Effect of fermentation system on the production and properties of tannase of Aspergillus niger van Tieghem MTCC 2425

The tannase-producing efficiency of liquid-surface fermentation (LSF) and solid-state fermentation (SSF) vis-à-vis submerged fermentation (SmF) was investigated in a strain of Aspergillus niger, besides finding out if there was a change in the activity pattern of tannase in these fermentation processes. The studies on the physicochemical properties were confined to intracellular tannase as only this form of enzyme was produced by A. niger in all three fermentation processes. In LSF and SmF, the maximum production of tannase was observed by 120 h, whereas in SSF its activity peaked at 96 h of growth. SSF had the maximum efficiency of enzyme production. Tannase produced by the SmF, LSF and SSF processes had similar properties except that the one produced during SSF had a broader pH stability of 4.5-6.5 and thermostability of 20 degrees-60 degrees C.     

Optimisation of extracellular tannase production from <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

The tannase production by Paecilomyces variotii was confirmed by high performance thin layer chromatography (HPTLC), and substrate specificity of the tannase was determined by zymogram analysis in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE). A clear band of activity observed after electrophoresis of culture filtrate in non-denaturing gels indicated the production of extracellular tannase by P. varoitii. HPTLC analysis revealed that gallic acid was the enzymatic degradation product of tannic acid during the fermentation process. The optimum condition for tannase production was at 72 h of incubation in shaking condition and addition of 1.5% tannic acid, 1% glucose and 0.2% sodium nitrate at temperature of 35°C and pH of 5–7. The production of extracellular tannase from Paecilomyces variotii was investigated under optimized conditions in solid-state fermentation (SSF), submerged fermentation (SmF) and liquid surface fermentation (LSF) processes. The maximum extracellular tannase production was obtained within 60 h of incubation under SSF followed by SmF and LSF.     

Production of tannase by <Emphasis Type="Italic">Aspergillus niger</Emphasis> HA37 growing on tannic acid and Olive Mill Waste Waters

Production of tannase (tannin acyl hydrolase, EC 3.1.1.20) by Aspergillus nigerHA37 on a synthetic culture medium containing tannic acid at different concentrations has been studied. Maximal enzymatic activity increased according to the initial concentration of tannic acid; respectively 0.6, 0.9 and 1.5 enzyme activity units (EU) ml⁻¹ medium in the presence of 0.2%, 0.5% and 1% of tannic acid. Tannase production by A. niger HA37 on fourfold diluted olive mill waste waters (OMWW) as substrate, was between 0.37 and 0.65 EU ml⁻¹. Enzyme production on the diluted OMWW remained globally stable during more than 30 h. Growth of A. niger HA37 on OMWW was correlated with about 70% degradation of phenolic compounds present in the waste. This strain has therefore the capacity to degrade complex wastewaters which cause environmental damage to aquatic streams.     

Exopectinases produced by Aspergillus niger in solid-state and submerged fermentation: a comparative study

Exopectinase production by Aspergillus niger was compared in submerged fermentation (SmF) and solid-state fermentation (SSF). SSF was carried out using polyurethane foam (PUF) as the solid support. The purpose was to study the effect of sucrose addition (0 or 40 g/l) and water activity level (A _w=0.99 or 0.96) on the level of enzyme activity induced by 15 g/l of pectin. Mycelial growth, as well as extracellular protease production, was also monitored. Sucrose addition in SmF resulted in catabolite repression of exopectinase activity. However, in SSF, an enhancement of enzyme activity was observed. Protease levels were minimal in SSF experiments with sucrose and maximal in SmF without sucrose. Exopectinase yields (IU/g X) were negligible in SmF with sucrose. The high levels of exopectinase with sucrose and high A _w in SSF can be explained by a much higher level of biomass production without catabolite repression and with lower protease contamination. Journal of Industrial Microbiology & Biotechnology (2001) 26, 271–275. Received 05 July 2000/ Accepted in revised form 27 January 2001     

Co-production of tannase and gallic acid by a novel Penicillium rolfsii (CCMB 714)

Abstract

A novel tannase and gallic acid-producing Penicillium rolfsii (CCMB 714) was isolated from cocoa leaves from the South of Bahia. The influence of nutritional sources and the simultaneous effect of parameters involved in the fermentation process were available. Tannase (9.97 U?mL^?1) and gallic acid (9?mg mL^?1) production were obtained in 48?h by submerged fermentation in non-optimized conditions. Among the carbon sources, tested gallic acid and tannic acid showed the highest tannase production (p<.05) when compared with methyl gallate and glucose. After optimization using the temperature and tannic acid concentration as variables with the Central Compound Rotational Design (CCRD), the maximal tannase production (25.6?U mL^?1) was obtained at 29.8?°C and 12.7%, respectively, which represents an increase of 2.56 times in relation to the initial activity. The parameters optimized for the maximum production of gallic acid (21.51?mg mL^?1) were 30?°C and 10% tannic acid. P. rolfsii CCMB 714 is a new strain with a high tannase and gallic acid production and the gallic acid produced is very important, mainly for its applications in the food and pharmaceutical industry.     

Effects of different carbon sources on the synthesis of pectinase by Aspergillus niger in submerged and solid state fermentations

A study was made to compare the production of pectinase by Aspergillus niger CH4 in solid-state (SSF) and submerged (SmF) fermentations. Production of endo- (endo-p) and exo-pectinase (exo-p) by SSF was not reduced when glucose, sucrose or galacturonic acid (up to 10%) were added to a culture medium containing pectin. Moreover, both activities increased when concentrations of the carbon sources were also increased. In SmF, these activities were strongly decreased when glucose or sucrose (3%) was added to culture medium containing pectin. The addition of galacturonic acid affected endo-p activity production to a lesser extend than exo-p. Final endo-p and exo-p activities in SSF were three and 11 times higher, respectively, than those obtained in SmF. The overall productivities of SSF were 18.8 and 4.9 times higher for endo-p and exo-p, respectively, than those in SmF. These results indicate that regulatory phenomena, such as induction-repression or activation-inhibition, related to pectinase synthesis by A. niger CH4 are different in the two types of fermentation. Correspondence to: E. Favela-Torres     

Tannase production by Aspergillus aculeatus DBF9 through solid-state fermentation

Tannase an industrially important enzyme was produced by Aspergillus aculeatus DBF9 through a solid-state fermentation (SSF). The organism produced good amount of enzyme and gallic acid in wheat bran among the solid substrate used in SSF. Maximum enzyme and gallic acid production occurred in 5% tannic acid after 72 h. Eighty percent initial substrate moisture and 30 degrees C temperature was found suitable for tannase production.     

Production of tannase from wood-degrading fungus using as substrate plant residues: purification and characterization

In the present study Lenzites elegans, Schizophyllum commune, Ganoderma applanatum and Pycnoporus sanguineus (wood-degrading fungi) were assayed for their tannase producing potential in culture media containing plant residues or/and tannic acid as carbon source. Aspergillus niger was used as positive control for tannase production. We also carried out the isolation, purification and characterization of the enzyme from the fungi selected as the major productor. The highest fungal growth was observed in A. niger and L. elegans in the media containing tannic acid + glucose + plant residues (Fabiana densa). A. niger and L. elegans reached the highest extracellular tannase production in a medium containing tannic acid + F. densa and in a medium supplemented with glucose + tannic acid + F. densa. The produced enzyme by L. elegans was purified by DEAE-Sepharose. Km value was 5.5 mM and relative molecular mass was about 163,000. Tannase was stable at a pH range 3.0–6.0 and its optimum pH was 5.5. The enzyme showed an optimum temperature of 60°C and was stable between 40 and 60°C. This paper is the first communication of tannase production by wood-degrading fungi. Fermentation technology to produce tannase using plant residues and xylophagous fungi could be very important in order to take advantage of plant industrial waste.     

Pestalotiopsis mangiferae isolated from cocoa leaves and concomitant tannase and gallic acid production

The enzyme tannase is of great industrial and biotechnological importance for the hydrolysis of vegetable tannins, reducing their undesirable effects and generating products for a wide range of processes. Thus, the search for new microorganisms that permit more stable tannase production is of considerable importance. A strain of P. mangiferae isolated from cocoa leaves was selected and investigated for its capacity to produce tannase enzymes and gallic acid through submerged fermentation. The assessment of the variables affecting tannase production by P. mangiferae showed that tannic acid, ammonium nitrate and temperature were the most significant (8.4 U/mL). The variables were analyzed using Response Surface Methodology - RSM (Box-Behnken design), with the best conditions for tannase production being: 1.9% carbon source, 1% nitrogen source and temperature of 23 °C. Tannase activity doubled (16.9 U/mL) after the optimization process when compared to the initial fermentation. A pH of 7.0 was optimal for the tannase and it presented stability above 80% with pH between 4.0 and 7.0 after 2h of incubation. The optimal temperature was 30 °C and activity remained at above 80% at 40–60 °C after 1 h. Production of gallic acid was achieved with 1% tannic acid (0.9 mg/mL) and P. mangiferae had not used up the gallic acid produced by tannic acid hydrolysis after 144 h of fermentation. A 5% tannic acid concentration was the best for gallic acid production (1.6 mg/mL). These results demonstrate P. mangiferae’s potential for tannase and gallic acid production for biotechnological applications.     

Indirect method for quantification of cellular biomass in a solidscontaining medium used as pre-culture for cellulase production

A process that combines the advantages of solid state fermentation (SSF) and submerged fermentation (SmF) could increase the efficiency of cellulase production required in the cellulosic ethanol industry. Due to the difficulty of measuring cellular biomass in the presence of solids, we developed a novel methodology for indirect quantification of biomass during production of the preculture for a combined fermentation process. Cultivation of Aspergillus niger was initiated as SSF using sugar cane bagasse as a solid substrate. Experiments were conducted in the absence of bagasse to determine growth kinetic parameters. Changes in glucose and biomass concentrations were measured. and the data were used for simulation employing a simple unstructured model. Parameters were estimated by applying a combination of Simulated Annealing (SA) and Levenberg-Marquardt (LM) algorithms to search for minimization of the error between model estimates and experimental data. Growth kinetics followed the Contois model, with a maximum specific growth rate (μ_max) of 0.042/h, a yield coefficient for biomass formation (Y_x/s) of 0.30 g/g and a death constant (k_D) of 0.005/h.These parameters were used to simulate cellular growth in the solids-containing medium. The proposed model accurately described the experimental data and succeeded in simulating the cell concentration profile. The selected pre-culture conditions (24 h as SSF followed by 48 h as SmF) were applied for cellulase production using the combined fermentation process and resulted in an endoglucanase activity (1,052 ± 34 U/L) greater than that obtained using the conventional SmF procedure (824 ± 44 U/L). Besides the standardization of pre-culture conditions, this methodology could be very useful in systems where direct measurement of cell mass is not possible.     

Biodegradation of hexadecane in liquid and solid-state fermentations by Aspergillus niger

The biodegradation and mineralisation of hexadecane (HXD) by Aspergillus niger were studied in SmF and Solid-state fermentation (SSF). HXD concentrations ranging from 45 to 180 g/l (SSF) and from 20 to 80 g/l (SmF) were tested. HXD consumption was three times higher and fungal growth was up to 30 times faster in SSF than in SmF. The maximum HXD consumption in SmF was 62% (18% mineralised) and in SSF 100% (52% mineralised) for initial HXD concentrations of 20 and 45 g/l, respectively. The respiratory quotient in SmF increased (from 0.85 to 1.08) with increase in HXD concentration, while it was independent (approximately 0.68) of the initial HXD concentration in SSF. These results showed that the consumption rate and biodegradation efficiency for HXD were higher in SSF than in SmF.     

Production and Regulation of Lipase Activity from <Emphasis Type="Italic">Penicillium restrictum</Emphasis> in Submerged and Solid-State Fermentations

Different carbon (C) sources, mainly carbohydrates and lipids, have been screened for their capacity to support growth and lipase production by Penicillium restrictum in submerged fermentation (SmF) and in solid-state fermentation (SSF). Completely different physiological behaviors were observed after the addition of easily (oleic acid and glucose) and complex (olive oil and starch) assimilable C sources to the liquid and solid media. Maximal lipolytic activities (12.1 U/mL and 17.4 U/g) by P. restrictum were obtained with olive oil in SmF and in SSF, respectively. Biomass levels in SmF (12.2–14.1 mg/mL) and SSF (7.0–8.0 mg/g) did not varied greatly with the distinct C sources used. High lipase production (12.3 U/g) using glucose was only attained in SSF, perhaps due to the ability of this fermentation process to minimize catabolite repression.     

Laccase production in solid‐state and submerged fermentation by Pleurotus ostreatus

A solid‐state fermentation (SSF) system for production of an industrially important enzyme laccase by Pleurotus ostreatus was developed by using potato dextrose yeast extract medium and polyurethane foam as a supporting material. The maximum laccase production in the SSF system was as high as 3×10⁵ U/L. Addition of inducers, such as copper and ferulic acid, further enhanced the laccase production in SSF. Moreover, the time required for the maximum laccase production was reduced to 6 days compared to 10 days reported earlier. The improvement achieved by the SSF system was investigated by comparing it to a submerged fermentation system (SmF), both experimentally and by using a standard theoretical model along with a parameter sensitivity analysis. Laccase production in SSF was found to be twice of that in SmF. One of the main reasons for higher laccase production in SSF compared to SmF was possibly due to the presence of higher proteolytic activity in SmF. Strong proteolytic activity in SmF presumably caused subsequent laccase degradation, which lowered the ultimate laccase production in SmF compared to SSF.     

Tamarind seed powder and palm kernel cake: two novel agro residues for the production of tannase under solid state fermentation by Aspergillus niger ATCC 16620

Palm kernel cake (PKC), the residue obtained after extraction of palm oil from oil palm seeds and tamarind seed powder (TSP) obtained after removing the fruit pulp from tamarind fruit pod were tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger ATCC 16620. The fungal strain was grown on the substrates without any pretreatment. In PKC medium, a maximum enzyme yield of 13.03 IU/g dry substrate (gds) was obtained when SSF was carried out at 30 degrees C, 53.5% initial substrate moisture, 33 x 10(9) spores/5 g substrate inoculum size and 5% tannic acid as additional carbon source after 96 h of fermentation. In TSP medium, maximum tannase yield of 6.44 IU/gds was obtained at 30 degrees C, 65.75% initial substrate moisture, 11 x 10(9) spores/5 g substrate inoculum, 1% glycerol as additional carbon source and 1% potassium nitrate as additional nitrogen source after 120 h of fermentation. Results from the study are promising for the economic utilization and value addition of these important agro residues, which are abundantly available in many tropical and subtropical countries.     

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications

This investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett–Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO₄ · 7H₂O. The use of response surface methodology (RSM) by Box–Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5–6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the “generally regarded as safe” (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).     

Purification,structural characterization and biotechnological potential of tannase enzyme produced by Enterobacter cloacae strain 41

Tannase production by Enterobacter cloacae strain 41 was investigated under submerged fermentation which was optimized at various circumstances such as pH, temperature, substrate, and agitation, carbon, and nitrogen sources. Tannase was purified by a two-step approach comprising of ion exchange and size exclusion chromatography, respectively. The maximum tannase production was achieved at 1.0% tannic acid concentration, incubation temperature of 50 °C, and initial pH 6.0. The molecular weight of purified tannase was 45 kDa on 10% SDS-PAGE, and it was confirmed by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS). The enzymatic products of purified tannase were characterized by HPLC, TLC and FT-IR spectroscopy which showed the functional groups such as OH, CO, and CC. The purified tannase retained the activity up to 90% under the condition at 50 °C and pH 6.0 after 1 h incubation. Enzyme kinetics and inhibition studies were also investigated. Cytotoxicity studies showed that the tannase has no cytotoxic effects on Vero cell line. The results indicated the E. cloacae strain 41 would give a potential source for the efficient production of tannase and can be used in tannery effluent degradation, food, and pharmaceutical industrial applications.     

Optimization of tannase production by Aspergillus niger in solid-state packed-bed bioreactor

Tannin acyl hydrolase, also known as tannase, is an enzyme with important applications in the food, feed, pharmaceutical, and chemical industries. However, despite a growing interest in the catalytic properties of tannase, its practical use is very limited owing to high production costs. Several studies have already demonstrated the advantages of solid-state fermentation (SSF) for the production of fungal tannase, yet the optimal conditions for enzyme production strongly depend on the microbial strain utilized. Therefore, the aim of this study was to improve the tannase production by a locally isolated A. niger strain in an SSF system. The SSF was carried out in packed-bed bioreactors using polyurethane foam as an inert support impregnated with defined culture media. The process parameters influencing the enzyme production were identified using a Plackett–Burman design, where the substrate concentration, initial pH, and incubation temperature were determined as the most significant. These parameters were then further optimized using a Box-Behnken design. The maximum tannase production was obtained with a high tannic acid concentration (50 g/l), relatively low incubation temperature (30°C), and unique low initial pH (4.0). The statistical strategy aided in increasing the enzyme activity nearly 1.97-fold, from 4,030 to 7,955 U/l. Consequently, these findings can lead to the development of a fermentation system that is able to produce large amounts of tannase in economical, compact, and scalable reactors.     

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 amyloglucosidase by Aspergillus niger under different cultivation regimens

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation.M. Ramadas is with the Department of Biochemistry, Faculty of Medicine, University of Jaffna, Kokuvil, Sri Lanka. O. Holst and B. Mattiasson are with the Department of Biotechnology, Chemical Center, Lund University, Box 124, S-221 00 Lund, Sweden     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.