Solid-state fermentation: a promising microbial technology for secondary metabolite production

Solid state (substrate) fermentation (SSF) has been used successfully for the production of enzymes and secondary metabolites. These products are associated with the stationary phase of microbial growth and are produced on an industrial scale for use in agriculture and the treatment of disease. Many of these secondary metabolites are still produced by submerged liquid fermentations (SmF) even though production by this method has been shown to be less efficient than SSF. As large-scale production increases further, so do the costs and energy demands. SSF has been shown to produce a more stable product, requiring less energy, in smaller fermenters, with easier downstream processing measures. In this article we review an important area of biotechnology, since the recent evidence indicates that bacteria and fungi, growing under SSF conditions, are more than capable of supplying the growing global demand for secondary metabolites.     

Complex dynamics in a model microbial system

The forced double-Monod model (for a chemostat with a predator, a prey and periodically forced inflowing substrate) displays quasiperiodicity, phase locking, period doubling and chaotic dynamics. Stroboscopic sections reveal circle maps for the quasiperiodic regimes and noninvertible maps of the interval for the chaotic regimes. Criticality in the circle maps sets the stage for chaos in the model. This criticality may arise with an increase in the period or amplitude of forcing.     

Characterization of proton production and consumption associated with microbial metabolism

Background  

Production or consumption of protons in growth medium during microbial metabolism plays an important role in determining the pH of the environment. Such pH changes resulting from microbial metabolism may influence the geochemical speciation of many elements in subsurface environments. Protons produced or consumed during microbial growth were measured by determining the amount of acid or base added in a 5 L batch bioreactor equipped with pH control for different species including Escherichia coli, Geobacter sulfurreducens, and Geobacter metallireducens.     

Scaled-up biotechnological production of individual betalains in a microbial system

The recent interest in plant pigment betalains as bioactive compounds and chemopreventive agents has led to the search for a reliable and scalable process to obtain them. The cloning of the novel and efficient enzyme 4,5-DOPA-extradiol dioxygenase from Gluconacetobacter diazotrophicus in an expression vector, and the subsequent heterologous expression in Escherichia coli cultures has led to the start-up of a biotechnological production system of individual pigments. The aim of this study was to search for the optimal conditions for the production of betalamic acid in microbial factories and the scaled-up obtention of the derived pigments. Four different betaxanthins and two betacyanins were obtained after the addition of non-transformable amines and amino acids and their condensation with the betalamic acid produced by the dioxygenase. The scaled-up obtention and purification of betalains improved the yields of the previous methodologies reaching quantities by up to 150 mg of pure compounds.     

Biodesulfurization: a model system for microbial physiology research

Biological desulfurization (biodesulfurization) of dibenzothiophene (DBT) by the 4S pathway is a model system for an enviromentally benign way to lower the sulfur content of petroleum. Despite a large amount of effort the efficiency of the 4S pathway is still too low for a commercial oil biodesulfurization process, but the 4S pathway could potentially be used now for commercial processes to produce surfactants, antibiotics, polythioesters and other chemicals and for the detoxification of some chemical warfare agents. Proteins containing disulfide bonds are resistant to temperature, pH, and solvents, but the production of disulfide-rich proteins in microbial hosts is challenging. The study of the 4S pathway can provide insights as to how to maximize the production of disulfide-rich proteins. Engineering of the operon encoding the 4S pathway to contain a greater content of methionine and cysteine may be able to link use of DBT as a sole sulfur source to increasing 4S pathway activity by increasing the nutritional demand for sulfur. This strategy could result in the development of biocatalysts suitable for use in an oil biodesulfurization process, but the study of the 4S pathway can also lead to a better understanding of microbial physiology to optimize activity of a mult-step co-factor-requiring pathway, as well as the production of highly stable industrially relevant enzymes for numerous applications.     

Wastewater treatment: a model system for microbial ecology

Biological wastewater treatment is among the most important biotechnological applications and, as drivers of the key processes, microorganisms are central to its success. Therefore, the study of wastewater microorganisms has obvious applied significance; however, the importance of wastewater treatment reactors as model systems for microbial ecology is often overlooked. Modern molecular techniques, including environmental genomics, have identified unexpected microbial key players for nutrient removal and sludge bulking and/or foaming, and provided many exciting insights into the diversity, functions and niche differentiations of these predominantly uncultivated microorganisms. It is now time for wastewater microbiology to be recognized as a mature and dynamic discipline in its own right, offering much toward a deeper understanding of life in complex microbial communities. Here, we consider selected key findings to illustrate the past and future roles of molecular ecophysiology and genomics in the development of wastewater microbiology as an important subdiscipline of microbial ecology.     

Diel and daily fluctuations in virioplankton production in coastal ecosystems

Viruses saturate the world around us, yet a basic understanding of how viral impacts on microbial host organisms vary over days to hours, which typify the replication cycles of aquatic viruses, remains elusive. Thus, diel patterns of viral production (VP) in Chesapeake Bay surface waters were examined on five sampling dates. Day-to-day variations in VP in the Chesapeake and coastal California surface waters were also investigated. Significant variations in VP were detected over 24 h cycles during four of five studies, but rates did not vary significantly over the course of a few days in either location. Diel patterns of VP displayed seasonality with shorter viral assemblage turnover times and shorter times to maximum viral abundance in summer, implying shorter replication cycles for virus–host systems in warmer months. No correlation was found between VP and time of day, likely due to seasonal changes in the diel patterns of VP. This analysis significantly increases our knowledge of the short-term patterning of in situ VP, and thus viral impacts, and suggests that variations in viral biology in response to changes in host communities or physio-chemical properties affect both diel and seasonal cycles and magnitudes of VP.     

Advances in microbial culturing conditions to activate silent biosynthetic gene clusters for novel metabolite production

Journal of Industrial Microbiology & Biotechnology - Natural products (NPs) produced by bacteria and fungi are often used as therapeutic agents due to their complex structures and wide range of...     

Conditions for a continuous production of transient microbial products in a two-stage fermentation system

Summary The optimal residence time in the inducing reactor of a continuous two-stage system has been studied in order to maximize the yield in such a process. The attention has been focused on the case in which the product (or one of its forms) appears in the culture only transiently after the induction. Whereas in some cases the two-stage system is able to improve the yield and to allow a continuous product concentration in the outgoing stream, in others, and depending on how the product is accumulated in the induced culture, no optimal residence time can be found, and a higher productivity will be achieved by using the one-stage continuous strategy or a batch fermentation.     

Measurement of O2 consumption, CO2 production, and water vapor production in a closed system

Equations for the calculation of O2 consumption, CO2 production, and water vapor production in a constant-volume, closed-system respirometer are presented. Necessary measurements include only the initial temperature, pressure, and gas volume in the respirometer chamber, and the fractional concentration of O2 in gas samples taken at the beginning and end of the period of measurement. Potential errors resulting from changes in CO2 and water vapor concentrations are identified. Ignoring CO2 effects can produce up to a 6.4% error in estimates of O2 consumption, and errors due to water vapor effects can exceed 100%. Techniques are presented for minimizing potential errors and for measuring CO2 and water vapor concentrations with an O2 analyzer so that potential errors can be eliminated.     

A model for improving microbial biofuel production using a synthetic feedback loop

Cells use feedback to implement a diverse range of regulatory functions. Building synthetic feedback control systems may yield insight into the roles that feedback can play in regulation since it can be introduced independently of native regulation, and alternative control architectures can be compared. We propose a model for microbial biofuel production where a synthetic control system is used to increase cell viability and biofuel yields. Although microbes can be engineered to produce biofuels, the fuels are often toxic to cell growth, creating a negative feedback loop that limits biofuel production. These toxic effects may be mitigated by expressing efflux pumps that export biofuel from the cell. We developed a model for cell growth and biofuel production and used it to compare several genetic control strategies for their ability to improve biofuel yields. We show that controlling efflux pump expression directly with a biofuel-responsive promoter is a straightforward way of improving biofuel production. In addition, a feed forward loop controller is shown to be versatile at dealing with uncertainty in biofuel production rates.     

Diel and density-related changes in food consumption and prey selection by brook charr in a New Hampshire stream

Synopsis We report the results of a field study testing influences of both density and changes over the diel cycle on food consumption and prey selection by brook charr,Salvelinus fontinalis. Charr density in replicate 35 m long sections of a New Hampshire stream was adjusted to either medium or high levels (relative to natural densities). Diets of charr and the availability of drifting prey were then sampled every four hours for 24 hours. There were no significant diel changes in the weight of prey consumed by charr per four hours, though there was some indication of reduced feeding at night. Chary fed selectively on different prey taxa, showing most preference for cased caddis larvae. Several species of mayflies and stoneflies were selected more strongly during the day than at night. Charr fed selectively on larger prey during the daytime but showed no size-selection at night. The density of charr had no significant effects on either their rate of food consumption or on selection for prey of different taxa or sizes.     

Satellite remote sensing data can be used to model marine microbial metabolite turnover

Sampling ecosystems, even at a local scale, at the temporal and spatial resolution necessary to capture natural variability in microbial communities are prohibitively expensive. We extrapolated marine surface microbial community structure and metabolic potential from 72 16S rRNA amplicon and 8 metagenomic observations using remotely sensed environmental parameters to create a system-scale model of marine microbial metabolism for 5904 grid cells (49 km²) in the Western English Chanel, across 3 years of weekly averages. Thirteen environmental variables predicted the relative abundance of 24 bacterial Orders and 1715 unique enzyme-encoding genes that encode turnover of 2893 metabolites. The genes'' predicted relative abundance was highly correlated (Pearson Correlation 0.72, P-value <10⁻⁶) with their observed relative abundance in sequenced metagenomes. Predictions of the relative turnover (synthesis or consumption) of CO₂ were significantly correlated with observed surface CO₂ fugacity. The spatial and temporal variation in the predicted relative abundances of genes coding for cyanase, carbon monoxide and malate dehydrogenase were investigated along with the predicted inter-annual variation in relative consumption or production of ∼3000 metabolites forming six significant temporal clusters. These spatiotemporal distributions could possibly be explained by the co-occurrence of anaerobic and aerobic metabolisms associated with localized plankton blooms or sediment resuspension, which facilitate the presence of anaerobic micro-niches. This predictive model provides a general framework for focusing future sampling and experimental design to relate biogeochemical turnover to microbial ecology.     

Mycelial morphology and metabolite production

Mycelial microorganisms are exploited extensively in the commercial production of a wide range of secondary metabolites. They can be cultured as free mycelia, as aggregated forms (pellets/flocs), or as artificially bound/entrapped cells, though problems are associated with the culture of each morphological type. Since the morphological type can strongly influence metabolite production, the methodology for inducing pellet formation, and the type of pellets produced are an important consideration for effective metabolite production.     

A model that links growth and secondary metabolite production in plant cell suspension cultures

Plant cell suspensions of grape cells (Vitis vinifera L. cv. Gamay Fréaux) were grown in shake flasks operated both in the batch and semicontinuous mode. A mathematical model was developed to describe grape cell growth, sucrose uptake, and secondary metabolite (anthocyanin) production. Parameters were estimated from batch studies data. The model was able to predict results for semicontinuous experiments by only modifying the value of four of these parameters. The modified parameters (maximum specific rate of biomass production, maximum specific rate of substrate consumption for maintenance, maximum specific rate of anthocyanin production, and degradation constant of anthocyanins) were related to the kinetics rather than to the yield of the process. The model introduces the concept of primary and secondary metabolism substrate concentration-dependent competition for precursors. Further, the model was able to predict the evolution of the cell system when substrate is scarce, as the value of the different kinetic constants determines the portion of substrate that is used for biomass production, secondary metabolite production, and cell maintenance. (c) 1995 John Wiley & Sons, Inc.     

Diel variation in surface and subsurface microbial activity along a gradient of drying in an Australian sand-bed stream

1. Microbes play key roles in nutrient transformation and organic matter mineralisation in the hyporheic zone but their short‐term responses to diel variations in discharge and temperature are unknown. Rates of microbial esterase activity were hypothesised to vary vertically and along a gradient of moisture in a drying sand‐bed stream where discharge fluctuated daily in response to evapotranspiration. 2. At ‘fully saturated’, ‘moist’ and ‘dry’ locations in three sites along a drying Australian sand‐bed stream, microbial activity at three depths (surface, 10 and 30 cm) was assessed using fluorescein diacetate hydrolysis. Samples were collected in mid‐summer in the late afternoon and again at dawn to assess diel variation in hydrolytic activity at each site and depth. Data loggers tracked diel variations in temperature at each depth. 3. Hydrolytic activity was up to 10‐fold greater in the surface sediments in late afternoon than at dawn in all habitats, and was correlated with surface sediment temperature. Diel differences in activity were not detected at 10 cm, although daily thermal cycles were evident at this depth. Unexpectedly, activity was marginally higher at dawn at 30 cm in all habitats, perhaps reflecting lags in temperature at that depth. 4. Overall, microbial activity declined with depth, strongly correlated with vertical trends in total organic matter and concentrations of dissolved phosphorus. Particulate organic matter, probably buried during a flood 35 days earlier, appeared largely responsible for these vertical trends. On the other hand, there was little evidence for hydrological exchange between much of the hyporheic zone and the surface stream, implying that processes in the subsurface zone of this stream are effectively isolated during baseflow in mid‐summer. 5. Diel cycles of wetting and drying in the moist habitats did not enhance esterase activity relative to the dry or fully saturated habitats. Sediment moisture was not correlated with microbial activity, and mats of senescent algae appeared to inhibit water loss from surface sediments in the moist habitat. In this sand‐bed stream, local diel fluctuations in water level appear to have less influence on microbial activity and mineralisation of organic matter in the sediments than occasional floods that bury leaf litter and renew many hyporheic zone functions. Subreach‐scale processes seem to be the major driving force of microbial processes and nutrient cycling in this sand‐bed river.     

Diel dualism in the energy consumption of the European catfish Silurus glanis

Twenty individuals of the largest European freshwater predator, the European catfish Silurus glanis, were tagged with electromyogram (EMG) physiological telemetry sensors. The fish were observed during diel cycles during the spring and summer in the Elbe and Berounka Rivers, Czech Republic. The purpose of this study was to determine whether diel dualism in the activity of S. glanis occurs naturally or is induced by the laboratory environment and by the conditions occurring in aquaculture. The results obtained from the riverine environment tended to show dualism in the use of the light and dark phases of the day because 35% of the individuals varied from a site‐specific common diel activity pattern. The EMG values increased in accordance with the mass (M) of the fish. To eliminate the influence of M on individual energy consumption, the EMG records were analysed in terms of the EMG:M ratios. High individual variability was found in these ratios. The diel activity pattern of the individuals with relatively high energy consumption differed from the common diel activity pattern. In contrast, the fish that adopted the common diel activity pattern displayed relatively low energy consumption. The results of this study indicated that dualism and energy consumption are related. The EMG values also varied with the values of the environmental variables. Increasing temperature was associated with high EMG values, whereas the EMG values decreased with increasing flow.