Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation

Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity of 5791 U/h.     

Invertase production on solid-state fermentation by Aspergillus niger strains improved by parasexual recombination

Invertase production by Aspergillus niger grown by solid-state fermentation was found to be higher than by conventional submerged fermentation. The haploid mutant strains Aw96-3 and Aw96-4 showed better productivity of various enzymes, as compared to wild-type parental strain A. niger C28B25. Here we use parasexual crosses of those mutants to increase further the productivity of invertase in solid-state fermentation. We isolated both a diploid (DAR2) and an autodiploid (AD96-4) strain, which were able to grow in minimal medium after mutation complementation of previously isolated haploid auxotrophic strains. Invertase production was measured in solid-state fermentation cultures, using polyurethane foam as an inert support for fungal growth. Water activity value (Aw) was adjusted to 0.96, since low Aw values are characteristic in some solid-state fermentation processes. Such diploid strains showed invertase productivity levels 5–18 times higher than levels achieved by the corresponding haploid strains. For instance, values for C28B25, Aw96-3, Aw96-4, DAR2, and AD96-4 were 441, 254, 62, 1324, and 2677 IU/(L·h), respectively. These results showed that genetic recombination, achieved through parasexual crosses in A. niger, results in improved strains with potential applications for solid-state fermentation processes.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Effect of operating conditions on biomass growth during citric acid production by solid-state fermentation

The effect of aeration, agitation, and bed loading on biomass and product concentration during citric acid biosynthesis by solid-state fermentation was investigated. For this purpose, Aspergillus niger S was cultivated on sugar beet pulp in a 4.5 dm³ horizontal rotating drum bioreactor. The results suggest that the parameters examined have a remarkable effect on the quantity of biomass being formed and on the product concentration. The maximum citric acid production (about 150 g per kg of the substrate dry matter) was obtained under the following conditions: aeration rate 0.2 dm³ kg⁻¹ min⁻¹, mixing (periodical) 1 min once an hour, and bed loading 30 % of the bioreactor working volume. However, these values did not favour biomass formation. Moreover, it was found that accumulation of the product reached its maximum when the amount of biomass was minimal (approximately 252-29 g per kg of the substrate dry matter) under the conditions involved. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Solid-state fermentation with Aspergillus niger for cellobiase production

Aspergillus niger NRRL3 was cultivated in a moist wheat bran and ground corncob solid medium supplemented with inorganic minerals for the production of cellobiase (β-1,4-glucosidase, EC 3.2.1.21). With this method, A. niger NRRL3 was able to produce a high concentration of cellobiase (215 IU/gofsolid substrate) after 96 h of incubation. Temperature and moisture content affected final cellobiase titers. The best conditions for cell obiase production from solid substrate by A. niger NRRL3 were determined to be 70% moisture and 35°C.     

Enzyme production and profile by Aspergillus niger during solid substrate fermentation using palm kernel cake as substrate

The oil palm sector is one of the major plantation industries in Malaysia. Palm kernel cake is a byproduct of extracted palm kernel oil. Mostly palm kernel cake is wasted or is mixed with other nutrients and used as animal feed, especially for ruminant animals. Recently, palm kernel cake has been identified as an important ingredient for the formulation of animal feed, and it is also exported especially to Europe, South Korea, and Japan. It can barely be consumed by nonruminant (monogastric) animals owing to the high percentages of hemicellulose and cellulose contents. Palm kernel cake must undergo suitable pretreatment in order to decrease the percentage of hemicellulose and cellulose. One of the methods employed in this study is fermentation with microorganisms, particularly fungi, to partially degrade the hemicellulose and cellulose content. This work focused on the production of enzymes by Aspergillus niger and profiling using palm kernel cake as carbon source.     

Culture conditions dictate protease and tannase production in submerged and solid-state cultures of Aspergillus niger Aa-20

Undesirable protease production by Aspergillus niger Aa-20 in submerged culture and solid-state culture was evaluated using different concentrations of tannic acid as sole carbon source in a model system designed for tannase production. Protease production was found to be dependent on the culture system used (submerged culture or solid-state culture) and on the initial tannic acid concentration. Expression of protease activity in submerged culture was higher (up to 10 times) than activity obtained in solid-state culture, using identical culture medium composition. In submerged culture, the lowest final protease activity (0.13 IU) was obtained with the highest tannic acid concentration, while in solid-state culture protease activity was not affected by changes in initial substrate concentration. Absence of detectable proteolytic activity in solid-state culture is related to high production of tannase enzyme. Hence, the use of solid-state culture for fungal enzyme production may allow for higher and more stable enzyme titers present in culture extracts.     

Enhanced production of xylanase from locally isolated fungal strain using agro-industrial residues under solid-state fermentation

This study is related to the isolation of fungal strain for xylanase production using agro-industrial residues. Forty fungal strains with xylanolytic potential were isolated by using xylan agar plates and quantitatively screened in solid-state fermentation. Of all the tested isolates, the strain showing highest ability to produce xylanase was assigned the code Aspergillus niger LCBT-14. For the enhanced production of the enzyme, five different fermentation media were evaluated. Out of all media, M4 containing wheat bran gave maximum enzyme production. Effect of different variables including incubation time, temperature, pH, carbon and nitrogen sources has been investigated. The optimum enzyme production was obtained after 72 h at 30°C and pH 4. Glucose as a carbon source while ammonium sulphate and yeast extract as nitrogen sources gave maximum xylanase production (946 U/mL/min). This study was successful in producing xylanase by A. niger LCBT-14 economically by utilising cheap indigenous substrate.     

Production of coconut aroma by fungi cultivation in solid-state fermentation

The production of 6-pentyl-α-pyrone (6-PP), an unsaturated d-lactone with a strong coconut-like aroma was studied and compared with liquid and solid substrates. A fungi strain that produces coconut aroma compound was selected. The liquid medium of the submerged culture was used to impregnate a solid support of sugarcane bagasse in SSF (Solid State Fermentation). This substrate was adequate for growth and aroma production; the concentration obtained using SSF was higher than using liquid fermentation process. In the present work, it is demonstrated that, by solid-state-fermentation process, it is possible to produce 6-PP. The amount of 6-PP produced using a solid state substrate, following a 5 d culture, was 3 mg/g dry matter. Therefore, the amount of 6-PP produced during solid-state-fermentation process is higher than that reported in literature for submerged process.     

Gibberellic acid production by solid-state fermentation in coffee husk

Five strains of Gibberella fujikuroi and one of Fusarium moniliforme were screened for the production of gibberellic acid (GA₃) in coffee husk, and based on the results, one strain, G. fujikuroi LPB-06, was selected. The comparative production of GA₃ by solid-state fermentation and submerged fermentation indicated better productivity with the former technique, mainly with pretreated substrate. The GA₃ accumulation was 6.1 times higher in the case of solid-state fermentation. Considering the C:N relation, higher yields of GA₃ were achieved using a mixed substrate comprising coffee husk and cassava bagasse (7:3, dry wt), increasing the results twice. Supplementation of an optimized saline solution containing 0.03% FeSO₄ and 0.01% (NH₄)₂SO₄ enhanced the accumulation of GA₃ 1.7 times in the fermented substrate. Under the finally optimized condition, the culture gave a maximum of 492.5 mg of GA₃/kg of dry substrate, with a pH of 5.3, moisture of 75%, and incubation temperature of 29°C. GA₃ yield was almost 13 times more than the initial results.     

Mutagenesis and analysis of mold Aspergillus niger for extracellular glucose oxidase production using sugarcane molasses

Aspergillus niger ORS-4.410, a mutant of A. niger ORS-4, was generated by repeated ultraviolet (UV) irradiation. Analysis of the UV treatment dose on wild-type (WT) A. niger ORS-4, conidial survival, and frequency of mutation showed that the maximum frequency of positive mutants (25.5%) was obtained with a 57% conidial survival rate after the second stage of UV irradiation. The level of glucose oxidase (GOX) production from mutant A. niger ORS-4.410 thus obtained was 149% higher than that for WT strain A. niger ORS-4 under liquid culture conditions using hexacyanoferrate (HCF)-treated sugarcane molasses (TM) as a cheaper carbohydrate source. When subcultured monthly for 24 mo, the mutant strain had consistent levels of GOX production (2.62±0.51 U/mL). Mutant A. niger ORS-4.410 was markedly different from the parent strain morphologically and was found to grow abundantly on sugarcane molasses. The mutant strain showed 3.43-fold increases in GOX levels (2.62±0.51 U/mL) using HCF-TM compared with the crude form of cane molasses (0.762±0.158 U/mL). The results reported herein were obtained while the author was working at the Department of Biotechnology, Indian Institute of Technology, Roorkee-247667, India.     

Effect of the oxygen transfer coefficient on xylitol production from sugarcane bagasse hydrolysate by continuous stirred-tank reactor fermentation

The effect of the oxygen transfer coefficient on the production of xylitol by biocon version of xylose present in sugarcane bagasse hemicellulosic hydrolysate using the yeast Candiada guilliermondii was investigated. Continuous cultivation was carried out in a 1.25-L fermentor at 30°C, pH 5.5, 300 rpm, and a dilution rate of 0.03/h, using oxygen transfer coefficients of 10,20, and 30/h. The results showed that the microbial xylitol production (11 g/L) increased by 108% with the decrease in the oxygen volumetric transfer coefficient from 30 to 20/h. The maximum values of xylitol productivity (0.7g/[L…h]) and yield (0.58 g/g) were obtained at k _L a 20/h.     

Solid-state fermentation for production of phytase by Rhizopus oligosporus

Solid-state fermentation of coconut oil cake has been carried out with Rhizopus oligosporus for the production of phytase. Phytase is used commercially in the animal feed industry to improve animal performance because there is a substantial and growing interest among swine and poultry producers in the application of phytase to improve the nutritional quality in animal feeds. Demonstrated benefits include improved feed yield ratios and reduction in the environmental costs associated with the disposal of animal wastes. We report the production of extracellular phytase by R. oligosporus under solid-state fermentation using coconut oil cake as substrate. Maximal enzyme production (14.29 U/g of dry substrate) occurred at pH 5.3, 30°C, and 54.5% moisture content after 96 h of incubation. The addition of extra nutrients to the substrate resulted in inhibition of product formation. The results indicate the scope for production of phytase using coconut oil cake as solid substrate without additional nutrients.     

Fungi allergens produced by solid-state fermentation process

Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate · d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16–160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.     

Xanthan gum production from cassava bagasse hydrolysate with Xanthomonas campestris using alternative sources of nitrogen

Cassava bagasse was hydrolyzed using HCl and the hydrolysate was used for the production of xanthan gum using a bacterial culture of Xanthomonas campestris. Cassava bagasse hydrolysate with an initial concentration of approx 20 g of glucose/L proved to be the best substrate concentration for xanthan gum production. Among the organic and inorganic nitrogen sources tested to supplement the medium—urea, yeast extract, peptone, potassium nitrate, and ammonium sulfate—potassium nitrate was most suitable. Ammonium sulfate was the least effective for xanthan gum production, and it affected sugar utilization by the bacterial culture. In media with an initial sugar concentration of 48.6 and 40.4 g/L, at the end of fermentation about 30 g/L of sugars was unused. Maximum xanthan gum (about 14 g/L) was produced when fermentation was carried out with a medium containing 19.8 g/L of initial reducing sugars supplemented with potassium nitrate and fermented for 72 h, and it remained almost the same until the end of fermentation (i.e., 96 h).     

Evaluation of different carbon and nitrogen sources in production of rhamnolipids by a strain of Pseudomonas aeruginosa

Culture conditions involving variations in carbon and nitrogen sources and different C:N ratios were examined with the aim of increasing productivity in the process of rhamnolipid synthesis by Pseudomonas aeruginosa. In addition to the differences in productivity, the use of different carbon sources resulted in several proportions related to the types of rhamnolipids synthesized (monorhamnolipids and dirhamnolipids). Furthermore, the variation in nutrients, mainly the nitrogen source, resulted in different amounts of virulence factors, as phenazines and extracellular proteins. The data point out a new concern in the choice of substrate to be used for rhamnolipid production by P. aeruginosa: toxic byproducts.     

Effect of span 20 concentration on oxalic acid production from post-refining fatty acids by Aspergillus niger XP

Submerged fermentation experiments were carried out to study the stimulating effects of the surfactant Span 20 on the growth of Aspergillus niger XP mutant and oxalic acid production from the post-refining fatty acids. Span 20 concentration of 0.75 g dm⁻³ was found to be the most suitable for oxalic acid production from fatty acids. Using this dose and a fermentation medium containing 30 g dm⁻³ of post-refining fatty acids, the oxalic acid production, oxalate yield, and overall oxalate productivity were the highest. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Effect of temperature, moisture, and carbon supplementation on lipase production by solid-state fermentation of soy cake by Penicillium simplicissimum

The production of lipases by Penicillium simplicissimum using solid-state fermentation and soy cake as substrate was investigated. The effects of temperature, cake moisture, and carbon supplementation on lipase production were studied using a two-level experimental plan. Moisture, pH, and lipase activity were followed during fermentation. Statistical analysis of the results was performed to evaluate the effect of the studied variables on the maximum lipase activity. Incubation temperature was the variable that most affected enzyme activity, showing a negative effect. Moisture and carbon supplementation presented a positive effect on activity. It was possible to obtain lipase activity as high as 21 U/g of dry cake in the studied range of process variables.     

Relationship between coffee husk caffeine degradation and respiration of Aspergillus sp. LPBx in solid-state fermentation

Studies were carried out in a packed-bed column fermentor using coffee husk as substrate in order to verify a relationship between caffeine degradation and the respiration of Aspergillus sp. LPBx. Fermentation conditions were optimized by using factorial design experiments. The kinetic study showed that the caffeine degradation was related to the development of mold and its respiration and also with the consumption of reducing sugars present in coffee husk. From the values obtained experimentally for oxygen uptake rate and CO₂ evolved, we determined a biomass yield of 3.811 g of biomass/g of consumed O₂ and a maintenance coefficient of 0.0031 g of consumed O₂/(g of biomass·h). The maximum caffeine degradation achieved was 90%.