Optimizing carbon dioxide utilization for microalgae biofilm cultivation.

The loss of carbon dioxide (CO2 ) to the environment during microalgae cultivation is undesirable for both environmental and economic reasons. In this study, a phototrophic biofilm growth model was developed and validated with the objective to maximize both CO2 utilization efficiency and production of microalgae in biofilms. The model was validated in growth experiments with CO2 as the limiting substrate. The CO2 utilization and biomass productivity were maximized by changing the gas flow rate, the number of biofilm reactors in series and gas composition. Based on simulations, the maximum CO2 utilization efficiency that was reached was 96% based on a process employing flue gas. The corresponding drop in productivity was only 2% in comparison to the non-CO2 limited reference situation. In order to achieve this, 25 biofilm reactors units, or more, must be operated in series. Based on these results, it was concluded that concentrated CO2 streams and plug flow behavior of the gaseous phase over the biofilm surface are essential for high productivity and CO2 utilization efficiency. Biotechnol. Bioeng. 2017;114: 769-776. © 2016 Wiley Periodicals, Inc.

Deceleration-stats save much time during phototrophic culture optimization.

In case of phototrophic cultures, photobioreactor costs contribute significantly to the total operating costs. Therefore one of the most important parameters to be determined is the maximum biomass production rate, if biomass or a biomass associated product is the desired product. This is traditionally determined in time consuming series of chemostat cultivations. The goal of this work is to assess the experimental time that can be saved by applying the deceleration stat (D-stat) technique to assess the maximum biomass production rate of a phototrophic cultivation system, instead of a series of chemostat cultures. A mathematical model developed by Geider and co-workers was adapted in order to describe the rate of photosynthesis as a function of the local light intensity. This is essential for the accurate description of biomass productivity in phototrophic cultures. The presented simulations demonstrate that D-stat experiments executed in the absence of pseudo steady-state (i.e., the arbitrary situation that the observed specific growth rate deviates <5% from the dilution rate) can still be used to accurately determine the maximum biomass productivity of the system. Moreover, this approach saves up to 94% of the time required to perform a series of chemostat experiments that has the same accuracy. In case more information on the properties of the system is required, the reduction in experimental time is reduced but still significant.

Identifying Variables Influencing Traditional Food Solid-State Fermentation by Statistical Modeling.

Solid-state fermentation is widely used in traditional food production, but most of the complex processes involved were designed and are carried out without a scientific basis. Often, mathematical models can be established to describe mass and heat transfer with the assistance of chemical engineering tools. However, due to the complex nature of solid-state fermentation, mathematical models alone cannot explain the many dynamic changes that occur during these processes. For example, it is hard to identify the most important variables influencing product yield and quality fluctuations. Here, using solid-state fermentation of Chinese liquor as a case study, we established statistical models to correlate the final liquor yield with available industrial data, including the starting content of starch, water and acid; starting temperature; and substrate temperature profiles throughout the process. Models based on starting concentrations and temperature profiles gave unsatisfactory yield predictions. Although the most obvious factor is the starting month, ambient temperature is unlikely to be the direct driver of differences. A lactic-acid-inhibition model indicates that lactic acid from lactic acid bacteria is likely the reason for the reduction in yield between April and December. Further integrated study strategies are necessary to confirm the most crucial variables from both microbiological and engineering perspectives. Our findings can facilitate better understanding and improvement of complex solid-state fermentations.

Modeling growth, lipid accumulation and lipid turnover in submerged batch cultures of Umbelopsis isabellina.

The production of lipids by oleaginous yeast and fungi becomes more important because these lipids can be used for biodiesel production. To understand the process of lipid production better, we developed a model for growth, lipid production and lipid turnover in submerged batch fermentation. This model describes three subsequent phases: exponential growth when both a C-source and an N-source are available, carbohydrate and lipid production when the N-source is exhausted and turnover of accumulated lipids when the C-source is exhausted. The model was validated with submerged batch cultures of the fungus Umbelopsis isabellina (formerly known as Mortierella isabellina) with two different initial C/N-ratios. Comparison with chemostat cultures with the same strain showed a significant difference in lipid production: in batch cultures, the initial specific lipid production rate was almost four times higher than in chemostat cultures but it decreased exponentially in time, while the maximum specific lipid production rate in chemostat cultures was independent of residence time. This indicates that different mechanisms for lipid production are active in batch and chemostat cultures. The model could also describe data for submerged batch cultures from literature well.

Modeling lipid accumulation in oleaginous fungi in chemostat cultures. II: Validation of the chemostat model using yeast culture data from literature.

A model that predicts cell growth, lipid accumulation and substrate consumption of oleaginous fungi in chemostat cultures (Meeuwse et al. in Bioproc Biosyst Eng. doi: 10.1007/s00449-011-0545-8 , 2011) was validated using 12 published data sets for chemostat cultures of oleaginous yeasts and one published data set for a poly-hydroxyalkanoate accumulating bacterial species. The model could describe all data sets well with only minor modifications that do not affect the key assumptions, i.e. (1) oleaginous yeasts and fungi give the highest priority to C-source utilization for maintenance, second priority to growth and third priority to lipid accumulation, and (2) oleaginous yeasts and fungi have a growth rate independent maximum specific lipid production rate. The analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al. (in Biotechnol Bioeng 34:1268-1276, 1989), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate.

Modeling lipid accumulation in oleaginous fungi in chemostat cultures: I. Development and validation of a chemostat model for Umbelopsis isabellina.

Lipid-accumulating fungi may be able to produce biodiesel precursors from agricultural wastes. As a first step in understanding and evaluating their potential, a mathematical model was developed to describe growth, lipid accumulation and substrate consumption of the oleaginous fungus Umbelopsis isabellina (also known as Mortierella isabellina) in submerged chemostat cultures. Key points of the model are: (1) if the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. Model parameters were obtained from chemostat cultures of U. isabellina grown on mineral media with glucose and NH(4) (+). The model describes the results of chemostat cultures well for D > 0.04 h(-1), but it has not been validated for lower dilution rates because of practical problems with the filamentous fungus. Further validation using literature data for oleaginous yeasts is described in part II of this paper. Our model shows that not only the C/N-ratio of the feed, but also the dilution rate highly influences the lipid yield in chemostat cultures.

Water dynamics during solid-state fermentation by Aspergillus oryzae YH6.

Water is crucial for microbial growth, heat transfer and substrate hydrolysis, and dynamically changes with time in solid-state fermentation. However, water dynamics in the solid substrate is difficult to define and measure. Here, nuclear magnetic resonance was used to monitor water dynamics during the pure culture of Aspergillus oryzae YH6 on wheat in a model system to mimic solid starter (Qu or Koji) preparation. During fermentation, overall water content gradually decreased from 0.84 to 0.36 g/g, and water activity decreased from 0.99 to 0.93. Water content in different state (bound, immobilized and free) changed differently and all moved to more "bound" direction. The internal water distribution over the substrate matrix also showed a faster reduction inward both in the radical and axial direction. Our findings provide the prerequisites for optimal processes where water dynamics in solid-state fermentation can be monitored and controlled.

Microbiology of synthesis gas fermentation for biofuel production.

A significant portion of biomass sources like straw and wood is poorly degradable and cannot be converted to biofuels by microorganisms. The gasification of this waste material to produce synthesis gas (or syngas) could offer a solution to this problem, as microorganisms that convert CO and H2) (the essential components of syngas) to multicarbon compounds are available. These are predominantly mesophilic microorganisms that produce short-chain fatty acids and alcohols from CO and H2. Additionally, hydrogen can be produced by carboxydotrophic hydrogenogenic bacteria that convert CO and H2O to H2 and CO2. The production of ethanol through syngas fermentation is already available as a commercial process. The use of thermophilic microorganisms for these processes could offer some advantages; however, to date, few thermophiles are known that grow well on syngas and produce organic compounds. The identification of new isolates that would broaden the product range of syngas fermentations is desirable. Metabolic engineering could be employed to broaden the variety of available products, although genetic tools for such engineering are currently unavailable. Nevertheless, syngas fermenting microorganisms possess advantageous characteristics for biofuel production and hold potential for future engineering efforts.

Modeling conversion and transport phenomena in solid-state fermentation: a review and perspectives.

Solid-state fermentation (SSF) is accompanied inevitably by development of concentration and temperature gradients within the substrate particles and microbial biofilms. These gradients are needed for driving the transport of substrates and products. In addition, concentration gradients have been suggested to be crucial for obtaining the characteristics that define the products of SSF; nevertheless, gradients are also known to result in reduced productivity and unwanted side reactions. Solid-state fermentations are generally batch processes and this further complicates their understanding as conditions change with time. Mathematical models are therefore needed for improving the understanding of SSF processes and allowing their manipulation to achieve the desired outcomes. Existing models of SSF processes describe coupled substrate conversion and diffusion and the consequent microbial growth. Existing models disregard many of the significant phenomena that are known to influence SSF. As a result, available models cannot explain the generation of the numerous products that form during any SSF process and the outcome of the process in terms of the characteristics of the final product. This review critically evaluates the proposed models and their experimental validation. In addition, important issues that need to be resolved for improved modeling of SSF are discussed.

Effect of low oxygen concentrations on growth and alpha-amylase production of Aspergillus oryzae in model solid-state fermentation systems.

Oxygen transfer in the fungal mat is a major concern in solid-state fermentation (SSF). Oxygen supply into the mycelial layers is hampered by diffusion limitation. For aerobic fungi, like Aspergillus oryzae, this oxygen depletion can be a severely limiting factor for growth and metabolite production. This paper describes the effects of a low oxygen concentration on growth at the levels of individual hyphae, colonies and overcultures, and on alpha-amylase production in overcultures. PDA medium was used to study the effect of a low oxygen concentration on hyphal elongation rate and branching frequency of hyphae, and radial extension rate of colonies of A. oryzae. We found similar saturation constants (K(O2)) of 0.1% (v/v in the gas phase) for oxygen concentration described with Monod kinetics, for branching frequency of hyphae and colony extension rate. When A. oryzae was grown as an over-culture on wheat-flour model substrate at 0.25% (v/v) oxygen concentration, the reduction in growth was more pronounced than as individual hyphae and a colony on PDA medium. Experimental results also showed that the specific alpha-amylase production rate under the condition of 0.25% (v/v) oxygen was reduced. Because the value of K(O2) is relatively low, it is reasonable to simplify the kinetics of growth of A. oryzae to zero-order kinetics in coupled diffusion/reaction models.

Significance of bed porosity, bran and specific surface area in solid-state cultivation of Aspergillus oryzae.

In this paper, the effects of bed porosity, bran and specific surface area on the oxygen uptake rate and alpha-amylase production during growth of Aspergillus oryzae on wheat grain and wheat-flour substrate are reported. The high oxygen uptake rate found during cultivation of A. oryzae on wheat-flour substrate was not reached on wheat grain. This is mainly due to the bran of the wheat grain. Using wheat-flour substrates, it was shown that extra bed porosity increased the alpha-amylase production and oxygen uptake rates. Furthermore, the peak oxygen uptake rate decreased with increasing surface area-volume ratio of the substrate particles, while the alpha-amylase production and the cumulative oxygen uptake per gram of initial substrate dry matter increased. The present work does not support a direct correlation between aerial mycelia and enzyme production. There is, however, a correlation between the alpha-amylase yield and the cumulative oxygen uptake (not the uptake rate). This implies that aerial mycelia could accelerate alpha-amylase production even if they do not increase the yield.

A model for customising biomass composition in continuous microalgae production.

A kinetic model is presented that describes functional biomass, starch and storage lipid (TAG) synthesis in the microalga Neochloris oleoabundans as a function of nitrogen and light supply rates to a nitrogen-limited turbidostat cultivation system. The model is based on the measured electron distribution in N. oleoabundans, which showed that starch is the primary storage component, whereas TAG was only produced after an excess of electrons was generated, when growth was limited by nitrogen supply. A fixed 8.6% of the excess electrons ended up in TAG, suggesting close metabolic interactions between nitrogen assimilation and TAG accumulation, such as a shared electron pool. The proposed model shows that by manipulating the cultivation conditions in a light or nitrogen limited turbidostat, algal biomass composition can be customised and the volumetric productivities and yields of the major biomass constituents can be changed on demand.

Polyol accumulation by Aspergillus oryzae at low water activity in solid-state fermentation.

Polyol accumulation and metabolism were examined in Aspergillus oryzae cultured on whole wheat grains or on wheat dough as a model for solid-state culture. In solid-state fermentation (SSF), water activity (a(w)) is typically low resulting in osmotic stress. In addition to a high level of mannitol, which is always present in the cells, A. oryzae accumulated high concentrations of glycerol, erythritol and arabitol at relatively low a(w) (0.96-0.97) in SSF. Accumulation of such a mixture of polyols is rather unusual and might be typical for SSF. A. oryzae mycelium accumulating various polyols at low a(w) contained at least four distinct polyol dehydrogenases with highest activities toward glycerol, erythritol, D-arabitol and mannitol. NADP(+)-dependent glycerol dehydrogenase activity correlated very well with glycerol accumulation. A similar correlation was observed for erythritol and NADP(+)-erythritol dehydrogenase suggesting that NADP(+)-dependent glycerol and erythritol dehydrogenases are involved in biosynthesis of glycerol and erythritol, respectively, and that these enzymes are induced by osmotic stress.

Validation of a model for process development and scale-up of packed-bed solid-state bioreactors.

We have validated our previously described model for scale-up of packed-bed solid-state fermenters (Weber et al., 1999) with experiments in an adiabatic 15-dm(3) packed-bed reactor, using the fungi Coniothyrium minitans and Aspergillus oryzae. Effects of temperature on respiration, growth, and sporulation of the biocontrol fungus C. minitans on hemp impregnated with a liquid medium were determined in independent experiments, and the first two effects were translated into a kinetic model, which was incorporated in the material and energy balances of the packed-bed model. Predicted temperatures corresponded well with experimental results. As predicted, large amounts of water were lost due to evaporative cooling. With hemp as support no shrinkage was observed, and temperatures could be adequately controlled, both with C. minitans and A. oryzae. In experiments with grains, strong shrinkage of the grains was expected and observed. Nevertheless, cultivation of C. minitans on oats succeeded because this fungus did not form a tight hyphal network between the grains. However, cultivation of A. oryzae failed because shrinkage combined with the strong hyphal network formed by this fungus resulted in channeling, local overheating of the bed, and very inhomogeneous growth of the fungus. For cultivation of C. minitans on oats and for cultivation of A. oryzae on wheat and hemp, no kinetic models were available. Nevertheless, the enthalpy and water balances gave accurate temperature predictions when online measurements of oxygen consumption were used as input. The current model can be improved by incorporation of (1) gas-solids water and heat transfer kinetics to account for deviations from equilibrium observed with fast-growing fungi such as A. oryzae, and (2) the dynamic response of the fungus to changes in temperature, which were neglected in the isothermal kinetic experiments.

Water and glucose gradients in the substrate measured with NMR imaging during solid-state fermentation with Aspergillus oryzae.

Gradients inside substrate particles cannot be prevented in solid-state fermentation. These gradients can have a strong effect on the physiology of the microorganisms but have hitherto received little attention in experimental studies. We report gradients in moisture and glucose content during cultivation of Aspergillus oryzae on membrane-covered wheat-dough slices that were calculated from (1)H-NMR images. We found that moisture gradients in the solid substrate remain small when evaporation is minimized. This is corroborated by predictions of a diffusion model. In contrast, strong glucose gradients developed. Glucose concentrations just below the fungal mat remained low due to high glucose uptake rates, but deeper in the matrix glucose accumulated to very high levels. Integration of the glucose profile gave an average concentration close to the measured average content. On the basis of published data, we expect that the glucose levels in the matrix cause a strong decrease in water activity. The results demonstrate that NMR can play an important role in quantitative analysis of water and glucose gradients at the particle level during solid-state fermentation, which is needed to improve our understanding of the response of fungi to this nonconventional fermentation environment.

Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system.

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.

Aspergillus oryzae in solid-state and submerged fermentations. Progress report on a multi-disciplinary project.

We report the progress of a multi-disciplinary research project on solid-state fermentation (SSF) of the filamentous fungus Aspergillus oryzae. The molecular and physiological aspects of the fungus in submerged fermentation (SmF) and SSF are compared and we observe a number of differences correlated with the different growth conditions. First, the aerial hyphae which occur only in SSFs are mainly responsible for oxygen uptake. Second, SSF is characterised by gradients in temperature, water activity and nutrient concentration, and inside the hyphae different polyols are accumulating. Third, pelleted growth in SmF and mycelial growth in SSF show different gene expression and protein secretion patterns. With this approach we aim to expand our knowledge of mechanisms of fungal growth on solid substrates and to exploit the biotechnological applications.