Gibberellic acid production by free and immobilized cells in different culture systems

Gibberellic acid production was studied in different fermentation systems. Free and immobilized cells of Gibberella fujikuroi cultures in shakeflask, stirred and fixed-bed reactors were evaluated for the production of gibberellic acid (GA₃). Gibberellic acid production with free cells cultured in a stirred reactor reached 0.206 g/L and a yield of 0.078 g of GA₃/g biomass.     

Amylase Production by Saccharomycopsis fibuligera A11 in Solid-State Fermentation for Hydrolysis of Cassava Starch

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD_600 nm = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 °C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.     

Solid-state Fermentation for Enhanced Production of Laccase using Indigenously Isolated <Emphasis Type="Italic">Ganoderma</Emphasis> sp.

Laccase production by solid-state fermentation (SSF) using an indigenously isolated white rot basidiomycete Ganoderma sp. was studied. Among the various agricultural wastes tested, wheat bran was found to be the best substrate for laccase production. Solid-state fermentation parameters such as optimum substrate, initial moisture content, and inoculum size were optimized using the one-factor-at-a-time method. A maximum laccase yield of 2,400 U/g dry substrate (U/gds) was obtained using wheat bran as substrate with 70% initial moisture content at 25°C and the seven agar plugs as the inoculum. Further enhancement in laccase production was achieved by supplementing the solid-state medium with additional carbon and nitrogen source such as starch and yeast extract. This medium was optimized by response surface methodology, and a fourfold increase in laccase activity (10,050 U/g dry substrate) was achieved. Thus, the indigenous isolate seems to be a potential laccase producer using SSF. The process also promises economic utilization and value addition of agro-residues.     

Production of Alkaline Protease by Bacillus altitudinis GVC11 using Castor Husk in Solid-State Fermentation

Castor (Ricinus communis L.) is an important oil seed crop having its main cultivated area in India, China, and Brazil in dry land farming. Castor husk is generated as waste in castor oil production. Use of castor husk waste as substrate is studied for alkaline protease production by Bacillus altitudinis GVC11 in solid-state fermentation. Various parameters like moisture content, incubation period, particle size, effect of carbon and nitrogen sources are studied and optimized for enzyme production. Highest enzyme production of 419,293 units per gram husk is obtained. Cost of enzyme production can be reduced by using castor husk as substrate.     

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%.     

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.     

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.     

High-Yield Hypocrellin A Production in Solid-State Fermentation by <Emphasis Type="Italic">Shiraia</Emphasis> sp. SUPER-H168

Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 × 10⁶ spores, substrate particle size 0.8–1 mm, initial moisture content 50%, and temperature 30 °C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO₃ were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO₃ were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.     

Development of a bionematicide with Paecilomyces lilacinus to control Meloidogyne incognita

Root-knot disease caused by Meloidogyne incognita is a matter of grave concern because it affects several economically important crop plants. The use of solid-state fermentation (SSF) may help to elaborate efficient formulations with fungi to be employed in the biologic control of nematodes. Attempts were made to select low-cost substrates for spore production of a strain of Paecilomyces lilacinus with known nematicide capacity. Coffee husks, cassava bagasse, and defatted soybean cake were utilized as substrates, and sugarcane bagasse was used as support. Fermentations were carried out in flasks covered with filter paper at 28°C for 10 d. The products obtained by SSF were evaluated for their nematicide activity in pot experiments containing one seedling of the plant Coleus inoculated with the nematode M. incognita. The plants were evaluated 2 mo after inoculation. Fermented products showed a reduction in the number of nematodes. The best results were obtained with defatted soybean cake, which showed almost 100% reduction in the number of nematodes; the reduction with coffee husk was 80% and with cassava bagasse was about 60%.     

Zymomonas mobilis as catalyst for the biotechnological production of sorbitol and gluconic acid

The conversion of glucose and fructose into gluconic acid (GA) and sorbitol (SOR) was conducted in a batch reactor with free (CTAB-treated or not) or immobilized cells of Zymomonas mobilis. High yields (more than 90%) of gluconic acid and sorbitol were attained at initial substrate concentration of 600 g/L (glucose plus fructose at 1:1 ratio), using cells with glucose-fructose-oxidoreductase activity of 75 U/L. The concentration of the products varied hyperbolically with time according to the equations (GA)=t(GA)_max/(W_GA +t), (SOR)=t (SOR)_max/(W_Sor+t), v_GA=[W_GA (GA)_max]/(W_GA+t)² and V_SOR=[W_SOR (SOR)_max]/(W_SOR+t)². Taking the test carried out with free CTAB-treated cells as an example, the constant parameters were (GA)_max= 541 g/L, (SOR)_max=552 g/L, W_GA=4.8h, W_SOR=4.9h, υ_GA=112.7 g/L· and υ_SOR=112.7 g/L·.     

Production of α-amylase with Aspergillus oryzae on spent brewing grain by solid substrate fermentation

Ten Aspergillus oryzae strains were screened in solid substrate fermentation for α-amylase production on spent brewing grain (SBG) and on corn fiber. SBG proved to be a better substrate for enzyme production than corn fiber. A Plackett-Burman experimental design was used to optimize the medium composition for the best strain. Solid substrate fermentation on optimized medium with A. oryzae NRRL 1808 (=ATCC 12892) strain in stationary 500-mL Erlenmeyer flask culture yielded 4519 U of α-amylase/g of dry matter substrate in 3 d. The whole solid substrate fermentation material (crude enzyme, in situ enzyme) may be considered a cheap biocatalytic material for animal feed rations and for bioalcohol production from starchy materials.     

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.     

Enhancement of L-Asparaginase Production by Isolated <Emphasis Type="Italic">Bacillus circulans</Emphasis> (MTCC 8574) Using Response Surface Methodology

L-asparaginase production was optimized using isolated Bacillus circulans (MTCC 8574) under solid-state fermentation (SSF) using locally available agricultural waste materials. Among different agricultural materials (red gram husk, bengal gram husk, coconut, and groundnut cake), red gram husk gave the maximum enzyme production. A wide range of SSF parameters were optimized for maximize the production of L-asparaginase. Preliminary studies revealed that incubation temperature, moisture content, inoculum level, glucose, and L-asparagine play a vital role in enzyme yield. The interactive behavior of each of these parameters along with their significance on enzyme yield was analyzed using fractional factorial central composite design (FFCCD). The observed correlation coefficient (R ²) was 0.9714. Only L-asparagine and incubation temperature, were significant in linear and quadratic terms. L-asparaginase yield improved from 780 to 2,322 U/gds which is more than 300% using FFCCD as a means of optimizing conditions.     

Gibberellin Promotes Cell Growth and Induces Changes in Fatty Acid Biosynthesis and Upregulates Fatty Acid Biosynthetic Genes in Chlorella vulgaris UMT-M1

Microalgae lipids and oils are potential candidates for renewable biofuels and nutritional inventions. Recent studies from our lab have shown that two plant hormones, auxin and jasmonic acid, influence microalgae growth and fatty acid accumulation. Therefore, in this study, a high oil-producing strain Chlorella vulgaris UMT-M1 was selected for hormonal study using gibberellin (GA). Exogenous GA₃ was applied to early stationary culture of C. vulgaris UMT-M1. Results showed that GA₃ gradually increases the cell density of C. vulgaris to up to 42% on days after treatment (DAT)-8 and also capable of delaying the algal senescence. However, the increment in cell density did not enhance the total oil production albeit transient modification of fatty acid compositions was observed for saturated (SFA) and polyunsaturated (PUFA) fatty acids. This illustrates that GA₃ only promotes cell division and growth but not the oil accumulation. In addition, application of GA₃ in culture medium was shown to promote transient increment of palmitic (C16:0) and stearic (C18:0) acids from DAT-4 to DAT-6 and these changes are correlated with the expression of β-ketoacyl ACP synthase I (KAS I) gene.

     

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.     

Improvement on Citric Acid Production in Solid-state Fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis> LPB BC Mutant Using Citric Pulp

Citric acid (CA) production has been conducted through a careful strain selection, physical–chemical optimization and mutation. The aim of this work was to optimize the physical–chemical conditions of CA production by solid-state fermentation (SSF) using the Aspergillus niger LPB BC strain, which was isolated in our laboratory. The parental and mutant strain showed a good production of CA using citric pulp (CP) as a substrate. The physical–chemical parameters were optimized and the best production was reached at 65% moisture, 30 °C and pH 5.5. The influence of the addition of commercial and alternative sugars, nitrogen sources, salts, and alcohols was also studied. The best results (445.4 g of CA/kg of CP) were obtained with sugarcane molasses and 4% methanol (v/w). The mutagenesis induction of LPB BC was performed with UV irradiation. Eleven mutant strains were tested in SSF where two mutants showed a higher CA production when compared to the parental strain. A. niger LPB B3 produced 537.6 g of CA/kg of CP on the sixth day of fermentation, while A. niger LPB B6 produced 616.5 g of CA/kg of CP on the fourth day of fermentation, representing a 19.5% and 37% gain, respectively.     

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.     

Production of bacterial cellulose by Gluconacetobacter sp. RKY5 isolated from persimmon vinegar

The optimum fermentation medium for the production of bacterial cellulose (BC) by a newly isolated Gluconacetobacter sp. RKY5 was investigated. The optimized medium composition for cellulose production was determined to be 15 g/L glycerol, 8 g/L yeast extract, 3 g/L K₂HPO₄, and 3 g/L acetic acid. Under these optimized culture medium, Gluconacetobacter sp. RKY5 produced 5.63 g/L of BC after 144 h of shaken culture, although 4.59 g/L of BC was produced after 144 h of static culture. The amount of BC produced by Gluconacetobacter sp. RKY5 was more than 2 times in the optimized medium found in this study than in a standard Hestrin and Shramm medium, which was generally used for the cultivation of BC-producing organisms.     

Continuous fermentation of cellulosic biomass to ethanol

Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H₂SO₄) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H₂SO₄). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.