Enhanced phytase production from Achromobacter sp. PB‐01 using wheat bran as substrate: Prospective application for animal feed

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB‐01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X‐100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB‐01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non‐starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro‐industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

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     

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.     

High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation

Bacillus pumilus ASH produced a high level of an extracellular and thermostable xylanase enzyme when grown using solid-state fermentation (SSF). Among a few easily available lignocellulosics tested, wheat bran was found to be the best substrate (5,300 U/g of dry bacterial bran). Maximum xylanase production was achieved in 72 h (5,824 U/g). Higher xylanase activity was obtained when wheat bran was moistened with deionized water (6,378 U/g) at a substrate-to-moisture ratio of 1:2.5 (w/v). The optimum temperature for xylanase production was found to be 37°C. The inoculum level of 15% was found to be the most suitable for maximum xylanase production (7,087 U/g). Addition of peptone stimulated enzyme production followed by yeast extract and mustard oil cake, whereas glucose, xylose and malt extract greatly repressed the enzyme activity. Repression by glucose was concentration-dependent, repressing more than 60% of the maximum xylanase production at a concentration of 10% (w/v). Cultivation in large enamel trays yielded a xylanase titre that was slightly lower to that in flasks. The enzyme activity was slightly lower in SSF than in SmF but the ability of the organism to produce such a high level of xylanase at room temperature and with deionized water without addition of any mineral salts in SSF, could lead to substantial reduction in the overall cost of enzyme production. This is the first report on production of such a high level of xylanase under SSF conditions by bacteria.     

Production of high activity thermostable phytase from thermotolerant Aspergillus niger in solid state fermentation

The thermotolerant fungus, Aspergillus niger NCIM 563, was used for production of extracellular phytase on agricultural residues: wheat bran, mustard cake, cowpea meal, groundnut cake, coconut cake, cotton cake and black bean flour in solid state fermentation (SSF). Maximum enzyme activity (108 U g⁻¹ dry mouldy bran, DMB) was obtained with cowpea meal. During the fermentation phytic acid was hydrolysed completely with a corresponding increase in biomass and phytase activity within 7 days. Phosphate in the form of KH₂PO₄ (10 mg per 100 g of agriculture residue) increased phytase activity. Among various surfactants added to SSF, Trition X-100 (0.5%) exhibited a 30% increase in phytase activity. The optimum pH and temperature of the crude enzyme were 5.0 and 50°C respectively. Phytase activity (86%) was retained in buffer of pH 3.5 for 24 h. The enzyme retained 75% of its activity on incubation at 55°C for 1 h. In the presence of 1 mM K⁺ and Zn²⁺, 95% and 55% of the activity were retained. Scanning electron microscopy showed a high density growth of fungal mycelia on wheat bran particles during SSF. Journal of Industrial Microbiology & Biotechnology (2000) 24, 237–243. Received 07 June 1999/ Accepted in revised form 18 December 1999     

Phytase isozymes from Aspergillus niger NCIM 563 under solid state fermentation: Biochemical characterization and their correlation with submerged phytases

Aspergillus niger NCIM 563 produces dissimilar phytase isozymes under solid state and submerged fermentation conditions. Biochemical characterization and applications of phytase Phy III and Phy IV in SSF and their comparison with submerged fermentation Phy I and Phy III were studied. SSF phytases have a higher metabolic potential as compared to SmF. Phy I is tetramer and Phy II, III and IV are monomers. Phy I and IV have pH optima of 2.5 and Phy II and III have pH optima of 5.0 and 5.6, respectively. Phy I, III and IV exhibited very broad substrate specificity while Phy II was more specific for sodium phytate. SSF phytase is less thermostable as compared to SmF phytase. Phy I and II show homology with other known phytases while Phy III and IV show no homology with SmF phytases and any other known phytases from the literature suggesting their unique nature. This is the first report about differences among phytase produced under SSF and SmF by A. niger and this study provides basis for explanation of the stability and catalytic differences observed for these enzymes. Exclusive biochemical characteristics and multilevel application of SSF native phytases determine their efficacy and is exceptional.     

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).     

Production and characterization of thermostable alkaline phytase from <Emphasis Type="Italic">Bacillus laevolacticus</Emphasis> isolated from rhizosphere soil

A novel phytase producing thermophilic strain of Bacillus laevolacticus insensitive to inorganic phosphate was isolated from the rhizosphere soil of leguminous plant methi (Medicago falacata). The culture conditions for production of phytase by B. laevolacticus under shake flask culture were optimized to obtain high levels of phytase (2.957 ± 0.002 U/ml). The partially purified phytase from B. laevolacticus strain was optimally active at 70 °C and between pH 7.0 and pH 8.0. The enzyme exhibited thermostability with ∼80% activity at 70 °C and pH 8.0 for up to 3 h in the presence/absence of 5 mM CaCl₂. The phytase from B. laevolacticus showed high specificity for phytate salts of Ca⁺ > Na⁺. The enzyme showed an apparent K _m 0.526 mM and V _max 12.3 μmole/min/mg of activity against sodium phytate.     

Advantages of fungal enzyme production in solid state over liquid fermentation systems

The present paper attempts to explain why enzyme production in solid-state fermentation (SSF) is higher than in submerged fermentation (SmF). Recent work done in our laboratory [Biotechnol. Lett. 22 (2000) 1255; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 271; J. Ind. Microbiol. Biotechnol. 26 (5) (2001) 296] related to the production of invertase, pectinases and tannases, by Aspergillus niger grown by SSF and SmF is reviewed. To do such a comparative study, logistic and Luedeking–Piret equations are used in order to estimate the values of the following coefficients: maximal specific growth rate (μ_M), maximal biomass level (X_M), enzyme/biomass yield (Y_P/X) and secondary rate of production, or breakdown (k). It is shown that enzyme productivity is proportional to group, μ_MY_P/XX_M, corrected by a function of ν=k/Y_P/Xμ_M. In all three cases of enzyme production studied, productivity using a SSF system was higher than in SmF. Studies with invertase resulted in higher values of μ_MX_M. Studies with pectinases resulted in higher values of Y_P/XX_M. Studies with tannases resulted in higher Y_P/X and less negative values of k. Finally, a reaction–diffusion model is presented to try to explain such differences based on micrographic measurements of mycelial aggregates for each kind of fermentation system.     

Oxidative state in idiophase links reactive oxygen species (ROS) and lovastatin biosynthesis: Differences and similarities in submerged- and solid-state fermentations

The present work was focused on finding a relationship between reactive oxygen species (ROS) and lovastatin biosynthesis (secondary metabolism) in Aspergillus terreus. In addition, an effort was made to find differences in accumulation and control of ROS in submerged (SmF) and solid-state fermentation (SSF), which could help explain higher metabolite production in the latter. sod1 expression, ROS content, and redox balance kinetics were measured during SmF and SSF. Results showed that A. terreus sod1 gene (oxidative stress defence enzyme) was intensely expressed during rapid growth phase (trophophase) of lovastatin fermentations. This high expression decreased abruptly, just before the onset of production (idiophase). However, ROS measurements detected high concentrations only in idiophase, suggesting a link between ROS and lovastatin biosynthesis. Apparently sod1 down regulation promotes the rise of ROS during idiophase. This oxidative state in idiophase was further supported by a high redox balance observed in trophophase that changed to a low value in idiophase (around six-fold lower). The patterns of ROS accumulation, sod1 expression, and redox balance behaviour were similar in SmF and SSF. However, sod1 expression and ROS concentration (ten-fold), were higher in SmF. Our results indicate a link between ROS and lovastatin biosynthesis. Also, showed differences of physiology in SSF that yield lower but more steady ROS concentrations, which could be associated to higher lovastatin production.     

Optimization of phytase production by solid substrate fermentation

The production of phytase by three feed-grade filamentous fungi (Aspergillus ficuum NRRL 3135, Mucor racemosus NRRL 1994 and Rhizopus oligosporus NRRL 5905) on four commonly used natural feed ingredients (canola meal, cracked corn, soybean meal, wheat bran) was studied in solid substrate fermentation (SSF). A. ficuum NRRL 3135 had the highest yield [15 IU phytase activity/g dry matter (DM)] on wheat bran. By optimizing the supplementation of wheat bran with starch and (NH₄)₂SO₄, phytase production increased to 25 IU/g DM. Optimization was carried out by Plackett-Burman and central composite experimental designs. Using optimized medium, phytase, phosphatase, alpha-amylase and xylanase production by A. ficuum NRRL 3135 was studied in Erlenmeyer flask and tray SSF. By scaling up SSF from flasks to stationary trays, activities of 20 IU phytase activity/g DM were reproducibly obtained. Electronic Publication     

Comparative production of cellulases by mutants of Penicillium janthinellum NCIM 1171 and its application in hydrolysis of Avicel and cellulose

Mutants of Penicillium janthinellum NCIM 1171 were evaluated for cellulase production using both submerged fermentation (SmF) and solid state fermentation (SSF). Mutant EU2D-21 gave highest yields of cellulases in both SmF and SSF. Hydrolysis of Avicel and cellulose were compared using SmF and SSF derived enzyme preparations obtained from EU2D-21. Surprisingly, the use of SSF derived preparation gave less hydrolysis compared to SmF derived enzymes. This may be due to inactivation of β-glucosidase at 50 °C in SSF derived enzyme preparations. SmF derived enzyme preparations contained both thermostable and thermosensitive β-glucosidases where as SSF derived enzyme preparations contained predominantly thermosensitive β-glucosidase. This is the first report on less thermostability of SSF derived β-glucosidase which is the main reason for getting less hydrolysis.     

Production and optimization of polygalacturonase from mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) peel using <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in solid state fermentation

Mango peel is one of the major wastes from fruit processing industries, which poses considerable disposal problems and ultimately leads to environmental pollution. The objective of the current research was to determine the significant parameters on the production of polygalacturonase from mango peel which is a major industrial waste. Solid state culture conditions for polygalacturonase production by Fusarium moniliforme from dried mango peel powder were optimized by Taguchi’s L-18 orthogonal array experimental design methodology. Eight fungal metabolic influencing variables, viz. temperature, mango peel, inoculum, peptone, ammonium nitrate (NH₄NO₃₎, magnesium sulphate (MgSO₄), zinc sulphate (ZnSO₄) and potassium dihydrogen phosphate (KH₂PO₄) were selected to optimize polygalacturonase production. The optimized parameters composed of temperature (30°C), mango peel (6.5%, g, w/v), inoculum (8%, ml, v/v), peptone (1%, g, w/v), NH₄NO₃ (0.60%, g, w/v), MgSO₄ (0.05%, g, w/v), ZnSO₄ (0.06%, g, w/v) and KH₂PO₄ (0.4%, g, w/v). Based on the influence of interaction of fermentation components of fermentation, these could be classified as the least significant and the most significant at individual and interaction levels. The temperature, inoculum level, mango peel substrate and KH₂PO₄ showed maximum production impact at optimized conditions. From the optimized conditions the polygalacturonase activity was maximized to 43.2 U g⁻¹.     

Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread

Aims: Phytase production by Sporotrichum thermophile in a cost‐effective cane molasses medium in submerged fermentation and its application in bread. Methods and Results: The production of phytase by a thermophilic mould S. thermophile was investigated using free and immobilized conidiospores in cane molasses medium in shake flasks, and stirred tank and air‐lift fermenters. Among surfactants tested, Tweens (Tween‐20, 40 and 80) and sodium oleate increased phytase accumulation, whereas SDS and Triton X‐100 inhibited the enzyme production. The mould produced phytase optimally at a_w 0·95, and it declined sharply below this a_w value. The enzyme production was comparable in air‐lift and stirred tank reactors with a marked reduction in fermentation time. Among the matrices tried, Ca‐alginate was the best for conidiospore immobilization, and fungus secreted sustained levels of enzyme titres over five cycles. The phytic acid in the dough was efficiently hydrolysed by the enzyme accompanied by the liberation of soluble phosphate in the bread. Conclusions: The phytase production by S. thermophile was enhanced in the presence of Tween‐80 in cane molasses medium. A peak in enzyme production was attained in 48 h in the fermenter when compared with that of 96 h in shake flasks. Ca‐alginate immobilized conidiospores germinated to produce fungal growth that secreted sustained levels of phytase over five cycles. The bread made with phytase contained reduced level of phytic acid and a high‐soluble phosphate. Significance and Impact of the Study: The phytase accumulation by S. thermophile was increased by the surfactants. The sustainability of enzyme production in stirred tank and air‐lift fermenters suggested the possibility for scaling up of phytase. The bread made with phytase contained low level of antinutrient, i.e. phytic acid.     

Solid-state fermentation increases secretome complexity in Aspergillus brasiliensis

Aspergillus is used for the industrial production of enzymes and organic acids, mainly by submerged fermentation (SmF). However, solid-state fermentation (SSF) offers several advantages over SmF. Although differences related to lower catabolite repression and substrate inhibition, as well as higher extracellular enzyme production in SSF compared to SmF have been shown, the mechanisms undelaying such differences are still unknown. To explain some differences among SSF and SmF, the secretome of Aspergillus brasiliensis obtained from cultures in a homogeneous physiological state with high glucose concentrations was analyzed. Of the regulated proteins produced by SmF, 74% were downregulated by increasing the glucose concentration, whereas all those produced by SSF were upregulated. The most abundant and upregulated protein found in SSF was the transaldolase, which could perform a moonlighting function in fungal adhesion to the solid support. This study evidenced that SSF: (i) improves the kinetic parameters in relation to SmF, (ii) prevents the catabolite repression, (iii) increases the branching level of hyphae and oxidative metabolism, as well as the concentration and diversity of secreted proteins, and (iv) favors the secretion of typically intracellular proteins that could be involved in fungal adhesion. All these differences can be related to the fact that molds are more specialized to growth in solid materials because they mimic their natural habitat.     

Algal biomass: A sustainable,economical and renewable approach for microbial production of pectinolytic enzymes using submerged and solid state fermentation techniques

In the past decade, algal waste has been used as useful natural resource for production of enormous range of products that have wide economical and commercial importance. Pectinases are group of enzymes that have wide commercial applications. Hence, current study was designed to utilize algal biomass for the production of pectinases using submerged (SmF) and solid state fermentation (SSF) techniques. Different algal sources including brown (Dictyopteris polypodioides, Sargassum wightii and Dictyopteris divaricata) and green algae (Ulva lactuca and Codium tomentosum) were used and U. lactuca was found to be the most suitable substrate. Several bacterial and fungal strains were screened and among them Bacillus licheniformis KIBGE-IB4 was selected based on maximum pectinase production. SmF and SSF were studied utilizing U. lactuca as a substrate and results revealed that enzyme production was favoured by SmF (2457?±?3.31?U?mg^?1) as compared to SSF (1432?±?1.46?U?mg^?1). Parametric optimization of pectinase production indicated that B. licheniformis KIBGE-IB4 requires 10.0?g L^–1 U. lactuca as a biomass in the medium with a pH 7.0 when incubated at 37?°C for 24 hours. Likewise, production of pectinase using algal resource was also compared with that of the conventional agricultural biomass and it was observed that when U. lactuca was used, the selected bacterial isolate produced a higher yield of enzyme than sugarcane bagasse and rice husk. Hence, it is anticipated that algal biomass can be efficiently utilized as an environmental friendly bioresource for the production of industrially important hydrolytic enzymes.     

Production of tannase by Aspergillus niger Aa-20 in submerged and solid-state fermentation: influence of glucose and tannic acid

Tannase production by Aspergillus niger Aa-20 was studied in submerged (SmF) and solid-state (SSF) fermentation systems with different tannic acid and glucose concentrations. Tannase activity and productivity were at least 2.5 times higher in SSF than in SmF. Addition of high tannic acid concentrations increased total tannase activity in SSF, while in SmF it was decreased. In SmF, total tannase activity increased from 0.57 to 1.03 IU/mL, when the initial glucose concentration increased from 6.25 to 25 g/L, but a strong catabolite repression of tannase synthesis was observed in SmF when an initial glucose concentration of 50 g/L was used. In SSF, maximal values of total tannase activity decreased from 7.79 to 2.51 IU when the initial glucose concentration was increased from 6.25 to 200 g/L. Kinetic results on tannase production indicate that low tannase activity titers in SmF could be associated to an enzyme degradation process which is not present in SSF. Tannase titers produced by A. niger Aa-20 are fermentation system-dependent, favoring SSF over SmF. Journal of Industrial Microbiology & Biotechnology (2001) 26, 296–302. Received 07 July 2000/ Accepted in revised form 15 February 2001     

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Novel Characteristics of Bifidobacterium bifidum in Solid State Fermentation System

Summary The characteristics of Bifidobacterium bifidum grown in solid state fermentation (SSF) system (water content of media 54.5 and 68.8%) was compared with the submerged fermentation (SmF) system (water content of medium: 89.8%). Besides lactic acid (lactate) and acetic acid (acetate), the bacterium was able to secrete propionic acid (propionate) and butyric acid (butyrate) under SSF conditions. However, it only produced lactate and acetate under SmF conditions. The ratio of lactate to acetate was 1.26–1.62:1 in SSF but it was 1:2 in SmF. A higher content of C16:0 and C18:1 as well as a lower content of C18:0 cell membrane fatty acids were observed in SSF than in SmF. There was a lower growth rate, a lower viable count and a longer logarithmic growth phase for B. bifidum cultivated in SSF than in SmF.     

Effect of nonionic surfactants on Rhizopus homothallicus lipase activity: a comparative kinetic study

Based on amino-terminal sequencing and mass spectrometry data on the Rhizopus homothallicus lipase extracted using solid (SSF) and submerged state fermentation (SmF) methods, we previously established that the two enzymes were identical. Differences were observed, however, in terms of the specific activity of these lipases and their inhibition by diethyl p-nitrophenyl phosphate (E600). The specific activity of the SSF lipase (10,700 μmol/min/mg) was found to be 1.2-fold that of SmF lipase (8600 μmol/min/mg). These differences might be the result of residual Triton X-100 molecules interacting with the SSF lipase. To check this hypothesis, the SmF lipase was incubated with submicellar concentrations of Triton X-100. The specific activity of the lipase increased after this treatment, reaching similar values to those measured with the SSF lipase. Preincubating SSF and SmF lipases with E600 at a molar excess of 100 for 1 h resulted in 80% and 60% enzyme inhibition levels, respectively. When the SmF lipase was preincubated with Triton X-100 for 1 h at a concentration 100 times lower than the Trition X-100 critical micellar concentration, the inhibition of the lipase by E600 increased from 60% to 80%. These results suggest that residual detergent monomers interacting with the enzyme may after the kinetic properties of the Rh. homothallicus lipase.