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.     

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。     

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 the Virtual Instrument (VI) concept, the performance of the 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. The text was submitted by the authors in English.     

Production of phytase by Mucor racemosus in solid-state fermentation

Phytase production was studied by three Mucor and eight Rhizopus strains by solid-state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosus NRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH(4))(2)SO(4), phytase production in solid-state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett-Burman and central composite experimental designs. Using the optimized medium phytase, alpha-amylase and lipase production of Mucor racemosus NRRL 1994 was compared in solid-state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food-grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.     

The influence of process parameters in production of lipopeptide iturin A using aerated packed bed bioreactors in solid-state fermentation

The strain Bacillus iso 1 co-produces the lipopeptide iturin A and biopolymer poly-γ-glutamic acid (γ-PGA) in solid-state fermentation of substrate consisting of soybean meal, wheat bran with rice husks as an inert support. The effects of pressure drop, oxygen consumption, medium permeability and temperature profile were studied in an aerated packed bed bioreactor to produce iturin A, diameter of which was 50 mm and bed height 300 mm. The highest concentrations of iturin A and γ-PGA were 5.58 and 3.58 g/kg-dry substrate, respectively, at 0.4 L/min after 96 h of fermentation. The low oxygen uptake rates, being 23.34 and 22.56 mg O₂/kg-dry solid substrate for each air flow rate tested generated 5.75 W/kg-dry substrate that increased the fermentation temperature at 3.7 °C. The highest pressure drop was 561 Pa/m at 0.8 L/min in 24 h. This is the highest concentration of iturin A produced to date in an aerated packed bed bioreactor in solid-state fermentation. The results can be useful to design strategies to scale-up process of iturin A in aerated packed bed bioreactors. Low concentration of γ-PGA affected seriously pressure drop, decreasing the viability of the process due to generation of huge pressure gradients with volumetric air flow rates. Also, the low oxygenation favored the iturin A production due to the reduction of free void by γ-PGA production, and finally, the low oxygen consumption generated low metabolic heat. The results show that it must control the pressure gradients to scale-up the process of iturin A production.     

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).     

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.     

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.     

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.     

Integrated Strategies to Enhance Cellulolytic Enzyme Production Using an Instrumented Bioreactor for Solid-State Fermentation of Sugarcane Bagasse

The conversion of agro-industrial residues, such as sugarcane bagasse, into high-value products and renewable energy, within the biorefinery concept, is a potential alternative towards the sustainable management of these resources. This work evaluates the production of cellulolytic enzymes by a selected strain of Aspergillus niger cultivated in sugarcane bagasse under solid-state fermentation using an instrumented lab-scale bioreactor. The effects of environmental factors including the type of substrate and medium composition, as well as the operational conditions (air flow rate, inlet air relative humidity, and initial substrate moisture content) on the production of the enzymatic complex were evaluated using statistical design tools. Significant increases in FPase, endoglucanase, and xylanase activities were achieved under the optimized conditions predicted by the models, with values of 0.88, 21.77, and 143.85 IU/g of dry solid substrate, respectively, representing around ten-, four-, and twofold increases compared to the activities obtained under the initial growth conditions. This demonstrates the importance of evaluating environmental and operational criteria in order to achieve efficient enzyme production. The crude enzymatic extract obtained under optimized conditions was employed for enzymatic hydrolysis of pretreated sugarcane bagasse. Approximately 13 % of total reducing sugars, and a glucose concentration of 2.54 g/L, were obtained after 22 h of hydrolysis of steam exploded sugarcane bagasse, indicating that the enzymatic cocktail produced has good potential for use in the conversion of biomass.     

Comparative studies on the production of cellulases by thermophilic fungi in submerged and solid-state fermentation

Summary Six thermophilic fungi were examined for their ability to produce cellulolytic enzymes in liquid (LF) and solid-state fermentation (SSF). The best cellulase activities were achieved by Thermoascus aurantiacus and Sporotrichum thermophile. Taking into consideration that solid-state medium obtained from 100 g of dry sugar-beet pulp occupies about 11 of fermentor volume equivalent to 11 of LF, it was confirmed that enzyme productivity per unit volume from both fungi was greater in SSF than in LF. The cellulase system obtained by SSF with T. aurantiacus contained 1.322 IU/1 of exo--d-glucanase, 53.269 IU/1 of endo--d-glucanase and 8.974 IU/1 of -d-glucosidase. The thermal and pH characteristics of cellulases from solid-state fermentation of T. aurantiacus and S. thermophile are described.     

Streptomyces sp. TEM 33 possesses high lipolytic activity in solid-state fermentation in comparison with submerged fermentation

Solid-state fermentation (SSF) is a bioprocess that doesn’t need an excess of free water, and it offers potential benefits for microbial cultivation for bioprocesses and product development. In comparing the antibiotic production, few detailed reports could be found with lipolytic enzyme production by Streptomycetes in SSF. Taking this knowledge into consideration, we prefer to purify Actinomycetes species as a new source for lipase production. The lipase-producing strain Streptomyces sp. TEM 33 was isolated from soil and lipase production was managed by solid-state fermentation (SSF) in comparison with submerged fermentation (SmF). Bioprocess-affecting factors like initial moisture content, incubation time, and various carbon and nitrogen additives and the other enzymes secreted into the media were optimized. Lipase activity was measured as 1.74 ± 0.0005 U/g dry substrate (gds) by the p-nitrophenylpalmitate (pNPP) method on day 6 of fermentation with 71.43% final substrate moisture content. In order to understand the metabolic priority in SSF, cellulase and xylanase activity of Streptomyces sp. TEM33 was also measured. The microorganism degrades the wheat bran to its usable form by excreting cellulases and xylanases; then it secretes the lipase that is necessary for degrading the oil in the medium.     

Performance of an intermittent agitation rotating drum type bioreactor for solid-state fermentation of wheat straw

A laboratory bioreactor, designed for solid-state fermentation of thermophilic microorganisms, was operated for production of cellulases and hemicellulases by the thermophilic fungus Thermoascus aurantiacus. The suitability of the apparatus for the effective control of important operating variables affecting growth of microbes in solid-state cultivation was determined. Application of the optimum conditions found for the moisture content of the medium, growth temperature and airflow rate produced enzyme yields of 1709 U endoglucanase, 4 U cellobiohydrolase, 79 U beta-glucosidase, 5.5 U FPA, 4490 U xylanase and 45 U beta-xylosidase per g of dry wheat straw. The correlation between microorganism growth and production of enzymes was efficiently described by the Le Duy kinetic model.     

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 of xylooligosaccharides in SSF by Bacillus subtilis KCX006 producing β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes

Xylanase and xylooligosaccharides (XOS) are employed in food and feed industries. Though xylanase production from lignocellulosic materials (LCMs) by solid-state fermentation (SSF) is well known, the XOS formed during growth is not recovered due to its conversion to xylose by β-xylosidase and subsequent bacterial metabolism. A new strain, Bacillus subtilis KCX006, was exceptionally found to synthesize β-xylosidase-free endo-xylanase and multiple xylan debranching enzymes constitutively in the presence of LCMs. Absence of β-xylosidase resulted in accumulation of XOS during growth of KCX006 on LCMs. Therefore, this strain was used for simultaneous production of xylanase and XOS from agro-residues in solid-state fermentation (SSF). Partial purification of XOS from culture supernatant using activated charcoal followed by high-performance liquid chromatography (HPLC) analysis showed xylobiose to xylotetraose formed as the major products. Among various LCM substrates, wheat bran and groundnut oil-cake supported highest xylanase and XOS production at 2158 IU/gdw and 24.92 mg/gdw, respectively. The levels of xylanase and XOS were improved by 1.5-fold (3102 IU/gdw) and 1.9-fold (48 mg/gdw), respectively, by optimization of culture conditions.     

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.     

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.     

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.     

Production of green biodegradable plastics of poly(3-hydroxybutyrate) from renewable resources of agricultural residues

This work describes potential opportunities for utilization of agro-industrial residues to produce green biodegradable plastics of poly(3-hydroxybutyrate) (PHB). Wheat straws were examined with good efficacy of carbon substrates using Cupriavidus necator. Production was examined in separate hydrolysis and fermentation (SHF) in the presence and absence of WS hydrolysis enzymes, and in simultaneous saccharification and fermentation (SSF) with enzymes. Results showed that production of PHB in SSF was more efficient in terms of viable cell count, cell dry weight, and PHB production and yield compared to those of SHF and glucose-control cultures. While glucose control experiment produced 4.6 g/L PHB; SSF produced 10.0 g/L compared to 7.1 g/L in SHF when utilizing enzymes during WS hydrolysis. Results showed that most of sugars produced during the hydrolysis were consumed in SHF (~98 %) compared to 89.2 % in SSF. Results also demonstrated that a combination of glucose and xylose can compensate for the excess carbon required for enhancing PHB production by C. necator. However, higher concentration of sugars at the beginning of fermentation in SHF can lead to cell inhibition and consequently catabolite repressions. Accordingly, results demonstrated that the gradual release of sugars in SSF enhanced PHB production. Moreover, the presence of sugars other than glucose and xylose can eliminate PHB degradation in medium of low carbon substrate concentrations in SSF.     

<Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> Spore Production Under Solid-State Fermentation of Lignocellulosic Residues

This study was conducted to elucidate cultivation conditions determining Bacillus amyloliquefaciens B-1895 growth and enhanced spore formation during the solid-state fermentation (SSF) of agro-industrial lignocellulosic biomasses. Among the tested growth substrates, corncobs provided the highest yield of spores (47?×?10¹⁰ spores g^?1 biomass) while the mushroom spent substrate and sunflower oil mill appeared to be poor growth substrates for spore formation. Maximum spore yield (82?×?10¹⁰ spores g^?1 biomass) was achieved when 15 g corncobs were moistened with 60 ml of the optimized nutrient medium containing 10 g peptone, 2 g KH₂PO₄, 1 g MgSO₄·7H₂O, and 1 g NaCl per 1 l of distilled water. The cheese whey usage for wetting of lignocellulosic substrate instead water promoted spore formation and increased the spore number to 105?×?10¹⁰ spores g^?1. Addition to the cheese whey of optimized medium components favored sporulation process. The feasibility of developed medium and strategy was shown in scaled up SSF of corncobs in polypropylene bags since yield of 10?×?10¹¹ spores per gram of dry biomass was achieved. In the SSF of lignocellulose, B. amyloliquefaciens B-1895 secreted comparatively high cellulase and xylanase activities to ensure good growth of the bacterial culture.