emphasis Type="Italic">nana</Emphasis>)

The dwarf pomegranate (Punica granatum L. var. nana) is a dwarf ornamental plant that has the potential to be the model plant of perennial fruit trees because it bears fruits within 1 year of seedling. We established an Agrobacterium-mediated transformation system for the dwarf pomegranate. Adventitious shoots regenerated from leaf segments were inoculated with A. tumefaciens strain EHA105 harboring the binary vector pBin19-sgfp, which contains neomycin phosphotransferase (npt II) and green fluorescent protein (gfp) gene as a selectable and visual marker, respectively. After co-cultivation, the inoculated adventitious shoots were cut into small pieces to induce regeneration, and then selected on MS medium supplemented with 0.5 μM α-naphthaleneacetic acid (NAA), 5 μM N⁶-benzyladenine (BA), 0.3% gellan gum, 50 mg/l kanamycin, and 10 mg/l meropenem. Putative transformed shoots were regenerated after 6–8 months of selection. PCR and PCR-Southern blot analysis revealed the integration of the transgene into the plant genome. Transformants bloomed and bore fruits within 3 months of being potted, and the inheritance of the transgene was confirmed in T₁ generations. The advantage of the transformation of dwarf pomegranate was shown to be the high transformation rate. The establishment of this transformation system is invaluable for investigating fruit-tree-specific phenomena.     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 under submerged fermentation conditions

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran–3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4°C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70°C, which will be useful for its spray drying.     

Characterisation of <Emphasis Type="Italic">Aspergillus niger</Emphasis> prolyl aminopeptidase

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.     

Regulatory activity of heterologous gene-activator <Emphasis Type="Italic">xlnR</Emphasis> of <Emphasis Type="Italic">Aspergillus niger</Emphasis> in <Emphasis Type="Italic">Penicillium canescens</Emphasis>

The gene encoding the xlnR xylanolytic activator of the heterologous fungus Aspergillus niger was incorporated into the Penicillium canescens genome. Integration of the xlnR gene resulted in the increase in a number of activities, i.e. endoxylanase, β-xylosidase, α-L-arabinofuranosidase, α-galactosidase, and feruloyl esterase, compared to the host P. canescens PCA 10 strain, while β-galactosidase, β-glucosidase, endoglucanase, and CMCase activities remained constant. Two different expression constructs were developed. The first consisted of the nucleotide sequence containing the mature P. canescens phytase gene under control of the axhA promoter region gene encoding A. niger (1,4)-β-D-arabinoxylan-arabinofuranohydrolase. The second construct combined the P. canescens phytase gene and the bgaS promoter region encoding homologous β-galactosidase. Both expression cassettes were transformed into P. canescens host strain containing xlnR. Phytase synthesis was observed only for strains with the bgaS promoter on arabinose-containing culture media. In conclusion, the bgaS and axhA promoters were regulated by different inducers and activators in the P. canescens strain containing a structural tandem of the axhA promoter and the gene of the xlnR xylanolytic activator.     

Changes in primary metabolism leading to citric acid overflow in <Emphasis Type="Italic">Aspergillus niger</Emphasis>

For citric acid-accumulating Aspergillus niger cells, the enhancement of anaplerotic reactions replenishing tricarboxylic acid cycle intermediates predisposes the cells to form the product. However, there is no increased citrate level in germinating spores and a complex sequence of developmental events is needed to change the metabolism in a way that leads to an increased level of tricarboxylic acid cycle intermediates in mycelia. A review of physiological events that cause such intracellular conditions, with the special emphasis on the discussion of hexose transport into the cells and regulation of primary metabolism, predominantly of glycolytic flux during the process, is presented.     

Lipase production by recombinant strains of <Emphasis Type="Italic">Aspergillus niger</Emphasis> expressing a lipase-encoding gene from <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis>

Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Y_{xp total}), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7±0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3±0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (r_{p total}, the sum of extracellular and intracellular lipase productivity) was found to be 1.60±0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h^–1, compared with a total specific lipase productivity of 1.10±0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall.     

Dynamic analysis of <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">bulgaricus</Emphasis> CFL1 physiological characteristics during fermentation

This study aimed at examining and comparing the relevance of various methods in order to discriminate different cellular states of Lactobacillus bulgaricus CFL1 and to improve knowledge on the dynamics of the cellular physiological state during growth and acidification. By using four fluorescent probes combined with multiparametric flow cytometry, membrane integrity, intracellular esterase activity, cellular vitality, membrane depolarization, and intracellular pH were quantified throughout fermentations. Results were compared and correlated with measurements of cultivability, acidification activity (Cinac system), and cellular ability to recover growth in fresh medium (Bioscreen system). The Cinac system and flow cytometry were relevant to distinguish different physiological states throughout growth. Lb. bulgaricus cells maintained their high viability, energetic state, membrane potential, and pH gradient in the late stationary phase, despite the gradual decrease of both cultivability and acidification activity. Viability and membrane integrity were maintained during acidification, at the expense of their cultivability and acidification activity. Finally, this study demonstrated that the physiological state during fermentation was strongly affected by intracellular pH and the pH gradient. The critical pHi of Lb. bulgaricus CFL1 was found to be equal to pH 5.8. Through linear relationships between dpH and cultivability and pHi and acidification activity, pHi and dpH well described the time course of metabolic activity, cultivability, and viability in a single analysis.