Response surface optimization of mixed substrate solid state fermentation for the production of lovastatin by Monascus purpureus

The objective of this work is to enhance the production of lovastatin using Monascus purpureus MTCC 369 in mixed substrate solid state fermentation using various solid substrates and to optimize the combination of the solid substrates by response surface methodology. Solid state fermentation was conducted in a 250 mL Erlenmeyer flask at 30°C for 14 days with initial moisture content of 40% and inoculum size of 10% active culture. Barley, long grain rice and sago starch were found to be the suitable substrates producing maximum lovastatin of 193.7 mg, 190.2 mg and 180.9 mg/g of dry solids. These substrates were further used in various combinations as designed by the central composite design for enhancing the lovastatin production using Monascus purpureus. To the best of our knowledge this is the first report on the production of lovastatin using a mixed substrate solid state fermentation using Monascus purpureus.     

Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material

Synthesis of amylase by Aspergillus niger strain UO-01 under solid-state fermentation with sugarcane bagasse was optimized by using response surface methodology and empirical modelling. The process parameters tested were particle size of sugarcane bagasse, incubation temperature and pH, moisture level of solid support material and the concentrations of inoculum, total sugars, nitrogen and phosphorous. The optimum conditions for high amylase production (457.82 EU/g of dry support) were particle size of bagasse in the range of 6–8 mm, incubation temperature and pH: 30.2°C and 6.0, moisture content of bagasse: 75.3%, inoculum concentration: 1 × 10⁷ spores/g of dry support and concentrations of starch, yeast extract and KH₂PO₄: 70.5, 11.59 and 9.83 mg/g of dry support, respectively. After optimization, enzyme production was assayed at the optimized conditions. The results obtained corroborate the effectiveness and reliability of the empirical models obtained.     

Development of a novel solid-state fermentation strategy for the production of poly-3-hydroxybutyrate using polyurethane foams by Bacillus sphaericus NII 0838

The extensive use of synthetic plastics has caused serious waste disposal problems in our environment. Poly-3-hydroxybutyrates (PHB) are eco-friendly bacterial polyesters which are produced under unbalanced nutrient conditions. Few reports are available on PHB production by solid state fermentation (SSF). We have developed a novel SSF bioprocess in which polyurethane foam (PUF) is used as a physical inert support for the production of PHB by Bacillus sphaericus NII 0838. Media engineering for optimal PHB production was carried out using response surface methodology (RSM) adopting a Box–Behnken design. The factors optimized by RSM were inoculum size, pH and (NH₄)₂SO₄ concentration. Under optimized conditions—6.5 % inoculum size, 1.7 % (w/v) (NH₄)₂SO₄ and pH 9.0—PHB production and biomass were 0.169?±?0.03 and 0.4?±?0.002 g/g PUF, respectively. This is the first report on PHB production by SSF using PUF as an inert support. Our results demonstrate that SSF can be used as an alternative strategy for the production of PHB.     

Exoglucanase production by Aspergillus niger grown on wheat bran

β-Exoglucanase production on the lignocellulosic material, wheat bran, by Aspergillus niger under solid state fermentation (SSF) on a laboratory scale was investigated. Different fermentation parameters, such as moisture content, initial pH, temperature, depth of the substrate, and inoculum size on exoglucanase production were optimized. Moisture content of 40 %, pH of 7.0, substrate depth of 1.0 cm, inoculum size of 2?×?10⁶ spores/g of wheat bran, and temperature at 30 °C were optimal for maximum production of exoglucanase. Maximum yields of exoglucanase with 28.60 FPU/g of wheat bran were obtained within 3 days of incubation under optimal conditions.     

Citric acid production by solid state fermentation using sugarcane bagasse

A solid state fermentation (SSF) method was used to produce citric acid by Aspergillus niger DS 1 using sugarcane bagasse as a carrier and sucrose or molasses based medium as a moistening agent. Initially bagasse and wheat bran were compared as carrier. Bagasse was the most suitable carrier, as it did not show agglomeration after moistening with medium, resulting in better heat and mass transfer during fermentation and higher product yield. Different parameters such as moisture content, particle size, sugar level and methanol concentration of the medium were optimised and 75% moisture level, 31.8 g sugar/100 g dry solid, 4% (v/w) methanol and particles of the size between 1.2 and 1.6 mm were found to be optimal. Sucrose and clarified and non-clarified molasses medium were also tested as moistening agents for SSF and under optimised conditions, 20.2, 19.8 and 17.9 g citric acid /100 g of dry solid with yield of 69.6, 64.5 and 62.4% (based on sugar consumed) was obtained in sucrose, clarified and non-clarified molasses medium respectively, after 9 days of fermentation.     

Improvement of β-glucosidase production by co-culture of Aspergillus niger and A. oryzae under solid state fermentation through feeding process

Eleven different Aspergillus strains were evaluated for their ability to produce β-glucosidase using sugar cane bagasse as a sole carbon source under solid state fermentation (SSF). The most potent strains, A. niger NRC 7 (674.6 U/g ds) and A. oryzae NRRL 447 (83 U/g ds), were used in a mixed culture to enhance β-glucosidase production by co-culturing under SSF. In mixed culture, β-glucosidase of the two strains (814 U/g ds) was nearly 1.2- and 9.8-fold than that of monocultures of A. niger NRC 7A and A. oryzae NRRL 447, respectively. Optimization of the culture parameters, initial pH value, moisture content, inoculum size and ratios of the two strains. and incubation time exhibited a significant increase in β-glucosidase production (1,893 U/g ds) than before optimization. Single feeding with citrate-phosphate buffer, succinate buffer, casein. and soybean flour individually after the third day of the fermentation time and controlling the moisture content at 90 % (w/w) induced β-glucosidase production. Maximum enzyme production increased up to 2.1-fold compared to 2,188 U/g ds during normal batch culture. Among nitrogen sources, soybean flour gave the highest β-glucosidase (4,578 U/g ds). while urea reduced β-glucosidase production (1,693 U/g ds). However, the combination of buffers with soybean flour through two fed cycles resulted in a decrease of the enzyme than single fed with buffers or soybean flour alone.     

Microbial production of extra-cellular phytase using polystyrene as inert solid support

Aspergillus ficuum TUB F-1165 and Rhizopus oligosporus TUB F-1166 produced extra-cellular phytase during solid-state fermentation (SSF) using polystyrene as inert support. Maximal enzyme production (10.07 U/g dry substrate (U/gds) for A. ficuum and 4.52 U/gds for R. oligosporus) was observed when SSF was carried out with substrate pH 6.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1.3 x 10(7) spores/5 g substrate, for 72 h for A. ficuum and with substrate pH 7.0 and moisture 58.3%, incubation temperature 30 degrees C, inoculum size of 1 x 10(6) spores/5 g substrate for 96 h for R. oligosporus. Results indicated scope for production of phytase using polystyrene as inert support.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

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.     

Polyurethane foam as an inert carrier for the production of L(+)-lactic acid by Lactobacillus casei under solid-state fermentation

AIM: Production of L-lactic acid in solid-state fermentation (SSF) using polyurethane foam (PUF) as inert support moistened with cassava bagasse starch hydrolysate. METHODS AND RESULTS: PUF impregnated with cassava bagasse starch hydrolysate as major carbon source was used for the production of L-lactic acid using Lactobacillus casei in solid-state condition. The key parameters such as reducing sugar, inoculum size and nutrient mixture were optimized by statistical approach using response surface methodology. More than 95% conversion of sugars to lactic acid from 4 g reducing sugar per gram dry support was attained after 72 h when the inert substrate was moistened with 6.5 ml of nutrient solution and inoculated with 1.5 x 10(9) CFU of L. casei. While considering the lactate yield based on the solid support used, a very high yield of 3.88 g lactic acid per gram PUF was achieved. CONCLUSION: PUF acted as an excellent inert support for L. casei and provided a platform for the utilization of starchy waste hydrolysate in a lower reactor volume. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a cost effective cultivation of lactic acid bacteria for producing lactic acid from agro based waste products such as cassava bagasse. This is the first report on the exploitation of PUF as an inert support for lactate production under SSF.     

Use of coconut coir fibers as an inert solid support for production of cyclosporin A

In the present study, coconut coir was evaluated as an inert support for the production of cyclosporin A (CyA) using Tolypocladium inflatum MTCC 557 by solid state fermentation. Initially, four different inert supports such as coconut coir, polyurethane foam, polystyrene beads, and sugarcane baggase were screened using different production media as moistening agents for the maximum production of CyA. Different parameters such as fermentation time, carbon sources, moisture content, pH, and inoculum size were optimized. It was observed that coconut coir impregnated with medium modified with glycerol as carbon source, pH 6, at 80% moisture content, and inoculum size of 2.5 mL/2.5 g support produced 2641 mg/kg of CyA after 12 days as compared to 998 mg/kg before optimization. The yields were further increased to 3597 mg/kg substrate with addition of combination of amino acids after 48 h of fermentation.     

Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water

The production of extracellular α-amylase by thermotolerant Bacillus subtilis was studied in solid state fermentation (SSF). The effect of wheat bran (WB) and rice husk (RH) was examined. The appropriate incubation period, moisture level, particle size and inoculum concentration was determined. Maximum yields of 159,520 and 21,760 U g⁻¹ were achieved by employing WB and RH as substrates in 0.1 M phosphate buffer at pH 7 with 30% initial moisture content at 24 and 48 h. Particle size and inoculum concentration were found to be 1000 μm, 20% and 500 μm, 15% for WB and RH, respectively. Enzyme yield was 7.3-fold higher with WB medium compared with RH.     

Efficient Acetone–Butanol–Ethanol Production from Corncob with a New Pretreatment Technology—Wet Disk Milling

Acetone–butanol–ethanol (ABE) production from corncob was achieved using an integrated process combining wet disk milling (WDM) pretreatment with enzymatic hydrolysis and fermentation by Clostridium acetobutylicum SE-1. Sugar yields of 71.3 % for glucose and 39.1 % for xylose from pretreated corncob were observed after enzymatic hydrolysis. The relationship between sugar yields and particle size of the pretreated corncob was investigated, suggesting a smaller particle size benefits enzymatic hydrolysis with the WDM pretreatment approach. Analysis of the correlation between parameters representing particle size and efficiency of enzymatic hydrolysis predicted that frequency 90 % is the best parameter representing particle size for the indication of the readiness of the material for enzymatic hydrolysis. ABE production from corncob was carried out with both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes using C. acetobutylicum SE-1. Interestingly, when considering the time for fermentation as the time for ABE production, a comparable rate of sugar consumption and ABE production in the SHF process (0.55 g/l·h sugar consumption and 0.20 g/l·h ABE production) could be observed when glucose (0.50 g/l·h sugar consumption and 0.17 g/l·h ABE production) or a mixture of glucose and xylose (0.68 g/l·h sugar consumption and 0.22 g/l·h ABE production) mimicking the corncob hydrolysate was used as the substrate for fermentation. This result suggested that the WDM is a suitable pretreatment method for ABE production from corncob owing to the mild conditions. A higher ABE production rate could be observed with the SSF process (0.15 g/l·h) comparing with SHF process (0.12 g/l·h) when combining the time for saccharification and fermentation and consider it as the time for ABE production. This is possibly a result of low sustained sugar level during fermentation. These investigations lead to the suggestion that this new WDM pretreatment method has the potentials to be exploited for efficient ABE production from corncob.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.     

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 °C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost.     

Bioindicator production with <Emphasis Type="Italic">Bacillus atrophaeus</Emphasis>’ thermal-resistant spores cultivated by solid-state fermentation

Bacillus atrophaeus’ spores are used in the preparation of bioindicators to monitor the dry heat, ethylene oxide, and plasma sterilization processes and in tests to assess sterilizing products. Earlier production methods involved culture in chemically defined medium to support sporulation with the disadvantage of requiring an extended period of time (14 days) besides high cost of substrates. The effect of cultivation conditions by solid-state fermentation (SSF) was investigated aiming at improving the cost–productivity relation. Initial SSF parameters such as the type of substrate were tested. Process optimization was carried out using factorial experimental designs and response surface methodology in which the influence of different variables—particle size, moisture content, incubation time, pH, inoculum size, calcium sources, and medium composition—was studied. The results have suggested that soybean molasses and sugarcane bagasse are potential substrate and support, respectively, contributing to a 5-day reduction in incubation time. Variables which presented significant effects and optimum values were mean particle size (1.0 mm), moisture content (93%), initial substrate pH (8.0), and water as a solution base. The high-yield spore production was about 3 logs higher than the control and no significant difference in dry heat resistance was observed.     

Production of polysaccharide from Agaricus subrufescens Peck on solid-state fermentation

The interest upon products obtained from fungi has increased during the recent years. Among the most noticeable, nutraceuticals, enzymes, and natural drugs occupy a privileged position. Fungal biomass for the obtainment of those products can be produced either by solid-state fermentation (SSF) or submersed fermentation. SSF has been employed for the production of spawn on pretreated wheat grains with the objective of increasing the fungal polysaccharide (glucomannans) contents. Among the important factors for the production of spawn, time of cooking, time of resting after grain cooking, consequently grain moisture, substrate pH, temperature of incubation, and initial inoculum amount are among the most significant. For wheat grains, cooking time of 21 min followed by a 24-min resting time has been shown as optimal for the production of glucomannans by the fungus Agaricus subrufescens (=Agaricus brasiliensis). Amendments of CaSO₄ (up to 3 %) and CaCO₃ (up to 1 %) had an important influence on the substrate pH. In general, better results for glucomannan production were obtained when no supplement was added or when up to 0.25 % CaCO₃ (pH 6.6) has been added to the mix. Our results demonstrate that the inoculum amount necessary for the best polysaccharide levels is around 10.3 %, while the best temperature is around 27.2 °C. Besides using the spawn for its main purpose, it could potentially and alternatively be used as nutraceutical due to the high levels of glucomannan observed (6.89 %), a compound technically proven to be a potent immunostimulatory and antitumoral agent.     

2-phenylethanol (rose aroma) production potential of an isolated pichia kudriavzevii through solid-state fermentation

2-phenylethanol (2-PE) is a higher alcohol widely used in industry that can be obtained by solid-state fermentation (SSF) using low-cost raw materials. This report describes the 2-PE production potential of an indigenous Pichia kudriavzevii isolated from solid-state fermented sugarcane bagasse that possesses attractive characteristics for processing waste streams such as its low-pH tolerance, high growth rate and temperature resistance. Besides, 2-PE production was optimized in batch-SSF using sugarcane bagasse supplemented with l-phenylalanine as substrate. Full factorial design allowed identifying the pH adjustment, micronutrient addition, inoculum and co-substrate load effects, and response surface methodology served to identify the maximum production based on temperature, initial moisture content (MC₀) and specific airflow rate (S_AFR). While the pH adjustment and micronutrient addition did not affect the 2-PE production, temperature and MC₀ resulted critical for the process. After optimization, the maximum 2-PE content was 27.2 ± 0.2 mg per gram of dry substrate at 31 °C, 76 % MC₀ and 0.129 L h⁻¹ g⁻¹ S_AFR. This result was 23.8 % higher than the sub-optimal condition, and it is the highest 2-PE production via SSF reported so far. These results confirm the ability of P. kudriavzevii for producing 2-PE, and its potential for using waste streams as substrate.     

Optimization of bagasse,nutrients and initial moisture ratios on the yield of penicillin in solid-state fermentation

An advanced solid-state fermentation (SSF) system (liquid medium absorbed on an inert support) has been applied to antibiotic production. The main components of this solid medium are: support (sugarcane bagasse), nutrients and water. The first two are solids and have to be considered to calculate the initial moisture content (IMC) of the medium. Earlier work indicated the importance of using high IMCs and concentrated media to obtain high penicillin yields in SSF. Nevertheless, the present work shows that high values of IMC or nutrients content can stimulate or inhibit penicillin production, depending on the strategy used to compensate this change (i.e. the proportions of the other two components). Conversely, increasing bagasse content always showed an inhibitory effect on the production. Since penicillin production depends on the combinations of these components, a global approach was used. The effect of the proportions of the three components on penicillin production was studied by means of a triangle of combinations and a 3D graph. It was possible to establish that high penicillin production is only obtained in a zone of low support content (10–12.5%). Surprisingly, one production maximum was observed in a zone of low moisture and high nutrients content (62 and 25.5% respectively); and another one in a zone of high moisture and a relatively low nutrients concentration (75.5 and 12.4% respectively).     

Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution

Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films.