Operating characteristics of solid-state fermentation bioreactor with air pressure pulsation

The development of solid state fermentation (SSF) technology is very important to the production of cellulase and ultimately to the utilization of natural cellulose. However, inadequate dissipation of heat generated by biological activities has prevented solid state fermentation from large-scale applications. The paper deals with the development of a novel SSF bioreactor with air pressure pulsation. By developing a measurement and control system under Virtual Instrument (VI) concept, performance of SSF bioreactor with pressure pulsation was studied by cultivating Trichoderma koningii in solid medium made of wheat bran and corncob. The cooling effects of pressure pulsation on solid porous beds are discussed. Experimental results show that pressure pulsation enhances medium moisture evaporation, and hence, heat dissipation. Furthermore, through changing the pressure pulsation directions, it is able to mitigate the temperature gradients in the bioreactor. To sum up, pressure pulsation can provide the microbes with a growing environment at optimal temperature and medium water content.     

A novel structured bioreactor for solid-state fermentation

A novel patented solid-state bioreactor (251 L) with honeycomb loading device was designed and its performance was tested. First, this apparatus gave a 66.87 % of calculated loading coefficient (volume ratio), which was almost twofold compared with conventional fermenters. Next, considering the crucial effect of heat transfer on bed loading and microbial growth, the performance was validated by temperature variance during fermentation and spore viability of Bacillus cereus DM423. Air pressure pulsation or external water jacket was used to control temperature; the maximal temperature variation was 7.7 versus 19.8 °C, respectively during fermentation. The difference was mainly due to the continuous gas phase characterized by solid-state fermentation (SSF). The average living spores of (1.50 ± 0.07) × 10¹¹ cfu/g at 40 h obtained from the device was higher than (0.70 ± 0.03) × 10¹¹ cfu/g from flask at 48 h. The results indicated that this new loading bioreactor with air pressure pulsation could be a good prospect for industrialization of SSF employing bacterial cultures.     

Hyperactive α-amylase production by Aspergillus oryzae IFO 30103 in a new bioreactor

Aims: To improve the α‐amylase production in solid‐state fermentation (SSF) condition utilizing a new bioreactor (NB) system. Methods and Results: In NB system, 20 g of wheat bran moistened with liquid medium in 1 : 1 ratio (w/v) was taken on the tray present inside the upper vessel and an additional 80 ml medium was supplemented into the lower vessel. Oxygen uptake rate was improved by supplying compressed air that lifted the liquid medium into the upper vessel and touched the substrate bed. This condition probably facilitated the heat transfer to liquid medium, reduce water loss and catabolite repression. With 1% glucose supplementation, maximum α‐amylase activity of 22 317 Ugds^?1 was produced by Aspergillus oryzae IFO 30103 within a very short incubation period (48 h) at 2‐cm bed height with air flow rate of 0·1 l min^?1 g^?1 wheat bran at 32°C and initial medium pH of 6. Conclusions: Within a short incubation period, significantly high α‐amylase activity was obtained and it is higher than those reported to date at bioreactor scale operating with a fungal strain. Significance and Impact of the Study: The reactor is novel and can overcome some of the major problems associated with SSF process. A. oryzae IFO 30103 is reported as the best fungal source for α‐amylase production.     

Deep-bed solid state fermentation of sweet sorghum stalk to ethanol by thermotolerant Issatchenkia orientalis IPE 100

A solid state fermentation (SSF) of sweet sorghum stalk to ethanol was conducted in 250-mL flask using thermotolerant Issatchenkia orientalis IPE 100, and the optimal operation parameters were determined as 42°C fermentation temperature, 75% (w/w) water content, 2mm particle size and 3% (w/w) inoculation rate in 250-mL conical flask. When the SSF was scaled up from the flask to a 10-L bioreactor, temperature gradient in the substrate bed was observed due to heat accumulation in the bioreactor. The temperature gradient was dependent on both substrate depth and operation temperature. Due to high thermotolerance of the strain IPE 100, a deep-bed SSF of sweet sorghum stalk was developed in the bioreactor. The highest ethanol yield of 0.25 g-ethanol/g-dry stalk was obtained at 37°C with 15-20 cm substrate depth in the bioreactor. These results provided a great potential for large-scale deep-bed SSF in practice.     

Air pressure pulsation solid state fermentation of feruloyl esterase by Aspergillus niger

Air pressure pulsation solid state fermentation (APP-SSF) was applied to produce feruloyl esterase (FAE) by Aspergillus niger. With the optimization of some variables by orthogonal design, the optimal condition obtained was 0.2 MPa (gauge pressure) of high pressure intensity, 30 min of low pressure duration and 20s of high pressure duration. Based on the optimized condition, the APP-SSF achieved the reasonable enzyme yield of 881 mU/g at 48 h, which was 58% more than that by static solid state fermentation (static SSF) at 72 h. By comparison of two fermentation methods in temperature, O(2) and CO(2) concentration, and respiration intensity, it was concluded that APP-SSF enhanced heat and mass transfer of fermentation system and strengthened the metabolism of microorganisms. The APP-SSF had a greatly positive effect on FAE production by A. niger, by enhancing mass and heat transfer and activating growth and metabolism.     

Impact of operating conditions on performance of a novel gas double-dynamic solid-state fermentation bioreactor (GDSFB)

A self-designed novel solid-state fermentation (SSF) bioreactor named “gas double-dynamic solid-state fermentation bioreactor (GDSFB)” showed great success in processes for the production of several valuable products. For the present study, a simple GDSFB (2 L in volume) was designed to investigate the impact of exhaust time on SSF performance. Both air pressure and vent aperture significantly influenced the exhaust time. The production of cellulase by Penicillium decumbens JUA10 was studied in this bioreactor. When the vent aperture was maintained at 0.2 cm, the highest FPA activity of 17.2 IU/g dry solid-state medium was obtained at an air pressure of 0.2 MPa (gauge pressure). When the air pressure was maintained at 0.2 MPa, a vent aperture of 0.3 cm gave the highest FPA activity of 18.0 IU/g dry solid-state medium. Further analysis revealed that the exhaust time was a crucial indicator of good performance in GDSFB.     

Enhanced lignin peroxidase synthesis by Phanerochaete Chrysosporium in solid state bioprocessing of a lignocellulosic substrate

Summary A solid state fermentation (SSF) process for the production of lignin peroxidase was optimized to enhance enzyme production by Phanerochaete chrysosporium. Optimization of the corncob SSF medium caused a significant reduction in fermentation time to give maximum lignin peroxidase yield. Supplementation of the SSF medium by low concentrations of peptone, yeast extract and Tween-80 enhanced lignin peroxidase production. Maximum yield of lignin peroxidase was 13.7 U/gds (units per gram dry substrate) noted after 5 days of SSF with 70% moisture and 20% (v/w) inoculum.     

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     

A comparative study of Taxol production in liquid and solid‐state fermentation with Nigrospora sp. a fungus isolated from Taxus globosa

Aims: To determine in liquid (LF) and solid‐state fermentation (SSF) the effect of medium concentration on growth and Taxol produced by Nigrospora sp., a fungus isolated from the Mexican yew. Methods and Results: Nigrospora sp. was grown at different concentrations of the base culture medium M1D, i.e. two (2×), four (4×), six (6×) and eight times (8×) the base concentration. The titres of Taxol determined by competitive inhibition enzyme immunoassay increased with increasing medium concentration in LF and SSF but were higher in SSF in every medium concentration. The Taxol produced in SSF and LF with 8× medium was 221 and 142 ng l^?1. The SSF gave also higher biomass, growth and sugar utilization than LF in every medium. The growth and sugar consumption were modelled by the logistic and the Pirt models, respectively. However, the Luedeking–Piret model was unsuitable for Taxol. Conclusions: The SSF surpassed LF in terms of Taxol, growth and sugar utilization; thus, it has significant advantages over LF. Significance and Impact of the Study: This is the first report on Taxol production by SSF and the first contribution to evaluate the influence of the medium on Taxol production in LF and SSF.     

Heat transfer simulation in solid substrate fermentation

A mathematical model was developed and tested to simulate the generation and transfer of heat in solid substrate fermentation (SSF). The experimental studies were realized in a 1-L static bioreactor packed with cassava wet meal and inoculated with Aspergillus niger. A simplified pseudohomogeneous monodimensional dynamic model was used for the energy balance. Kinetic equations taking into account biomass formation (logistic), sugar consumption (with maintenance), and carbon dioxide formation were used. Model verification was achieved by comparison of calculated and experimental temperatures. Heat transfer was evaluated by the estimation of Biot and Peclet heat dimensionless numbers 5-10 and 2550-2750, respectively. It was shown that conduction through the fermentation fixed bed was the main heat transfer resistance. This model intends to reach a better understanding of transport phenomena in SSF, a fact which could be used to evaluate various alternatives for temperature control of SSF, i.e., changing air flow rates and increasing water content. Dimensionless numbers could be used as scale-up criteria of large fermentors, since in those ratios are described the operating conditions, geometry, and size of the bioreactor. It could lead to improved solid reactor systems. The model can be used as a basis for automatic control of SSF for the production of valuable metabolites in static fermentors.     

Biotransformation of pentacyclic terpene isolated from Alstonia scholaris (R.BR.)

Abstract

Betulin (1) a pentacyclic triterpene was isolated from medicinal plant Alstonia scholaris (R.BR.) and its structural modification by five filamentous fungi was investigated using flask shake and stirred bioreactor methods. Screening-scale and preparative-scale biotransformation with a standard two-stage protocol yielded betulinic acid (2). Out of five fungal strains (Microsporum canis, Trichophyton tonsurans, Aspergillus niger, A. niger NIAB-280 and Penicillium spp.) only two strains, M. canis and T. tonsurans, showed significant yield of (2). Samples withdrawn from fermentation medium were extracted with ethyl acetate and purified using column chromatography. Compound 2 was extracted from fermentation medium after 5–10 days. Flasks and the bioreactor were stirred at 250 rpm and 28°C. The yield of (2) gradually increased with incubation time. A stirred bioreactor was found to be convenient and simple for compound 1 oxidation. A validated analytical HPLC method was employed to confirm the biotransformation of compound 1 to 2.     

Comparison of SHF and SSF processes for the bioconversion of steam-exploded wheat straw

Two processes for ethanol production from wheat straw have been evaluated — separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). The study compares the ethanol yield for biomass subjected to varying steam explosion pretreatment conditions: temperature and time of pretreatment was 200°C or 217°C and at 3 or 10 min. A rinsing procedure with water and NaOH solutions was employed for removing lignin residues and the products of hemicellulose degradation from the biomass, resulting in a final structure that facilitated enzymatic hydrolysis. Biomass loading in the bioreactor ranged from 25 to 100 g l⁻¹ (dry weight). The enzyme-to-biomass mass ratio was 0.06. Ethanol yields close to 81% of theoretical were achieved in the two-step process (SHF) at hydrolysis and fermentation temperatures of 45°C and 37°C, respectively. The broth required addition of nutrients. Sterilisation of the biomass hydrolysate in SHF and of reaction medium in SSF can be avoided as can the use of different buffers in the two stages. The optimum temperature for the single-step process (SSF) was found to be 37°C and ethanol yields close to 68% of theoretical were achieved. The SSF process required a much shorter overall process time (≈30 h) than the SHF process (96 h) and resulted in a large increase in ethanol productivity (0.837 g l⁻¹ h⁻¹ for SSF compared to 0.313 g l⁻¹ h⁻¹ for SHF). Journal of Industrial Microbiology & Biotechnology (2000) 25, 184–192. Received 02 December 1999/ Accepted in revised form 20 July 2000     

Cost-effective production of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> using simple netting bag solid bioreactor

A novel simple solid state fermentation method, netting bag bioreactor (Φ 120 × 800 mm), was developed and used to cultivate Bacillus licheniformis as probiotics. High spore yield (1.2 × 10¹¹ CFU/g dry substrate) has been obtained by using this method. Comparing to the tray bioreactor and the packed bed bioreactor for Bacillus fermentation, the netting bag method was more cost-effective, time- and space-saving and the material cost is also as low as ca. US $293 per 1,000 kg spores. Thus, netting bag SSF can be widely applied to produce probiotic bacteria in developing areas.     

Mathematical modeling of Kluyveromyces marxianus growth in solid-state fermentation using a packed-bed bioreactor

This work investigated the growth of Kluyveromyces marxianus NRRL Y-7571 in solid-state fermentation in a medium composed of sugarcane bagasse, molasses, corn steep liquor and soybean meal within a packed-bed bioreactor. Seven experimental runs were carried out to evaluate the effects of flow rate and inlet air temperature on the following microbial rates: cell mass production, total reducing sugar and oxygen consumption, carbon dioxide and ethanol production, metabolic heat and water generation. A mathematical model based on an artificial neural network was developed to predict the above-mentioned microbial rates as a function of the fermentation time, initial total reducing sugar concentration, inlet and outlet air temperatures. The results showed that the microbial rates were temperature dependent for the range 27–50°C. The proposed model efficiently predicted the microbial rates, indicating that the neural network approach could be used to simulate the microbial growth in SSF.     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

Caffeic acid production by simultaneous saccharification and fermentation of kraft pulp using recombinant Escherichia coli

Caffeic acid (3,4-dihydroxycinnamic acid) serves as a building block for thermoplastics and a precursor for biologically active compounds and was recently produced from glucose by microbial fermentation. To produce caffeic acid from inedible cellulose, separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) reactions were compared using kraft pulp as lignocellulosic feedstock. Here, a tyrosine-overproducing Escherichia coli strain was metabolically engineered to produce caffeic acid from glucose by introducing the genes encoding a 4-hydroxyphenyllactate 3-hydroxylase (hpaBC) from Pseudomonas aeruginosa and tyrosine ammonia lyase (fevV) from Streptomyces sp. WK-5344. Using the resulting recombinant strain, the maximum yield of caffeic acid in SSF (233 mg/L) far exceeded that by SHF (37.9 mg/L). In the SSF with low cellulase loads (≤2.5 filter paper unit/g glucan), caffeic acid production was markedly increased, while almost no glucose accumulation was detected, indicating that the E. coli cells experienced glucose limitation in this culture condition. Caffeic acid yield was also negatively correlated with the glucose concentration in the fermentation medium. In SHF, the formation of by-product acetate and the accumulation of potential fermentation inhibitors increased significantly with kraft pulp hydrolysate than filter paper hydrolysate. The combination of these inhibitors had synergistic effects on caffeic acid fermentation at low concentrations. With lower loads of cellulase in SSF, less potential fermentation inhibitors (furfural, 5-hydroxymethyfurfural, and 4-hydroxylbenzoic acid) accumulated in the medium. These observations suggest that glucose limitation in SSF is crucial for improving caffeic acid yield, owing to reduced by-product formation and fermentation inhibitor accumulation.

     

固态发酵的参数周期变化及对微生物发酵的影响

研究了压力脉动固态发酵反应器内环境参数的周期性变化以及这些周期性的环境刺激对于固态培养的斜卧青霉发酵的影响。研究结果显示:在这种反应器中,在空气压力脉动的带动下,反应器内的温度和空气湿度也会发生较大幅度的周期性变化,变化的周期和空气压力脉动的周期相同,变化的幅度随着压力脉动的幅度增大而增加;在外界周期刺激的条件下,较未加外界周期刺激斜卧青霉的生物量增加了104% ,二氧化碳的产生总量增加了229%和纤维素酶的产量增加了320 % ,数据显示外界周期刺激不仅增加了菌体的生物量同时增加了其代谢活性。     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

Solid-state fermentation-supported enhanced production of α-galactosidase by a deoxyglucose-resistant mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> and thermostabilization of the production process

The aim of the present investigation was to comparatively evaluate the behaviour of A. niger and its derepressed mutant in production of α-galactosidase in submerged (SmF) and solid state fermentation (SSF) using basal Vogel’s medium or corn steep liquor as nitrogen source and observe the response of latter source under both cultural techniques under different temperature regimes, and determine if SSF can be exploited in a wide range of temperature expected to vary in this fermentation system. All studies were performed in both systems under pre-optimized cultural conditions. Higher melting temperature and negative values of entropy of activation in SSF indicated that the genetic system of both organisms was thermodynamically resistant in the presence of corn steep liquor but sensitive to inactivation in the presence of Vogel’s nitrogen sources in submerged fermentation. This was reflected as the organisms demanded higher magnitudes of energy for product formation in the presence of ammonium salts. Studies on influence of corn steep liquor revealed that it had stabilizing effect too in both fermentation systems but the mutant strain was more stable in both fermentation systems. Because of these properties, the mutant organism may be exploited for bulk production of α-galactosidase in SSF under condition where temperature may fluctuate during fermentation.     

Utilization of palm kernel cake for production of β-mannanase by Aspergillus niger FTCC 5003 in solid substrate fermentation using an aerated column bioreactor

The production of β-mannanase from palm kernel cake (PKC) as a substrate in solid substrate fermentation (SSF) was studied using a laboratory column bioreactor. The simultaneous effects of three independent variables, namely incubation temperature, initial moisture content of substrate and airflow rate, on β-mannanase production were evaluated by response surface methodology (RSM) on the basis of a central composite face-centered (CCF) design. Eighteen trials were conducted in which Aspergillus niger FTCC 5003 was cultivated on PKC in an aerated column bioreactor for seven days under SSF process. The highest level of β-mannanase (2117.89 U/g) was obtained when SSF process was performed at incubation temperature, initial moisture level and aeration rate of 32.5°C, 60% and 0.5 l/min, respectively. Statistical analysis revealed that the quadratic terms of incubation temperature and initial moisture content had significant effects on the production of β-mannanase (P < 0.01). A similar analysis also demonstrated that the linear effect of initial moisture level and an interaction effect between the initial moisture content and aeration rate significantly influenced the production of β-mannanase (P < 0.01). The statistical model suggested that the optimal conditions for attaining the highest level of β-mannanase were incubation temperature of 32°C, initial moisture level of 59% and aeration rate of 0.5 l/min. A β-mannanase yield of 2231.26 U/g was obtained when SSF process was carried out under the optimal conditions described above.