The bright frontiers of microbial metabolic optogenetics

In recent years, light-responsive systems from the field of optogenetics have been applied to several areas of metabolic engineering with remarkable success. By taking advantage of light's high tunability, reversibility, and orthogonality to host endogenous processes, optogenetic systems have enabled unprecedented dynamical controls of microbial fermentations for chemical production, metabolic flux analysis, and population compositions in co-cultures. In this article, we share our opinions on the current state of this new field of metabolic optogenetics.We make the case that it will continue to impact metabolic engineering in increasingly new directions, with the potential to challenge existing paradigms for metabolic pathway and strain optimization as well as bioreactor operation.     

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.     

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.     

Contamination of a high-cell-density continuous bioreactor

Continuous fermentations were carried out with a recombinant flocculent Saccharomyces cerevisiae strain in an airlift bioreactor. Once operating under steady state at a dilution rate of 0.45 h(-1), the bioreactor was contaminated with Escherichia coli cells. The faster growing E. coli strain was washed out of the bioreactor and the recombinant, slower growing flocculating S. cerevisiae strain remained as the only species detected in the bioreactor. Flocculation, besides allowing for the realization of high-cell-density systems with corresponding unusual high productivity, may be used as a selective property for controlling some contamination problems associated with prolonged continuous operation.     

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.     

Periodic operation of immobilized live cell bioreactor for the production of candicidin

A lumped model for cell growth and secondary metabolite production in an immobilized live cell bioreactor has been developed. This model is applied here to simulate the performance of an immobilized bioreactor under steady-state conditions and under conditions of periodically varying concentration of a growth-limiting substrate. The results of the simulation study were experimentally verified in the case of the production of the antibiotic candicidin by Streptomyces griseus in an immobilized bioreactor with forced periodic operation. The results of the studies suggest that periodically operated immobilized live cell bioreactors can provide a potent alternative for the production of non-growth-associated biochemicals, as compared to free cell fermentations, pulsed fermentations with process cycle regeneration, and nonregenerated bioreactors. This work has demonstrated that by frequent pulsing of the growth limiting nutrient, stable extended production can be obtained at high specific cellular productivities.     

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.     

An ethanol-auxostat with on-line gas sensing

Summary Ethanol concentration in a bioreactor was held constant by using the signal from a commercial gas-sensor for organic vapors. The auxostat principle was demonstrated by runs of several hundred hours. All fermentations were open, and only the feed was sterile. The effect of stripping of volatile substrate from the liquid was investigated and compared with the growth curve.     

Solid-state fermentation: a continuous process for fungal tannase production

Truly continuous solid-state fermentations with operating times of 2-3 weeks were conducted in a prototype bioreactor for the production of fungal (Penicillium glabrum) tannase from a tannin-containing model substrate. Substantial quantities of the enzyme were synthesized throughout the operating periods and (imperfect) steady-state conditions seemed to be achieved soon after start-up of the fermentations. This demonstrated for the first time the possibility of conducting solid-state fermentations in the continuous mode and with a constant noninoculated feed. The operating variables and fermentation conditions in the bioreactor were sufficiently well predicted for the basic reinoculation concept to succeed. However, an incomplete understanding of the microbial mechanisms, the experimental system, and their interaction indicated the need for more research in this novel area of solid-state 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.     

Scale up studies for the production of biosurfactant in packed column bioreactor

Biosurfactants capable of emulsifying pesticides have great potential to assist in microbial degradation of the pesticides. Solid State Fermentation (SSF) due to several advantages, is one of the efficient ways of producing these surfactants and seldom receives attention for commercial exploitation. In this study, a packed column bioreactor with wheat bran as the raw material and Bacillus subtilis has been used to produce a biosurfactant specific to disperse Fenthion, an organophosphrous pesticide. The emulsifier activity (EA) and surface tension from the packed column bioreactor were compared with flask fermentation experiments, which served as control. Airflow rate in the packed column bioreactor was varied from 10-20 l/min. Maximum EA and minimum surface tension occurred at airflow rate of 20 l/min. Peak EA in the control was 1.2 at 29 h while it was 1.9 in the bioreactor. The least surface tension of 24 dynes/cm was noticed at 54 h in the bioreactor, which was 33% better than the control at the same time period. The results indicate that the packed column bioreactor can become a more acceptable solid state fermentation system for commercial exploitation of Fenthion specific biosurfactant production.     

An inverse correlation between stress resistance and stuck fermentations in wine yeasts. A molecular study.

During alcoholic fermentations yeast cells are subjected to several stress conditions and, therefore, yeasts have developed molecular mechanisms in order to resist this adverse situation. The mechanisms involved in stress response have been studied in Saccharomyces cerevisiae laboratory strains. However a better understanding of these mechanisms in wine yeasts could open the possibility to improve the fermentation process. In this work an analysis of the stress response in three wine yeasts has been carried out by studying the expression of several representative genes under several stress conditions which occur during fermentation. We propose a simplified method to study how these stress conditions affect the viability of yeast cells. Using this approach an inverse correlation between stress-resistance and stuck fermentations has been found. We also have preliminary data about the use of the HSP12 gene as a molecular marker for stress-resistance in wine yeasts.     

Morphological measurements on filamentous microorganisms by fully automatic image analysis

Characterization of mycelial morphology is important for physiological and engineering studies of filamentous fermentations, and in the design and operation of such fermentations. Image analysis has been developed as a method for this characterization, and has been shown to be faster and generally more accurate than previous methods. A fully automatic system has been developed, in which speed is gained, but with loss of accuracy in some cases. The method has been tested on Streptomyces clavuligerus and Penicillium chrysogenum P1 batch fermentations. It has also been tested on a fed-batch Penicillium chrysogenum P2 fermentation, in which the medium contained solid ingredients. Fully automatic image analysis for morphological characterization of filamentous microorganisms is an important development which will make practical many engineering and physiological studies of such fermentations that have so far not been completely satisfactory.     

Solid state fermentation—An overview

Since ancient times many solid state fermentations have utilized fungi and bacteria, almost always in mixed culture. The discovery and development of penicillin led to extensive use of liquid fermentations using actinomycetes and fungi and to subsequent neglect of research on solid state fermentations and the use of mixed cultures. This paper reviews the types of solid state fermentations, equipment used, products made, as well as the advantages and disadvantages of solid state fermentations. Products resulting from old and new solid and liquid substrate fermentations are enumerated.     

Proof-of-concept of a novel micro-bioreactor for fast development of industrial bioprocesses

The experimental performance of a novel micro-bioreactor envisaged for parallel screening and development of industrial bioprocesses has been tested in this work. The micro-bioreactor with an internal volume of 4.5 mL is operated under oscillatory flow mixing (OFM), where a controllable mixing and mass transfer rates are achieved under batch or continuous laminar flow conditions. Several batch fermentations with a flocculent Saccharomyces cerevisiae strain were carried out at initial glucose concentrations (S(0)) range of approximately 5-20 g/L and compared to yeast growth kinetics in a stirred tank (ST) bioreactor. Aerobic fermentations were monitored ex situ in terms of pH, DO, glucose consumption, and biomass and ethanol production (wherever applicable). An average biomass production increase of 83% was obtained in the micro-bioreactor when compared with the ST, with less 93.6% air requirements. It also corresponded to a 214% increase on biomass production when compared with growth in a shaken flask (SF) at S(0) = 20 g/L. Further anaerobic fermentations at the same initial glucose concentration ranges gave the opportunity to use state-of-the-art fiber optics technology for on-line and real-time monitoring of this bioprocess. Time profiles of biomass concentration (measured as optical density (OD)) were very similar in the ST bioreactor and in the micro-bioreactor, with a highly reproducible yeast growth in these two scale-down platforms.     

A two-reservoir, hollow-fiber bioreactor for the study of mixed-population dynamics: design aspects and validation of the approach

A two-reservoir, membrane bioreactor for carrying out studies of mixed-population dynamics in batch fermentations is presented. Mixing requirements and design aspects for the validity of the approach are given and discussed. Equations describing mixing times between the reservoirs are presented and compared to the experimental results. The validity of the approach is demonstrated by the study of an amensalistic-type interaction, the protein-mediated killer phenomenon between two Saccharomyces cerevisiae strains. The validation consisted in the comparison between the results obtained in actual mixed culture and the results obtained by keeping the strains separated. A good agreement was found which demonstrates the viability of the designed bioreactor.     

Rheology of filamentous fermentations

The performance of a bioreactor containing a filamentous fermentation broth is greatly influenced by the rheological properties of the broth. These properties are determined mainly by the concentration of biomass, its growth rate and morphology. Included in the morphology are such factors as the geometry of hyphae (length, diameter, branching frequency), hyphal flexibility and hyphal-hyphal interactions, which can all be affected by the operational design of the reactor. Thus, correlations describing viscosity as a function of biomass only are of limited value. A better understanding of the relations between morphology and rheology may be achieved by a combination of rheological and morphological studies.Rheological properties are normally determined using off-line measurements in-spite of associated problems with sample treatment influencing the results. Equipment for dynamic, on-line, measurement of morphology and rheology is available, but little used in filamentous fermentations. Controlling the rheological properties of mycelial fermentations may be difficult because of the great number of factors influencing mycelial development and/or hyphal-hyphal interactions.Polymer solutions are often used to simulate flow behaviour of filamentous fermentations and scale-up and mass transfer considerations are based on these studies. Although much information has been gained this way, the predictions developed do not include the effect of an active biomass on the mass transfer and flow properties of the culture. It is important to carry out studies on the non-homogeneous fermentation fluids, and develop correlations based on these studies.     

Physiologically Motivated Monitoring of Fermentation Processes by Means of an Electronic Nose

An on‐line approach of non‐invasive monitoring of the physiological changes in fermentation processes is presented. In yeast batch and bacterial fed‐batch fermentations it is shown that metabolic state changes can be revealed using an electronic nose. The transient responses of the gas sensors to the changes in the composition of the volatiles emitted from the cell cultures during fermentation are used to retrieve a semi‐quantitative representation of the physiological state of the cultures. With the sensor responses of the electronic nose it is shown that physiological variables such as rates of growth, substrate uptake and product formation can be depicted. The non‐invasive method thus seems as a pertinent alternative to conventional bioreactor monitoring methods.     

Alcoholic fermentation: modelling based on sole substrate and product measurement

Optimal automatic bioreactor control requires a mathematical model adapted to the potency of reliable sensors. A new relationship describing the kinetic behavior of alcoholic fermentation is discussed. By analogy with chemical kinetics, the biological rate of substrate consumption is related to substrate and product concentration by the following equation: \documentclass{article}\pagestyle{empty}\begin{document}$$r_s = kS;\alpha P;\beta$$\end{document} Using the well known yield relation between product and substrate, it is possible to describe in both batch and continuous cultures the ethanol and sugar concentrations versus time. This pattern has been successfully tested on several fermentations performed by yeasts (S. cerevisiae, S. bayanus, and S. cerevisiae sake) and a bacterium (Z. mobilis). This simple relationship is proposed as a tool for process control alcoholic fermentation.     

Particulate bioprocessing: A novel process strategy for biorefineries

A novel process strategy based on particulate bioprocessing has been developed for the production of value-added chemicals and biofuels. The process, which involves two main steps, fungal fermentation and discontinuous extraction, leads to the production of generic fermentation feedstocks from cereals. Partially pearled whole wheat grains were used as substrate for the growth of Aspergillus awamori in a packed bed bioreactor. Water was trickled through the bed of particles intermittently every 6 h to extract glucose and other nutrients and to maintain moisture and temperature levels. The feedstocks obtained through this system have been used for subsequent fermentations by Wautersia eutropha to produce the biodegradable plastic PHB (polyhydroxybutyrate) and by Saccharomyces cerevisiae for ethanol production. These preliminary results demonstrate the potential suitability of the novel concept of particulate bioprocessing in the development of biorefineries.