Multicomponent analysis of fermentation growth media using the electronic tongue (ET)

A potentiometric electronic tongue (ET) consisting of eight cross-sensitive chemical sensors and a standard pH electrode has been applied for analysis of simulated fermentation solutions typical for fermentation processes with Aspergillus niger. The electronic tongue has been found capable of simultaneous determination of ammonium, citrate and oxalate in complex media with good precision (typical error within 8%). The system preserved high sensitivity to the targeted substances also in the presence of sodium azide, which is commonly used for suppressing microbial activity in real-world fermentation samples. Sensor performance was fast and reproducible which promises well for routine application of the electronic tongue for fermentation process monitoring.     

Optimization of Xylanase Production by Filamentous Fungi in Solid-State Fermentation and Scale-up to Horizontal Tube Bioreactor

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504?±?7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO₃, 1.3 g/L (NH₄)₂SO₄, 4.5 g/L KH₂PO₄, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube bioreactor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.     

Effect of Acetic Acid on Citric Acid Fermentation in an Integrated Citric Acid–Methane Fermentation Process

An integrated citric acid–methane fermentation process was proposed to solve the problem of extraction wastewater in citric acid fermentation process. Extraction wastewater was treated by anaerobic digestion and then recycled for the next batch of citric acid fermentation to eliminate wastewater discharge and reduce water resource consumption. Acetic acid as an intermediate product of methane fermentation was present in anaerobic digestion effluent. In this study, the effect of acetic acid on citric acid fermentation was investigated and results showed that lower concentration of acetic acid could promote Aspergillus niger growth and citric acid production. 5-Cyano-2,3-ditolyl tetrazolium chloride (CTC) staining was used to quantify the activity of A. niger cells, and the results suggested that when acetic acid concentration was above 8 mM at initial pH 4.5, the morphology of A. niger became uneven and the part of the cells’ activity was significantly reduced, thereby resulting in deceasing of citric acid production. Effects of acetic acid on citric acid fermentation, as influenced by initial pH and cell number in inocula, were also examined. The result indicated that inhibition by acetic acid increased as initial pH declined and was rarely influenced by cell number in inocula.     

Characterization of a novel <Emphasis Type="Italic">Aspergillus niger</Emphasis> beta-glucosidase tolerant to saccharification of lignocellulosic biomass products and fermentation inhibitors

Properties of beta-glucosidase produced by Aspergillus niger URM 6642 recently isolated from the Atlantic rainforest biome and its potential tolerance to saccharification of lignocellulosic biomass products and fermentation inhibitors was evaluated. The fungus was cultivated under solid state culture conditions at 37°C with different agro-industrial wastes. High levels of beta-glucosidase (3778.9 U g^?1)from A. niger were obtained with rice meal as substrate under solid state culture conditions after ten days. Optimum pH for this particular beta-glucosidase activity was 4.0 although it was stable in the range of 4.0 to 7.0. The half-life (T_½) of beta-glucosidase at 55°C is 3 h. However, at the optimum temperature of the enzyme, 65°C, T_½ is 20 min. The enzyme showed tolerance to various compounds such as glucose, xylose, 5-hydroxymethyl furfural, furfural, coumarin, ethanol and acetic acid. Therefore, beta-glucosidase from the novel A. niger species may be of potential use in the saccharification of lignocellulosic biomass, as well as an additional enzyme supplement in cellulase cocktails used to increase the yield of fermentable sugars.     

Microbial Leaching of Waste Solder for Recovery of Metal

This study proposes an environment-friendly bioleaching process for recovery of metals from solders. Tin-copper (Sn-Cu), tin-copper-silver (Sn-Cu-Ag), and tin-lead (Sn-Pb) solders were used in the current study. The culture supernatant of Aspergillus niger removed metals faster than the culture supernatant of Acidithiobacillus ferrooxidans. Also, the metal removal by A. niger culture supernatant is faster for Sn-Cu-Ag solder as compared to other solder types. The effect of various process parameters such as shaking speed, temperature, volume of culture supernatant, and increased solder weight on bioleaching of metals was studied. About 99 (±1.75)?% metal dissolution was achieved in 60 h, at 200-rpm shaking speed, 30 °C temperature, and by using 100-ml A. niger culture supernatant. An optimum solder weight for bioleaching was found to be 5 g/l. Addition of sodium hydroxide (NaOH) and sodium chloride (NaCl) in the bioleached solution from Sn-Cu-Ag precipitated tin (85?±?0.35 %) and silver (80?±?0.08 %), respectively. Passing of hydrogen sulfide (H₂S) gas at pH 8.1 selectively precipitated lead (57.18?±?0.13 %) from the Sn-Pb bioleached solution. The proposed innovative bioleaching process provides an alternative technology for recycling waste solders to conserve resources and protect environment.     

Effect of media composition and growth conditions on production of β-glucosidase by Aspergillus niger C-6

The hydrolytic activity of fungal originated β-glucosidase is exploited in several biotechnological processes to increase the rate and extent of saccharification of several cellulosic materials by hydrolyzing the cellobiose which inhibits cellulases. In a previous presentation, we reported the screening and liquid fermentation with Aspergillus niger, strain C-6 for β-glucosidase production at shake flask cultures in a basal culture medium with mineral salts, corn syrup liquor, and different waste lignocellulosic materials as the sole carbon source obtaining the maximum enzymatic activity after 5–6 d of 8.5 IU/mL using native sugar cane bagasse. In this work we describe the evaluation of fermentation conditions: growth temperature, medium composition, and pH, also the agitation and aeration effects for β-glucosidase production under submerged culture using a culture media with corn syrup liquor (CSL) and native sugar cane bagasse pith as the sole carbon source in a laboratory fermenter. The maximum enzyme titer of 7.2 IU/mL was obtained within 3 d of fermentation. This indicates that β-glucosidase productivity by Aspergillus niger C-6 is function of culture conditions, principally temperature, pH, culture medium conditions, and the oxygen supply given in the bioreactor. Results obtained suggest that this strain is a potential microorganism that can reach a major level of enzyme production and also for enzyme characterization.     

Production of single-cell protein with two-step fermentation for treatment of potato starch processing waste

Potato starch processing waste is causing serious environmental problems. This study aimed to convert potato starch processing waste into single-cell protein as high-quality feed using a two-step fermentation process. The mutant strain Aspergillus niger H3 was selected after UV irradiation and ethyl methyl sulfone mutagenesis for more cellulase production. The activities of sodium carboxymethyl cellulase and filter paperase of strain H3 were 8.86 and 4.79 U, respectively, which were much higher than the parent strain (1.18 and 0.62 U). After treatment with strain H3, the cellulose degradation rate of potato residue was 80.54 %. A liquid fermentation using Bacillus licheniformis was performed as the second step. The optimized fermentation conditions were temperature of 32.8 °C, pH 6.67, and inoculum concentration of 1.78 % using the response surface method. Results of this study showed a potential application in large-scale industrial conversion.     

Simultaneous Production of Amyloglucosidase and Exo-Polygalacturonase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a Rotating Drum Reactor

Simultaneous production of amyloglucosidase (AMG) and exo-polygalacturonase (exo-PG) was carried out by Aspergillus niger in substrate of defatted rice bran in a rotating drum bioreactor (RDB) and studied by a 3¹ × 2² factorial experimental design. Variables under study were A. niger strains (A. niger NRRL 3122 and A. niger t0005/007-2), types of inoculum (spore suspension and fermented bran), and types of inducer (starch, pectin, and a mix of both). Solid-state fermentation process (SSF) was conducted at 30 °C under 60-vvm aeration for 96 h in a pilot scale. Production of AMG and exo-PG was significantly affected by the fungal strain and the type of inoculum, but inducers did not trigger any significant effect, an evidence of the fact that these enzymes are constitutive. The maximum activity of exo-PG was 84 U g_dm ^?1 whereas the maximum yield of AMG was 886.25 U g_dm ^?1.     

Fermentation monitoring using multisensor systems: feasibility study of the electronic tongue

The electronic tongue based on an array of 30 non-specific potentiometric chemical sensors has been applied to qualitative and quantitative monitoring of a batch fermentation process of starting culture for light cheese production. Process control charts were built by using PLS regression and data from fermentations run under normal operating conditions. Control charts allow discrimination of samples from fermentation batches run under abnormal operating conditions from normal ones at as early as 30–50% of fully evolved fermentations. The capability of the electronic tongue to quantify concentrations of important organic acids (citric, lactic and orotic) in the present type of fermentation media was demonstrated. Average prediction errors were assessed in the range 5–13% based on test set validation. Correlation between peptide profiles determined using HPLC and the electronic tongue output was also established. The electronic tongue was demonstrated to be a promising tool for fermentation process monitoring and quantitative analysis of growth media.http://www.electronictongue.com     

Protection of aspergillus niger cellulases by urea during growth on glucose or glycerol supplemented media

The cellulase enzymes ofAspergillus niger were found to undergo catabolite repression in the presence of glucose and glycerol accompanied by sudden drop in pH of the fermentation medium below 2.0. This sudden drop in pH caused inactivation of cellulolytic enzymes produced byAspergillus niger. The supplementation of nitrogen sources, especially urea, protectsA. niger cellulases from inactivation caused by a sudden drop in pH, since urea helped to maintain the pH of the fermentation medium between 3.5 and 4.5. The role of urea in the protection of cellulase was more prominent when it was used in combination with glycerol (5%).     

Evaluation for Rock Phosphate Solubilization in Fermentation and Soil–Plant System Using A Stress-Tolerant Phosphate-Solubilizing Aspergillus niger WHAK1

A strain WHAK1, identified as Aspergillus niger, was isolated from Yichang phosphate mines in Hubei province of China. The fungus developed a phosphate solubilization zone on modified National Botanical Research Institute’s phosphate growth (NBRIP) agar medium, supplemented with tricalcium phosphate. The fungus was applied in a repeated-batch fermentation process in order to test its effect on solubilization of rock phosphate (RP). The results showed that A. niger WHAK1 could effectively solubilize RP in NBRIP liquid medium and released soluble phosphate in the broth, which can be illustrated by the observation of scanning electron microscope, energy-dispersive X-ray microanalysis, and Fourier transform infrared spectroscopy. Acidification of the broth seemed to be the major mechanism for RP solubilization by the fungus. Indeed, multiple organic acids (mainly gluconic acid) were detected in the broth by high-performance liquid chromatography analysis. These organic acids caused a significant drop of pH and an obvious rise of titratable acidity in the broth. The fungus also exhibited high levels of tolerance against temperature, pH, salinity, and desiccation stresses, although a significant decline in the fungal growth and release of soluble phosphate was marked under increasing intensity of stress parameters. Further, the fungus was introduced into the soil supplemented with RP to analyze its effect on plant growth and phosphate uptake of wheat plants. The result revealed that inoculation of A. niger WHAK1 significantly increased the growth and phosphate uptake of wheat plants in the RP-amended soil compared to the control soil.     

Determination of indoor air quality of a phytosanitary plant

Effective fermentation monitoring is a growing need due to the rapid pace of change in the wine industry, which calls for fast methods providing real time information in order to assure the quality of the final product. The objective of this work is to investigate the potential of non-destructive techniques associated with chemometric data analysis, to monitor time-related changes that occur during red wine fermentation. Eight micro-fermentation trials conducted in the Valtellina region (Northern Italy) during the 2009 vintage, were monitored by a FT-NIR and a FT-IR spectrometer and by an electronic nose and tongue. The spectroscopic technique was used to investigate molecular changes, while electronic nose and electronic tongue evaluated the evolution of the aroma and taste profile during the must-wine fermentation. Must-wine samples were also analysed by traditional chemical methods in order to determine sugars (glucose and fructose) consumption and alcohol (ethanol and glycerol) production. Principal Component Analysis was applied to spectral, electronic nose and electronic tongue data, as an exploratory tool, to uncover molecular, aroma and taste modifications during the fermentation process. Furthermore, the chemical data and the PC1 scores from spectral, electronic nose and electronic tongue data were modelled as a function of time to identify critical points during fermentation. The results showed that NIR and MIR spectroscopies are useful to investigate molecular changes involved in wine fermentation while electronic nose and electronic tongue can be applied to detect the evolution of taste and aroma profile. Moreover, as demonstrated through the modeling of NIR, MIR, electronic nose and electronic tongue data, these non destructive methods are suitable for the monitoring of must-wine fermentation giving crucial information about the quality of the final product in agreement with chemical parameters. Although in this study the measurements were carried out in off-line mode, in future these non destructive techniques could be valid and simple tools, able to provide in-time information about the fermentation process and to assure the quality of wine.     

Highly Thermostable and pH-Stable Cellulases from Aspergillus niger NS-2: Properties and Application for Cellulose Hydrolysis

Optimization of cultural conditions for enhanced cellulase production by Aspergillus niger NS-2 were studied under solid-state fermentation. Significant increase in yields (CMCase 463.9?±?20.1 U/g, FPase 101.1?±?3.5 U/g and β-glucosidase 99?±?4.0 U/g) were obtained under optimized conditions. Effect of different nutritional parameters was studied to induce the maximum production of cellulase complex. Scale-up studies for enzyme production process were carried out. Characterization studies showed that enzymes produced by A. niger NS-2 were highly temperature- and pH stable. At 50 °C, the half life for CMCase, FPase, β-glucosidase were approximately 240 h. Cellulases from A. niger NS-2 were stable at 35 °C for 24 h over a broader pH range of 3.0–9.0. We examined the feasibility of using steam pretreatment to increase the saccharification yields from various lignocellulosic residues for sugar release which can potentially be used in bioethanol production. Saccharification of pretreated dry potato peels, carrot peels, composite waste mixture, orange peels, onion peels, banana peels, pineapple peels by crude enzyme extract from A. niger NS-2, resulted in very high cellulose conversion efficiencies of 92–98 %.     

Synthesis, characterization and antimicrobial activity of some new 1-(fluorobenzoyl)-3-(fluorophenyl)thioureas

Synthesis of a variety of new 1-(isomeric fluorobenzoyl)-3-(isomeric fluorophenyl)thioureas (1a-t) was accomplished in two steps. The synthetic route involves the reaction of equimolar quantities of isomeric fluorobenzoyl chlorides with potassium thiocyanate in anhydrous acetone to afford the corresponding isothiocyantes in situ, followed by treatment with equimolar quantities of isomeric fluoroanilines. All of the synthesized compounds (1a-t) were screened for their in vitro antibacterial activity using Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Pseudomonas aureginosa). The minimum inhibitory concentration (MIC) was also determined for the most active compounds. In vitro antifungal activity was also determined against the five fungal species (Rhizopus oryzae, Aspergillus tereus, Fusarium oxysporum, Aspergillus niger, Aspergillus fumigatus). In general, the antifungal activity of compounds was better than their antibacterial activity.     

Highly enantioselective bioreduction of ethyl 3-oxohexanoate

Seven wild-type microorganism strains were used to reduce ethyl 3-oxohexanoate to ethyl (R)-3-hydroxyhexanoate. Free cells of Kluyveromyces marxianus and Aspergillus niger led to higher than 99% of conversion with higher than 99% ee. After immobilization in calcium alginate spheres, cells of K. marxianus exhibited high enantioselectivity (>99% ee) and conversion level (99%) within 24 h even if substrate was added at concentration of 10 g/L (62 mM).     

A spectral study of 2-formylimidazole 4N-substituted thiosemicarbazones and their copper(II) complexes

Copper(II) complexes of 2-formylimidazole ⁴N-methyl-, ⁴N-dimethyl- ⁴N-ethyl- and 3-hexa-methyleneiminylthiosemicarbazone, along with two nickel(II) complexes of the latter thiosemicarbazone, have been synthesized. Infrared, electronic, NMR and ESR spectra have been used to characterize the complexes and the uncomplexed thiosemicarbazones. None of the complexes or thiosemicarbazones possess growth inhibitory activity against Aspergillus niger and Paecilomyces variotii.     

Bioleaching of Nickel and Cobalt from Lateritic Chromite Overburden Using the Culture Filtrate of Aspergillus niger

Extraction of metals (Ni, Co) from chromite overburden of Sukinda mines of Orissa, India, with the culture filtrate of Aspergillus niger was studied. Results showed that the amounts of metals leached varied directly with reaction temperature and period of fermentation. The culture filtrate was analyzed for citric and oxalic acids, and contained only oxalic acid—the concentration of which increased with time. Although this acid played the major role in leaching of metals, other unidentified metabolites present in the culture filtrate influenced the dissolution of the metals significantly. Maximum recovery of metals from raw and roasted ore samples was achieved at 80 °C with the 21-day culture filtrate containing the highest amount of oxalic acid. Under identical experimental conditions, much higher amounts of the metals were leached from roasted ore. Microstructures of the ore particles were studied by scanning electron microscopy and transmission electron microscopy; the bonding behaviors of metal compounds were identified by Fourier transform infrared spectroscopy which showed that the metals were leached after chelation with oxalic acid.     

Synthesis of lsopropyl-1-thio-Β-D-glucopyranoside (IPTGlc), an inducer ofaspergillus niger b1 Β-glucosidase production

Production of Β-glucosidase inAspergillus niger Bl is subjected to catabolic repression by glucose.Aspergillus niger Bl grown on bran as a carbon source secreted Β-glucosidase. The maximum level of the enzyme was reached after 7 d of fermentation. Addition of 1% glucose to the medium suppressed Β-glucosidase production to undetectable levels. In this study, the organic synthesis of a potential inducer of Β-glucosidase production by A.niger Bl’s reported. Isopropyl-1-thio-Β-D-glucopyranoside (IPTGlc) was synthesized using a two-step organic synthesis protocol. The H-NMR data agreed with those reported previously for the galactoside analog. When IPTGlc was added 24 h after inoculation at a final concentration of 0.4 mM, similar levels of Β-glucosidase were reached 3 to 4 d earlier as compared to fermentation without IPTGlc induction. In practice, this may translate to a more efficient method of producing Β-glucosidase from this fungus.     

Onsite Enzyme Production During Bioethanol Production from Biomass: Screening for Suitable Fungal Strains

Cellulosic ethanol production from biomass raw materials involves process steps such as pre-treatment, enzymatic hydrolysis, fermentation, and distillation. Use of streams within cellulosic ethanol production was explored for onsite enzyme production as part of a biorefinery concept. Sixty-four fungal isolates were in plate assays screened for lignocellulolytic activities to discover the most suitable fungal strain with efficient hydrolytic enzymes for lignocellulose conversion. Twenty-five were selected for further enzyme activity studies using a stream derived from the bioethanol process. The filter cake left after hydrolysis and fermentation was chosen as substrate for enzyme production. Five of the 25 isolates were further selected for synergy studies with commercial enzymes, Celluclast 1.5L and Novozym 188. Finally, IBT25747 (Aspergillus niger) and strain AP (CBS 127449, Aspergillus saccharolyticus) were found as promising candidates for onsite enzyme production where the filter cake was inoculated with the respective fungus and in combination with Celluclast 1.5L used for hydrolysis of pre-treated biomass.