Isolation of Penicillium expansum WH-3 for the production of L(+)-tartaric acid / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

The L(+)-form of tartaric acid (L(+)-TA) exists extensively in nature, and is widely used in the food, chemical, textile, building, and pharmaceutical industries (Su et al., 2001). The main method for L(+)-TA production is microbial transformation by cis-epoxysuccinate hydrolase (CESH), which can catalyze the asymmetric hydrolysis of cis-epoxysuccinic acid or its salts to TA or tartrate (Bao et al., 2019). Seventeen species containing CESH have been isolated so far. However, most species for L(+)-TA production have been reported from bacteria (Xuan and Feng, 2019). The only fungus isolated from soil by our lab recently, that could be used as catalyst for the process under acidic condition, is Aspergillus niger WH-2 (Bao et al., 2020). In order to find strains with new characteristics, this study attempted to isolate a new CESH source from fungi and investigate its application value.

Efecto antifúngico y antimicotoxigénico de Lactobacillus plantarum CRL 778 a diferentes valores de actividad de agua / Antifungal and antimycotoxigenic effect of Lactobacillus plantarum CRL 778 at different water activity values

Ochratoxin A (OTA) is a mycotoxin produced by filamentous fungi with high impact Lactic acid bacteria; in food safety due to its toxicity. In the last decade, the presence of OTA was widely reported in different foods. In this study, the ability of Lactobacillus (L.) plantarum CRL 778 to control growth and OTA production by Aspergillus (A.) niger 13D strain, at different water activity (a w) values (0.955, 0.964, 0.971, 0.982, and 0.995) was determined in vitro. Both parame ters were significantly (p<0.05) reduced by the lactobacilli and the effect depended on a w. Greatest growth rate inhibition (46.9%) was obtained at a w = 0.995, which is the most suitable value for growth and production of antifungal metabolites (lactic acid, acetic acid, phenyllac-tic and hydroxyl-phenyllactic acids) by L. plantarum CRL 778. Besides, morphological changes and inhibition of melanin synthesis were observed in colonies of A. niger 13D in presence of L. plantarum CRL 778 at a w ranged between 0.971 and 0.995. In addition, maximum reduction (90%) of OTA production took place at a w = 0.971, while inhibition of fungi growth was more evident at a w =0.995. These findings suggest that L. plantarum CRL 778 could be used for control of ochratoxigenic fungal growth and OTA contamination in different fermented foods with a w values between 0.971 and 0.995.

Ocratoxina A (OTA) es una micotoxina producida por hongos filamentosos con un alto impacto en la seguridad alimentaria debido a su toxicidad. En la última década se ha reportado ampliamente a nivel mundial, la presencia de OTA en diversos alimentos. En este estudio se evaluó in vitro, la capacidad de Lactobacillus (L.) plantarum CRL 778 de controlar el crecimiento y la producción de OTA por Aspergillus (A.) niger 13D, a diferentes valores de actividad de agua (a w): 0.955, 0.964, 0.971,0.982 y 0.995). La cepa láctica redujo significativamente (p <0.05) ambos parámetros, siendo el efecto dependiente del valor de a w. La mayor inhibición del crecimiento (46.9%) se obtuvo a a w =0.995, valor más adecuado para el crecimiento y producción de metabolitos antifúngicos (ácido láctico, ácido acético, ácidos fenil-láctico e hidroxi-fenil láctico) por la cepa láctica. Además, se observaron cambios morfológicos en las colonias de A. niger 13D, crecidas en presencia de L. plantarum CRL 778 a valores de a w de 0.971 y 0.995. El porcentaje máximo de reducción en la producción de OTA (90%) por la cepa láctica se observó a un valor de a w = 0.971, mientras la inhibición del crecimiento fúngico fue mayor cuando a w = 0.995. Estos hallazgos sugieren que L. plantarum CRL 778 podría emplearse para el control de la contaminación por hongos ocratoxigénicos en alimentos con valores de aw comprendidos entre 0.971-0.995.

Optimizing culture conditions for production of intra and extracellular inulinase and invertase from Aspergillus niger ATCC 20611 by response surface methodology (RSM)

Abstract The aim of this study was obtain a model that maximizes growth and production of inulinase and invertase by Aspergillus niger ATCC 20611, employing response surface methodology (RSM). The RSM with a five-variable and three-level central composite design (CCD) was employed to optimize the medium composition. Results showed that the experimental data could be appropriately fitted into a second-order polynomial model with a coefficient of determination (R2) more than 0.90 for all responses. This model adequately explained the data variation and represented the actual relationships between the parameters and responses. The pH and temperature value of the cultivation medium were the most significant variables and the effects of inoculum size and agitation speed were slightly lower. The intra-extracellular inulinase, invertase production and biomass content increased 10-32 fold in the optimized medium condition (pH 6.5, temperature 30 °C, 6% (v/v), inoculum size and 150 rpm agitation speed) by RSM compared with medium optimized through the one-factor-at-a-time method. The process development and intensification for simultaneous production of intra-extracellular inulinase (exo and endo inulinase) and invertase from A. niger could be used for industrial applications.

Surface response methodology for the optimization of lipase production under submerged fermentation by filamentous fungi

Abstract A Plackett–Burman Factorial Design of 16 experiments was conducted to assess the influence of nine factors on the production of lipases by filamentous fungi. The factors investigated were bran type (used as the main carbon source), nitrogen source, nitrogen source concentration, inducer, inducer concentration, fungal strain (Aspergillus niger or Aspergillus flavus were selected as good lipase producers via submerged fermentation), pH and agitation. The concentration of the yeast extract and soybean oil and the pH had a significant effect (p < 0.05) on lipase production and were consecutively studied through a Full Factorial Design 23, with the concentration of yeast extract and pH being significant (p < 0.05). These variables were optimized using a central composite design, obtaining maximum lipolytic activities with the use of 45 g/L of yeast extract and pH 7.15. The statistical model showed a 94.12% correlation with the experimental data.

Enhancing inulinase yield by irradiation mutation associated with optimization of culture conditions

A new inulinase-producing strain was isolated from rhizosphere soils of Jerusalem artichoke collected from Shihezi (Xinjiang, China) using Jerusalem artichoke power (JAP) as sole carbon source. It was identified as an Aspergillus niger strain by analysis of 16S rRNA. To improve inulinase production, this fungus was subjected to mutagenesis induced by ⁶⁰Co γ-irradiation. A genetically stable mutant (designated E12) was obtained and it showed 2.7-fold higher inulinase activity (128 U/mL) than the parental strain in the supernatant of a submerged culture. Sequential methodology was used to optimize the inulinase production of stain E12. A screening trial was first performed using Plackett-Burman design and variables with statistically significant effects on inulinase bio-production were identified. These significant factors were further optimized by central composite design experiments and response surface methodology. Finally, it was found that the maximum inulinase production (185 U/mL) could be achieved under the optimized conditions namely pH 7.0, yeast extract concentration of 5.0 g/L, JAP concentration of 66.5 g/L, peptone concentration of 29.1 g/L, solution volume of 49.4 mL in 250-mL shake flasks, agitation speed of 180 rpm, and fermentation time of 60 h. The yield of inulinase under optimized culture conditions was approximately 1.4-fold of that obtained by using basal culture medium. These findings are of significance for the potential industrial application of the mutant E12.

Thermotolerant and mesophylic fungi from sugarcane bagasse and their prospection for biomass-degrading enzyme production

Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best β-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For β-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0–8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.

Bioleaching of gold, copper and nickel from waste cellular phone PCBs and computer goldfinger motherboards by two Aspergillus nigerstrains

In an effort to develop alternate techniques to recover metals from waste electrical and electronic equipment (WEEE), this research evaluated the bioleaching efficiency of gold (Au), copper (Cu) and nickel (Ni) by two strains of Aspergillus niger in the presence of gold-plated finger integrated circuits found in computer motherboards (GFICMs) and cellular phone printed circuit boards (PCBs). These three metals were analyzed for their commercial value and their diverse applications in the industry. Au-bioleaching ranged from 42 to 1% for Aspergillus niger strain MXPE6; with the combination of Aspergillus niger MXPE6 + Aspergillus niger MX7, the Au-bioleaching was 87 and 28% for PCBs and GFICMs, respectively. In contrast, the bioleaching of Cu by Aspergillus niger MXPE6 was 24 and 5%; using the combination of both strains, the values were 0.2 and 29% for PCBs and GFICMs, respectively. Fungal Ni-leaching was only found for PCBs, but with no significant differences among treatments. Improvement of the metal recovery efficiency by means of fungal metabolism is also discussed.

Hydroxylation of 1,8-cineole by Mucor ramannianus and Aspergillus niger

The monoterpenoid 1,8-cineole is obtained from the leaves of Eucalyptus globulus and it has important biological activities. It is a cheap natural substrate because it is a by-product of the Eucalyptus cultivation for wood and pulp production. In this study, it was evaluated the potential of three filamentous fungi in the biotransformation of 1,8-cineole. The study was divided in two steps: first, reactions were carried out with 1,8-cineole at 1 g/L for 24 h; afterwards, reactions were carried out with substrate at 5 g/L for 5 days. The substrate was hydroxylated into 2-exo-hydroxy-1,8-cineole and 3-exo-hydroxy-1,8-cineole by fungi Mucor ramannianus and Aspergillus niger with high stereoselectivity. Trichoderma harzianum was also tested but no transformation was detected. M. ramannianus led to higher than 99% of conversion within 24 h with a starting high substrate concentration (1 g/L). When substrate was added at 5 g/L, only M. ramannianus was able to catalyze the reaction, but the conversion level was 21.7% after 5 days. Both products have defined stereochemistry and could be used as chiral synthons. Furthermore, biological activity has been described for 3-exo-hydroxy-1,8-cineol. To the best of our knowledge, this is the first report on the use of M. ramannianus in this reaction.

Characterization of succinic semialdehyde dehydrogenase from Aspergillus niger.

The catabolism of fungal 4-aminobutyrate (GABA) occurs via succinic semialdehyde (SSA). Succinic semialdehyde dehydrogenase (SSADH) from the acidogenic fungus Aspergillus niger was purified from GABA grown mycelia to the highest specific activity of 277 nmol min-1 mg-1, using phenyl Sepharose and DEAE Sephacel chromatography. The purified enzyme was specific for its substrates SSA and NAD+. The substrate inhibition observed with SSA was uncompetitive with respect to NAD+. While product inhibition by succinate was not observed, NADH inhibited the enzyme competitively with respect to NAD+ and noncompetitively with respect to SSA. Dead-end inhibition by AMP and p-hydroxybenzaldehyde (pHB) was analyzed. The pHB inhibition was competitive with SSA and uncompetitive with NAD+; AMP competed with NAD+. Consistent with the kinetic data, a sequential, ordered Bi Bi mechanism is proposed for this enzyme.

Bioremediation of dyes by fungi isolated from contaminated dye effluent sites for bio-usability

Biodegradation and detoxification of dyes, Malachite green, Nigrosin and Basic fuchsin have been carried out using two fungal isolates Aspergillus niger, and Phanerochaete chrysosporium, isolated from dye effluent soil. Three methods were selected for biodegradation, viz. agar overlay and liquid media methods; stationary and shaking conditions at 25 °C. Aspergillus niger recorded maximum decolorization of the dye Basic fuchsin (81.85%) followed by Nigrosin (77.47%), Malachite green (72.77%) and dye mixture (33.08%) under shaking condition. Whereas, P. chrysosporium recorded decolorization to the maximum with the Nigrosin (90.15%) followed by Basic fuchsin (89.8%), Malachite green (83.25%) and mixture (78.4%). The selected fungal strains performed better under shaking conditions compared to stationary method; moreover the inoculation of fungus also brought the pH of the dye solutions to neutral from acidic. Seed germination bioassay study exhibited that when inoculated dye solutions were used, seed showed germination while uninoculated dyes inhibited germination even after four days of observation. Similarly, microbial growth was also inhibited by uninoculated dyes. The excellent performance of A. niger and P. chrysporium in the biodegradation of textile dyes of different chemical structures suggests and reinforces the potential of these fungi for environmental decontamination.

Utilization of molasses and sugar cane bagasse for production of fungal invertase in solid state fermentation using Aspergillus niger GH1

Agro-industrial wastes have been used as substrate-support in solid state fermentation for enzyme production. Molasses and sugarcane bagasse are by-products of sugar industry and can be employed as substrates for invertase production. Invertase is an important enzyme for sweeteners development. In this study, a xerophilic fungus Aspergillus niger GH1 isolated of the Mexican semi-desert, previously reported as an invertase over-producer strain was used. Molasses from Mexico and Cuba were chemically analyzed (total and reducer sugars, nitrogen and phosphorous contents); the last one was selected based on chemical composition. Fermentations were performed using virgin and hydrolyzate bagasse (treatment with concentrated sulfuric acid). Results indicated that, the enzymatic yield (5231 U/L) is higher than those reported by other A. niger strains under solid state fermentation, using hydrolyzate bagasse. The acid hydrolysis promotes availability of fermentable sugars. In addition, maximum invertase activity was detected at 24 h using low substrate concentration, which may reduce production costs. This study presents an alternative method for invertase production using a xerophilic fungus isolated from Mexican semi-desert and inexpensive substrates (molasses and sugarcane bagasse).

Guava Psidium guajava seed flour and dry Aspergillus niger mycelium as nitrogen sources for the production of biomass and antimicrobial compounds produced by Weissella confusa

Background: The fermentation substrate efficiency of glucose supplemented with guava seed flour (GGSF) or glucose supplemented with dry Aspergillus niger mycelium (GANM) was evaluated during the production of biomass and antimicrobial compounds by the lactic acid bacteria Weissella confusa. Results: The fermentation substrate efficiency was measured by comparing the biomass formation, substrate consumption, substrate conversion, antimicrobial activity and product yield. The antimicrobial activity was measured against a commercial Staphylococcus aureus strain. The results were compared against fermentations performed in a commercial substrate (CS), the MRS (Man-Rogosa-Sharpe) substrate. The fermentations were performed discontinuously for 4 hrs at 100 rpm and 32ºC. The biomass production exhibited a statistically significant difference (P ≤ 0.05) between treatments. The biomass production was 13.98% higher in the CS than in the GGSF and GANM substrates; however, there were no statistically significant differences for the specific growth rate. Conclusions: The GGSF and GANM substrates favored an antimicrobial activity against Staphylococcus aureus during the second and third hours of fermentation (inhibition diameter was 6.11% and 4.72%, respectively). The GGSF, GANM and CS substrates did not present statistically significant differences for the production of antimicrobial substances against Staphylococcus aureus. Therefore, GGSF and GANM can be considered as viable and economical alternative nitrogen sources for the production of the antimicrobial compounds formed by Weissella confusa in submerged fermentations.

Aislamiento de cepas de Aspergillus sección Nigri en la yerba mate comercializada en Posadas (Misiones, Argentina) y evaluación de su potencial ocratoxigénico / [Isolation of Aspergillus section Nigri strains in yerba mate in Posadas (Misiones, Argentina) and evaluation of their ochratoxigenic potential].

The objectives of the present work were to investigate the isolation frequency of genus Aspergillus in canchada yerba mate (YMCH) and elaborated yerba mate (YME) (Ilex paraguariensis) and the proportion of section Nigri isolates, as well as to determine ochratoxin A production by Aspergillus species section Nigri. Three hundred twenty eight Aspergillus strains from 20 samples of YMCH and 1306 Aspergillus strains from 36 samples of YME were isolated; of the total, 279 from the first group of strains and 1215 from the latter group, belonged to section Nigri. For the detection of ochratoxin A production, the strains were cultivated on Czapeck yeast extract agar and the toxin was detected by thin layer chromatography under UV light. Uniserate species predominance was observed in the 1494 strains of Aspergillus section Nigri obtained (Aspergillus japonicus var. japonicus and Aspergillus japonicus var. aculeatus), whereas none of the strains analysed showed ochratoxin A production in vitro at the detection level of the methodology employed.

Effect of extrinsic and intrinsic parameters on inulinase production by Aspergillus niger ATCC 20611

Background: Inulinase is a versatile enzyme from glycoside hydrolase family which targets the beta-2, 1 linkage of fructopolymers. In the present study, the effect of medium composition and culture conditions on inulinase production by Aspergillus niger ATCC 20611 was investigated in shake-flasks. Results: The highest extracellular inulinase (3199 U/ ml) was obtained in the presence of 25 percent (w/v) sucrose, 0.5 percent (w/v) meat extract, 1.5 percent (w/v) NaNO3 and 2.5 mM (v/v) Zn2+, at initial pH of 6.5, temperature 35ºC and 6 percent (v/v) of spores suspension in the agitation speed of 100 rpm. Surfactants showed an inhibitory effect on enzyme production. The optimum temperature for inulinase activity was found to be 50ºC. TLC analysis showed the presence of both exo- and endo-inulinase. Conclusion: Sucrose, Zn2+, and aeration were found to be the most effective elements in inulinase production by A. niger ATCC 20611. TLC analysis also showed that the crude enzyme contained both endo and exo-inulinases. The strain is suggested as a potential candidate for industrial enzymatic production of fructose from inulin.

Bioconversion of apple pomace into a multienzyme bio-feed by two mixed strains of Aspergillus niger in solid state fermentation

Apple pomace is a wasted resource produced in large quantities and its deposit has caused serious environmental problems, so it is significance to make the full utilization of the residues. The objectives of this work were to produce multienzyme bio-feed, biodegrade the anti-nutritional factors such as pectin and tannins in apple pomace, and obtain the nutritional enrichment of the fermented substrate. The mixture of apple pomace and cottonseed powder (1:1, w/w), supplemented with 1 percent (w/w) (NH4)2SO4 and 0.1 percent (w/w) KH2PO4, was proved to be the optimum medium for the mixed strains of Aspergillus niger M2 and M3 (2:1, w/w). The activities of pectinase, proteinase and cellulase achieved 21168 u/g, 3585 u/g and 1208u/g, and the biodegradation rates of pectin and tannins reached 99.0 percent and 66.1 percent, respectively, when 0.4 percent(w/w) of the test fungiwere inoculated and incubated at 30ºC for 48 hrs in solid state fermentation. The utilization of apple pomace in the paper can be served as a model for the similar waste recycling.

Production of calcium gluconate by fermentation.

Calcium gluconate production by Aspergillus niger was investigated in shake flask, rolling shaker, air-lift reactor and stirred reactor. Growth pattern of the organism and fermentation conditions determined the yield of the product. High calcium gluconate production was achieved in air-lift reactor with pellet form of cell growth at moderate specific growth rate and biomass concentration. In another variation of air-lift reactor, when calcium carbonate was confined to a cellulose membrane, calcium gluconate production was maximum (149 g/L). At higher specific growth rate, obtained in shake flask, despite the formation of cell pellets, product formation was low. Physical separation of particulate calcium carbonate and growing cells favoured product formation. In stirred reactor pulpy mycelial growth was obtained and calcium gluconate production was poor.

Intra and extracellular nuclease production by Aspergillus niger and Aspergillus nidulans

Intra and extracellular nuclease production by strains of "Aspergillus niger"and "Aspergillus nidulans" was estimated using a modified DNAse test agar and cell-0free extract assays. Differences in the production of nucleases by A. niger and A. nidulans were observed. These observations suggest that the DNAse test agar can be helpful for a quick screening for some types of nucleases in filamentous fungi. The assays using cell-free extracts can also be useful for initial characterization of other types of nucleases.

A phytoalexin is modified to less fungitoxic substances by crown rot pathogen in groundnut Arachis hypogaea L.

Phytoalexins from four different treatments viz. control, AF 1-treated, A. niger-treated, and dual inoculated were separated by TLC showed that one phytoalexin with an Rf value of 0.628 (P1) appeared on 2nd day only in dual-inoculated seeds of groundnut (A. hypogaea). By 3rd day three additional phytoalexins were visualized in response to A. niger-treatment with lower Rf values 0.485 (P2), 0.388 (P3) and 0.314 (P4) as compared to P1. In dual inoculated seedlings, P1 and P3 could be visualized while only P1 appeared in response to AF 1 on 3rd day. All the compounds lost fluorescence on exposure to light, got converted to pale yellow colour. In all the treatments no phytoalexin accumulation was observed after 3rd day. All the four phytoalexins had a major peak between 338 and 339.5 nm. In potato dextrose broth, the growth of A. niger showed a steady increase up to 32 hr while it was significantly inhibited with P1 in microtiter plates. P2, P3 and P4 (in the same order) had significantly less antifungal activity as compared to P1. The antifungal activity of the phytoalexins decreased with decrease in their Rf value.

Removal of gold, silver and copper by living and nonliving fungi from leach liquor obtained from the gold mining industry

Três linhagens de fungos, identificadas como Aspergillus flavus, Aspergillus fumigatus e Aspergillus niger, isoladas de amostras da planta de extraçäo de ouro da Mineraçäo Morro Velho (Nova Lima, Brasil), foram testadas quanto à capacidade de remover metais pesados em uma soluçäo obtida do circuito de beneficiamento de minério de ouro da mineraçäo (liquor de lixiviaçäo). Foi investida a habilidade desses fungos em remover ouro, prata e cobre via processos independentes do metabolismo (biomassa seca) e células cultivadas no liquor de lixiviaçäo. A biomassa seca das três linhagens estudadas apresentou uma baixa capacidade de bissorçäo de metais, provavelmente devido a elevada concentraçäo de cianeto no liquor de lixiviaçäo. Os fungos A. fumigatus e A. niger, quando metabolicamente ativos, apresentaram uma elevada habilidade de remoçäo desses metais. A presença de atividade metabólica nas células fúngicas