Aspects of fermenter design for solid-state fermentations

Solid-state fermentation has gained importance recently due to several advantages over submerged fermentations. Bioreactor design aspects, which are important criteria, however, have not been given enough attention by researchers of solid-state fermentations and the present state of knowledge does not indicate an ideal type of bioreactor for solid state fermentations. This paper reviews different types of bioreactor which have been described and used for various purposes, incorporating several modification for improved operation and performance.     

Effects of intermittent mechanical mixing on solid-state fermentation of wet corn distillers grain with Trichoderma reesei

The influence of mixing on microorganism integrity and product formation is a critical design parameter for solid-state fermentation bioreactors. The effects of intermittent mechanical mixing on the solid-state fermentation of wet corn distillers grain with Trichoderma reesei NRRL 11460 for the production of cellulase were investigated. Experiments were conducted using the unbuffered media at mixing frequencies of 0, 1, 2, 3, and 6 d⁻¹ at 27.5 °C with an initial moisture content of 50%. The results indicate that mixing caused about a tenfold increase in spore production compared to fermentations at static conditions. The cellulase enzyme activity produced was minimally affected by mixing with only a 5–10% decrease in filter paper activity for mechanically mixed fermentations compared to static fermentations. Mixing at lower frequencies of 1, 2, and 3 d⁻¹ caused an increase in CO₂ evolution compared to static conditions and higher mixing frequencies of 6 d⁻¹. A correlation between substrate weight loss and cumulative CO₂ evolution was established. The ability to intermittently mix a solid-state fermentation bioreactor with minimal detrimental effects increases the feasibility of onsite production of enzymes at biofuel facilities to lower the overall production costs of cellulosic biofuels.     

Vitis vinifera culture in a non-conventional bioreactor: the reciprocating plate bioreactor

This paper is concerned with the potential use of a reciprocating plate bioreactor (RPB) for suspended plant cell cultures. The agitation mechanism of the RPB system, a plate stack, was first evaluated in pure water and in pseudocells medium of 20, 40 and 60% of PCV. As the pseudocell concentration increases, the oxygen mass transfer coefficient, K_La, significantly decreases. Correlations were established for each plate stack and concentration with good prediction of K_La. Three fermentations were performed with Vitis vinifera cells, two in the RPB system and one in shake flasks. Shake flask cultures showed better performance whereas the first fermentation performed with the RPB showed the lowest performance. The lower growth observed was attributed to the operating conditions for aeration and the dissolved oxygen control strategy. CO₂ stripping in the initial portion of the fermentation led to lower biomass growth. The second fermentation, with more appropriate operating conditions, appears to follow the trend of shake flask cultures but was terminated after 5 days due to contamination. The RPB has the potential to be used for suspended plant cell cultures but significant research needs to be performed to find optimal operating conditions but, more importantly, to make appropriate modifications to ensure the sterility of the bioreactor over long time periods.     

Applicability of a single‐use bioreactor compared to a glass bioreactor for the fermentation of filamentous fungi and evaluation of the reproducibility of growth in pellet form

The implementation of single‐use technologies offers several major advantages, e.g. prevention of cross‐contamination, especially when spore‐forming microorganisms are present. This study investigated the application of a single‐use bioreactor in batch fermentation of filamentous fungus Penicillium sp. (IBWF 040‐09) from the Institute of Biotechnology and Drug Research (IBWF), which is capable of intracellular production of a protease inhibitor against parasitic proteases as a secondary metabolite. Several modifications to the SU bioreactor were suggested in this study to allow the fermentation in which the fungus forms pellets. Simultaneously, fermentations in conventional glass bioreactor were also conducted as reference. Although there are significant differences in the construction material and gassing system, the similarity of the two types of bioreactors in terms of fungal metabolic activity and the reproducibility of fermentations could be demonstrated using statistic methods. Under the selected cultivation conditions, growth rate, yield coefficient, substrate uptake rate, and formation of intracellular protease‐inhibiting substance in the single‐use bioreactor were similar to those in the glass bioreactor.     

Production of carboxylic acids from hydrolyzed corn meal by immobilized cell fermentation in a fibrous-bed bioreactor

Corn meal hydrolyzed with amylases was used as the carbon source for producing acetic, propionic, and butyric acids via anaerobic fermentations. In this study, corn meal, containing 75% (w/w) starch, 20% (w/w) fibers, and 1.5% (w/w) protein, was first hydrolyzed using amylases at 60 degrees C. The hydrolysis yielded approximately 100% recovery of starch converted to glucose and 17.9% recovery of protein. The resulting corn meal hydrolyzate was then used, after sterilization, for fermentation studies. A co-culture of Lactococcus lactis and Clostridium formicoaceticum was used to produce acetic acid from glucose. Propionibacterium acidipropionici was used for propionic acid fermentation, and Clostridium tyrobutylicum was used for butyric acid production. These cells were immobilized on a spirally wound fibrous matrix packed in a fibrous-bed bioreactor (FBB) developed for multi-phase biological reactions or fermentation. The bioreactor was connected to a stirred-tank fermentor that provided pH and temperature controls via medium circulation. The fermentation system was operated at the recycle batch mode. Temperature and pH were controlled at 37 degrees C and 7.6, respectively, for acetic acid fermentation, 32 degrees C and 6.0, respectively, for propionic acid fermentation, and 37 degrees C and 6.0, respectively, for butyric acid production. The fermentation demonstrated a yield of approximately 100% and a volumetric productivity of approximately 1 g/(1 h) for acetic acid production. The propionic acid fermentation achieved an approximately 60% yield and a productivity of 2.12 g/(1 h), whereas the butyric acid fermentation obtained an approximately 50% yield and a productivity of 6.78 g/(1 h). These results were comparable to, or better than those fermentations using chemically defined media containing glucose as the substrate, suggesting that these carboxylic acids can be efficiently produced from direct fermentation of corn meal hydrolyzate. The corn fiber present as suspended solids in the corn meal hydrolyzate did not cause operating problem to the immobilized cell bioreactor as is usually encountered by conventional immobilized cell bioreactor systems. It is concluded that the FBB technology is suitable for producing value-added biochemicals directly from agricultural residues or commodities such as corn meal.     

Twenty-four-well plate miniature bioreactor high-throughput system: assessment for microbial cultivations

High-throughput (HT) miniature bioreactor (MBR) systems are becoming increasingly important to rapidly perform clonal selection, strain improvement screening, and culture media and process optimization. This study documents the initial assessment of a 24-well plate MBR system, Micro (micro)-24, for Saccharomyces cerevisiae, Escherichia coli, and Pichia pastoris cultivations. MBR batch cultivations for S. cerevisiae demonstrated comparable growth to a 20-L stirred tank bioreactor fermentation by off-line metabolite and biomass analyses. High inter-well reproducibility was observed for process parameters such as on-line temperature, pH and dissolved oxygen. E. coli and P. pastoris strains were also tested in this MBR system under conditions of rapidly increasing oxygen uptake rates (OUR) and at high cell densities, thus requiring the utilization of gas blending for dissolved oxygen and pH control. The E. coli batch fermentations challenged the dissolved oxygen and pH control loop as demonstrated by process excursions below the control set-point during the exponential growth phase on dextrose. For P. pastoris fermentations, the micro-24 was capable of controlling dissolved oxygen, pH, and temperature under batch and fed-batch conditions with subsequent substrate shot feeds and supported biomass levels of 278 g/L wet cell weight (wcw). The average oxygen mass transfer coefficient per non-sparged well were measured at 32.6 +/- 2.4, 46.5 +/- 4.6, 51.6 +/- 3.7, and 56.1 +/- 1.6 h(-1) at the operating conditions of 500, 600, 700, and 800 rpm shaking speed, respectively. The mixing times measured for the agitation settings 500 and 800 rpm were below 5 and 1 s, respectively.     

Propionic acid fermentation by Propionibacterium freudenreichii CCTCC M207015 in a multi-point fibrous-bed bioreactor

Propionic acid was produced in a multi-point fibrous-bed (MFB) bioreactor by Propionibacterium freudenreichii CCTCC M207015. The MFB bioreactor, comprising spiral cotton fiber packed in a modified 7.5-l bioreactor, was effective for cell-immobilized propionic acid production compared with conventional free cell fermentation. Batch fermentations at various glucose concentrations were investigated in the MFB bioreactor. Based on analysis of the time course of production, a fed-batch strategy was applied for propionic acid production. The maximum propionic acid concentration was 67.05 g l⁻¹ after 496 h of fermentation, and the proportion of propionic acid to total organic acids was approximately 78.28% (w/w). The MFB bioreactor exhibited excellent production stability during batch fermentation and the propionic acid productivity remained high after 78 days of fermentation.     

Current industrial practice in solid state fermentations for secondary metabolite production: the Biocon India experience

Solid substrate fermentation at Biocon was originally envisaged for the production of enzymes, used in the food processing industry. The original process developed at Biocon was a hygienically designed automated tray culture process. Plants using this process still continue to run effectively at Biocon, and produce a variety of products meeting and exceeding FCC/JECFA specifications for food products. Biocon recently designed, developed and patented a new bioreactor, the PlaFractor™ (pronounced play-fractor) for carrying out fermentations that use solid matrices—a term covering both nutritive support matrices as well as non-nutritive matrices impregnated with medium.Using the PlaFractor™ process it is now possible to extend the use of solid matrix fermentation for the production of enzymes, biocontrol agents and pharmaceutical products, that require elaborate containment—under precisely defined conditions. The production takes place in computer controlled bioreactors, using complex fermentation control algorithms. All the operations of solid matrix fermentation, i.e. sterilization, cooling, inoculation, fermentation and process control, product recovery and post-fermentation sterilization, are all done in one single equipment, which was not hitherto possible. All the advantages of traditional solid state fermentation, over submerged fermentation, like low energy consumption, low water requirement, high mass transfer coefficient, no foaming, and high product concentrations are retained. In addition, techniques that are important to submerged fermentation, like fed-batch fermentation, process parameter profiling, air and media sterilization, operation under aseptic environments, and ease of handling, can now be easily applied to solid state fermentation, because of the way this bioreactor is designed.A production plant, built around this bioreactor has already been operating for more than a year.     

alpha-Amylase production by Bacillus subtilis with dregs in an external-loop airlift bioreactor

An external-loop airlift bioreactor, with a low ratio 2.9 of height-to-diameter of the riser and a ratio 6.6 of riser-to-downcomer diameter, was used to produce alpha-amylase from fermentation with dregs by Bacillus subtilis. The effects of gas flow rate and liquid volume on alpha-amylase production were investigated. After a 36-h fermentation time, an average of 432.3U/ml alpha-amylase activity was obtained under the conditions of liquid volume 8.5l and gas flow rate 1.2vvm for the first 12h of fermentation, 1.4vvm from 12 to 27h, and 1.2vvm from 27h to the end. The activity was higher than that obtained in shaking flasks (409.0U/ml) and in a mechanically stirred tank bioreactor (397.2U/ml) under optimized operating conditions. The fermentation cycle of the airlift bioreactor was shorter than the 48h required for the shaking flasks and close to the 36h of the mechanically stirred tank bioreactor. It was demonstrated that the external-loop airlift bioreactor could substitute for the traditional mechanically stirred tank bioreactor to produce alpha-amylase from fermentation by Bacillus subtilis with dregs.     

Enhancement glucose oxidase production by solid-state fermentation of Aspergillus niger on polyurethane foams using mussel processing wastewaters

In the present work, studies of glucose oxidase (GOD) production in solid-state fermentation on polyurethane foams, using mussel processing wastewaters (MPW) as culture media, were carried out. Initially, the results generated in this experimental modality were compared with those that were obtained in conventional submerged fermentation. Later on, cultures were tested in solid state with fed-batch performance, defining suitable conditions of operation. These conditions were finally corroborated in experiments with a bioreactor of own design, in which GOD production was improved 13 times regarding to the initial cultures.     

Solid-state ethanol fermentation by means of inert gas circulation

A new method for solid-state ethanol fermentation (the SSEF system) was experimented on for the ethanol production from solid starchy materials, where a packedbed-type fermentor was used. Both cultivation of Aspergillus saitoi and enrichment of a saccharifying enzyme were effective for hydrolysis of the starch. Ethanol production was set in by a form of parallel fermentation using a respiration-deficient mutant of Saccharomyces cerevisiae. Produced ethanol was simultaneously stripped by circulating inert gas and separated in a condenser. Average ethanol concentration in the condensate was over 200 g/L, and over 90% of produced ethanol was recovered from the packed bed during 15 or 16 days of stripping. The fermentation efficiency was about 80%, which was evaluated much higher than those of conventional solid-state fermentations. The residue had lesser volume and a higher solids content compared with the distillery wastewaters of conventional liquid-state fermentations. This means an advantage for the treatment and the effective conversion of the residue into fetilizers or animal feeds.     

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.     

Use of Direct-Infusion Electrospray Mass Spectrometry To Guide Empirical Development of Improved Conditions for Expression of Secondary Metabolites from Actinomycetes

A major barrier in the discovery of new secondary metabolites from microorganisms is the difficulty of distinguishing the minor fraction of productive cultures from the majority of unproductive cultures and growth conditions. In this study, a rapid, direct-infusion electrospray mass spectrometry (ES-MS) technique was used to identify chemical differences that occurred in the expression of secondary metabolites by 44 actinomycetes cultivated under six different fermentation conditions. Samples from actinomycete fermentations were prepared by solid-phase extraction, analyzed by ES-MS, and ranked according to a chemical productivity index based on the total number and relative intensity of ions present in each sample. The actinomycete cultures were tested for chemical productivity following treatments that included nutritional manipulations, autoregulator additions, and different agitation speeds and incubation temperatures. Evaluation of the ES-MS data from submerged and solid-state fermentations by paired t test analyses showed that solid-state growth significantly altered the chemical profiles of extracts from 75% of the actinomycetes evaluated. Parallel analysis of the same extracts by high-performance liquid chromatography–ES-MS–evaporative light scattering showed that the chemical differences detected by the ES-MS method were associated with growth condition-dependent changes in the yield of secondary metabolites. Our results indicate that the high-throughput ES-MS method is useful for identification of fermentation conditions that enhance expression of secondary metabolites from actinomycetes.     

Characterization and application of a miniature 10 mL stirred-tank bioreactor, showing scale-down equivalence with a conventional 7 L reactor

The aim of this study was to characterize the engineering environment of an instrumented 10 mL miniature stirred-tank bioreactor and evaluate its potential as a scale-down device for microbial fermentation processes. Miniature bioreactors such as the one detailed in this work have been developed by several research groups and companies and seek to address the current bottleneck at the screening stage of bioprocess development. The miniature bioreactor was characterized in terms of overall volumetric oxygen transfer coefficient and mixing time over a wide range of impeller speeds. Power input to the miniature bioreactor was directly measured, and from this the power number of each impeller was calculated and specific power input estimated, allowing the performance of the miniature bioreactor to be directly compared with that of a conventional 7 L bioreactor. The capability of the miniature bioreactor to carry out microbial fermentations was also investigated. Replicate batch fermentations of Escherichia coli DH5alpha producing plasmid DNA were performed at equal specific power input, under fully aerobic and oxygen-limiting conditions. The results showed a high degree of equivalence between the two scales with regard to growth and product kinetics. This was underlined by the equal maximum specific growth rate and equal specific DNA product yield on biomass obtained at the two scales of operation, demonstrating the feasibility of scaling down to 10 mL on the basis of equivalent specific power input.     

Thermal profiling for parallel on-line monitoring of biomass growth in miniature stirred bioreactors

Recently we have described the design and operation of a miniature bioreactor system in which 4-16 fermentations can be performed (Gill et al., Biochem Eng J 39:164-176, 2008). Here we report on the use of thermal profiling techniques for parallel on-line monitoring of cell growth in these bioreactors based on the natural heat generated by microbial culture. Results show that the integrated heat profile during E. coli TOP10 pQR239 fermentations followed the same pattern as off-line optical density (OD) measurements. The maximum specific growth rates calculated from off-line OD and on-line thermal profiling data were in good agreement, at 0.66+/-0.04 and 0.69+/-0.05 h(-1) respectively. The combination of a parallel miniature bioreactor system with a non-invasive on-line technique for estimation of culture kinetic parameters provides a valuable approach for the rapid optimisation of microbial fermentation processes.     

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.     

Fermentative biohydrogen production: trends and perspectives

Biologically produced hydrogen (biohydrogen) is a valuable gas that is seen as a future energy carrier, since its utilization via combustion or fuel cells produces pure water. Heterotrophic fermentations for biohydrogen production are driven by a wide variety of microorganisms such as strict anaerobes, facultative anaerobes and aerobes kept under anoxic conditions. Substrates such as simple sugars, starch, cellulose, as well as diverse organic waste materials can be used for biohydrogen production. Various bioreactor types have been used and operated under batch and continuous conditions; substantial increases in hydrogen yields have been achieved through optimum design of the bioreactor and fermentation conditions. This review explores the research work carried out in fermentative hydrogen production using organic compounds as substrates. The review also presents the state of the art in novel molecular strategies to improve the hydrogen production.     

Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors

In this study, the optimal fermentation type and the operating conditions of anaerobic process in continuous-flow acidogenic reactors was investigated for the maximization of bio-hydrogen production using mixed cultures. Butyric acid type fermentation occurred at pH>6, propionic acid type fermentation occurred at pH about 5.5 with E(h) (redox potential) >-278mV, and ethanol-type fermentation occurred at pH<4.5. The representative strains of these fermentations were Clostridium sp., Propionibacterium sp. and Bacteriodes sp., respectively. Ethanol fermentation was optimal type by comparing the operating stabilities and hydrogen production capacities between the fermentation types, which remained stable when the organic loading rate (OLR) reached the highest OLR at 86.1kgCOD/m(3)d. The maximum hydrogen production reached up to 14.99L/d.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Solid-state fermentation in rotating drum bioreactors: operating variables affect performance through their effects on transport phenomena

Aspergillus oryzae ACM 4996 was grown on an artificial gel-based substrate and on steamed wheat bran during solid-state fermentations in 18.7 L rotating drum bioreactors. For gel fermentations fungal growth decreased as rotational speed increased, presumably due to increased shear. For wheat bran fermentations fungal growth improved under agitated compared to static culture conditions, due to superior heat and mass transfer. We conclude that the effects of operational variables on the performance of SSF bioreactors are mediated by their effects on transport phenomena such as mixing, shear, heat transfer, and mass transfer within the substrate bed. In addition, the substrate characteristics affect the need for and the rates of these transport processes. Different transport phenomena may be rate limiting with different substrates. This work improves understanding of the effects of bioreactor operation on SSF performance.