Effect of impeller type on cellular morphology and production of clavulanic acid by Streptomyces clavuligerus.

It is essential to evaluate the effects of operating conditions in submerged cultures of filamentous microorganisms. In particular, the impeller type influences the flow pattern, power consumption, and energy dissipation, leading to differences in the hydrodynamic environment that affect the morphology of the microorganism. This work investigated the effect of different impeller types, namely the Rushton turbine (RT-RT) and Elephant Ear impellers in up-pumping (EEUP) and down-pumping (EEDP) modes, on cellular morphology and clavulanic acid (CA) production by Streptomyces clavuligerus in a stirred-tank bioreactor. At 800 rpm and 0.5 vvm, the cultivations performed using RT-RT and EEUP impellers provided higher shear conditions and oxygen transfer rates than those observed with EEDP. These conditions resulted in higher clavulanic acid production using RT-RT (380.7 mg/L) and EEUP (453.3 mg/L) impellers, compared to EEDP (196.6 mg/L). Although the maximum CA concentration exhibited the same order of magnitude for RT-RT and EEUP impellers, the latter presented 40% of the specific power consumption (4.9 kW/m3) compared to the classical RT-RT (12.0 kW/m3). The specific energy for CA production ( E CA ), defined as the energy cost to produce 1 mg of CA, was 3.5 times lower using the EEUP impeller (1.91 kJ/mgCA) when compared to RT-RT (5.91 kJ/mgCA). Besides, the specific energy for O2 transfer ( E O 2 ), the energy required to transfer 1 mmol of O2, was 2.3 times lower comparing the EEUP impeller (3.28 kJ/mmolO2) to RT-RT (7.65 kJ/mmolO2). The results demonstrated the importance of choosing the most suitable impeller configuration in conventional bioreactors to manufacture bioproducts.

An innovative multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells.

The use of multi-enzymatic systems for the industrial production of chemical compounds is currently considered an important green tool in synthetic organic chemistry. Gluconic acid is a multi-functional organic acid widely used in the chemical, pharmaceutical, food, textile, and construction industries. Its industrial production from glucose by fermentation using Aspergillus niger has drawbacks including high costs related to cell growth and maintenance of cell viability. This study presents an innovative one-step multi-enzymatic system for gluconic acid production from starch using Aspergillus niger whole-cells in association with amylolytic enzymes. Using soluble starch as substrate, the following results were achieved for 96 h of reaction: 134.5 ± 4.3 g/L gluconic acid concentration, 98.2 ± 1.3 % gluconic acid yield, and 44.8 ± 1.4 gGA/gwhole-cells biocatalyst yield. Although the process has been developed using starch as raw material, the approach is feasible for any substrate or residue that can be hydrolyzed to glucose.

Red biocolorant from endophytic Talaromyces minnesotensis: production, properties, and potential applications.

Fungal colorants are gradually entering the global color market, given their advantages of being less harmful to human health, as well as having greater stability and biotechnological potential, compared to other natural sources. The present work concerns the isolation and identification of an endophytic filamentous fungus, together with the chemical characterization and assessment of the fluorescence, toxicity, stability, and application potential of its synthesized red colorant. The endophytic fungus was isolated from Hymenaea courbaril, a tree from the Brazilian savannah, and was identified as Talaromyces minnesotensis by phenotypic and genotypic characterization. Submerged cultivation of the fungus resulted in the production of approximately 12 AU500 of a red biocolorant which according to LC-DAD-MS analysis is characterized by being a complex mixture of molecules of the azaphilone class. Regarding cytotoxicity assays, activity against human hepatoblastoma (HepG2) cells was only observed at concentrations above 5.0 g L-1, while antimicrobial effects against pathogenic bacteria and yeast occurred at concentrations above 50.0 g L-1. The biocolorant showed high stability at neutral pH values and low temperatures (10 to 20 °C) and high half-life values (t1/2), which indicates potential versatility for application in different matrices, as observed in tests using detergent, gelatin, enamel, paint, and fabrics. The results demonstrated that the biocolorant synthesized by Talaromyces minnesotensis has potential for future biotechnological applications. KEY POINTS: • An endophytic fungus, which was isolated and identified, synthesize a red colorant. • The colorant showed fluorescence property, low toxicity, and application potential. • The red biocolorant was highly stable at pH 8.0 and temperatures below 20°C.

Exploring the roles of starch for microbial encapsulation through a systematic mapping review.

Microorganism encapsulation protects them from stressful conditions and assists in maintaining their viability, being especially beneficial when the carrier material is a renewable and biodegradable biopolymer, such as starch. Here, a systematic mapping was performed to provide a current overview on the use of starch-based systems for microbial encapsulation. Following well-established guidelines, a systematic mapping was conducted and the following could be drawn: 1) there was a significant increase in publications on microbial encapsulation using starch over the past decade, showing interest from the scientific community, 2) ionotropic gelation, emulsification and spray drying are the most commonly used techniques for starch-based microbial encapsulation, and 3) starch play important functions in the encapsulation matrix such as assisting in the survival of the microorganisms. The information gathered in this systematic mapping can be useful to guide researchers and industrial sectors on the development of innovative starch-based systems for microbial encapsulation.

Mathematical Modeling of Fed-Batch Ethanol Fermentation Under Very High Gravity and High Cell Density at Different Temperatures.

The use of more appropriate kinetic models can assist in improving ethanol fermentation under conditions of very high gravity (VHG) and high cell density (HCD), in order to obtain higher amounts of ethanol in the broth combined with high productivity. The aim of this study was to model fed-batch ethanol fermentation under VHG/HCD conditions, at different temperatures, considering three types of inhibition (substrate, ethanol, and cells). Fermentations were carried out using different temperatures (28 ≤ [Formula: see text] (°C) ≤ 34), inoculum sizes (50 ≤ [Formula: see text] (g L-1) ≤ 125), and substrate concentrations in the must (258 ≤ [Formula: see text] (g L-1) ≤ 436). In the proposed model, the cell inhibition power parameter varied with the temperature and inoculum size, while the cell yield coefficient varied with inoculum size and substrate concentration in the must. Hence, it was possible to propose correlations for the cell inhibition power parameter ([Formula: see text]) and for the cell yield coefficient ([Formula: see text]), as functions of the fermentation conditions. Simulations of fed-batch ethanol fermentations at different temperatures, under VHG/HCD conditions, were performed using the proposed correlations. Experimental validation showed that the model was able to accurately predict the dynamic behavior of the fermentations in terms of the concentrations of viable cells, total cells, ethanol, and substrate.

Ethanol yield calculations in biorefineries.

The ethanol yield on sugar during alcoholic fermentation allows for diverse interpretation in academia and industry. There are several different ways to calculate this parameter, which is the most important one in this industrial bioprocess and the one that should be maximized, as reported by Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91). On the one hand, the various methods currently employed in industry provide dissimilar results, and recent evidence shows that yield has been consistently overestimated in Brazilian sugarcane biorefineries. On the other hand, in academia, researchers often lack information on all the intricate aspects involved in calculating the ethanol yield in industry. Here, we comment on these two aspects, using fuel ethanol production from sugarcane in Brazilian biorefineries as an example, and taking the work of Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91.) as a starting point. Our work is an attempt to demystify some common beliefs and to foster closer interaction between academic and industrial professionals from the fermentation sector. Pereira, Rodrigues, Sonego, Cruz and Badino (A new methodology to calculate the ethanol fermentation efficiency at bench and industrial scales. Ind Eng Chem Res 2018; 57: 16182-91).

Cellulolytic enzymes production guided by morphology engineering.

Endoglucanase and xylanase are critical enzymes for liquefaction and enzyme hydrolysis of high solids lignocellulosic biomass to facilitate its transport and production of desired derived products. Here is reported how combinations of different spore concentrations and pH influence microbial morphology, and how this may be used to direct expression and secretion of enzymes by Aspergillus niger. While xylanase production is not affected by A. niger morphology changes, endoglucanase production is enhanced under conditions of lower stress and by morphology that results in pellets. ß-glucosidase production is enhanced under dispersed morphology, which results in up to fourfold increase of this enzyme production under the tested experimental conditions. A morphologic scale (Y) is proposed based on a form factor that considers the size and frequency of each morphology class, and that points to conditions that result in high selectivity for either endoglucanase or ß-glucosidase production. An equation proposed to relate enzyme activity to morphology provides a useful tool for tuning enzyme production of A. niger, where morphology is a first indication of relative enzyme activities in a fermentation broth.

Relation between pellet fragmentation kinetics and cellulolytic enzymes production by Aspergillus niger in conventional bioreactor with different impellers.

The hydrodynamic environment in bioreactors affects the oxygen transfer rate and the shear conditions during microbial cultivations. Therefore, assessment of the effect of the hydrodynamic environment on cellular morphology can contribute to favoring the production of metabolites of interest. The aim of this work was to use image analysis in order to quantify the fragmentation of Aspergillus niger pellets in a conventional bioreactor operated using different impeller speeds, air flow rates, and impeller configurations including Rushton turbines and Elephant Ear impellers, with evaluation of the influence of the hydrodynamic environment on the production of cellulolytic enzymes. An empirical kinetic model was proposed to describe the dynamics of pellet fragmentation and quantify the shear conditions. The results showed that the agitation speed affected the dynamics of pellet fragmentation in two ways, by accelerating the damage process and by increasing the magnitude of the fragmentation. Both endoglucanase and ß-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, ß-glucosidase production was favored under high shear conditions, while the highest endoglucanase production occurred under low shear conditions. These findings may be useful for defining suitable systems and operating conditions for the production of metabolites including enzymes in bioreactors, as well as defining conditions that favour a specific pre-determined enzyme cocktail.

Improvement of ethanol production by extractive fed-batch fermentation in a drop column bioreactor.

The use of fed-batch extractive fermentation can overcome inhibitory effects caused by the substrate and ethanol to the yeast cells, since it allows regulate the substrate concentration and remove the product as it is produced. The present study describes the modelling and experimental validation of ethanol production in fed-batch extractive fermentation with in situ ethanol removal by oleic acid in a non-conventional drop column bioreactor (DCB) operated under industrial conditions. The model developed using the hybrid Andrews-Levenspiel equation and ethanol distribution coefficient parameter (KDE) provided an excellent description of the fed-batch extractive ethanol fermentation process with oleic acid. Furthermore, extractive fed-batch fermentation allowed the feed up to 306.6 kg m-3 of substrate (total reducing sugars), with total ethanol concentration in extractive fermentation in the ranging 100.3-139.8 kg m-3 (12.7-17.7 ºGL), 19.9-67.2% higher when compared with the conventional process without ethanol removal. Moreover, this process has the advantage of less effluent generated and energy consumption for ethanol recovery when compared to the conventional process.

Anthraquinone encapsulation into polymeric nanocapsules as a new drug from biotechnological origin designed for photodynamic therapy.

Photodynamic therapy has been applied for the treatment of many diseases, especially skin diseases. However, poor aqueous solubility and toxicity of some photosensitizer drugs are the main disadvantages for their direct clinical applications. Thus, biotechnology and nanotechnology are important tools in the development of new ways of obtaining photoactive compounds that are biocompatible. We investigated the potential of a new nanostructured photosensitizer, an anthraquinone derivative produced by biotechnological process; then we associated nanotechnology to obtain a nanostructured anthraquinone active molecule. For this, it was prepared a classical nanocapsule formulations containing poly(lactide-co-glycolide) (PLGA) coating for encapsulation of anthraquinone derivative. These formulations were characterized by their physicochemical, morphological, photophysical properties, and stability. We performed in vitro biocompatibility and photodynamic activity assays of free and nanostructured anthraquinone. Nanocapsule formulations containing anthraquinone derivative showed a nanometric profile with particle size around 250 nm, negative zeta potential around -30 mV, and partially monodisperse. Besides that, characteristic spherical morphology of nanocapsules and homogeneous particle surface were observed by AFM analyses. The in vitro biocompatibility assay showed absence of cytotoxicity for all tested RD/NC concentrations and also for unloaded/NC in NIH3T3 cells. In vitro photoactivation assay using NIH3T3 cells showed that nanocapsules promoted greater drug uptake by NIH3T3 cells, around of 87%, of cell death compared to free drug showed around 48% of cell death. The anthraquinone derivative showed potential for use in PDT. Besides the association with nanocapsules improved cell uptake of photosensitizer resulting in increased cell death compared to free anthraquinone.

Average shear rate in airlift bioreactors: searching for the true value.

The shear rate is an important bioreactor parameter that needs to be evaluated due to its impact on microorganism morphology and viability, and consequently on bioproduct formation. Airlift bioreactors, classified as low-shear devices, are used as an alternative to conventional stirred-tank reactors. Considerable efforts have been made to characterize the shear environments in airlift bioreactors, using the average shear rate ([Formula: see text]) as a key parameter. However, there is no agreement among the values obtained in different studies, which can differ even in orders of magnitude. The methodologies used to obtain [Formula: see text] in the different studies could be the reason for the lack of agreement among them. In this work, [Formula: see text] in a concentric tube airlift bioreactor was evaluated using computational fluid dynamics (CFD), as well as based on universal velocity profiles for liquid flows in smooth pipes and annuli. Good agreement was obtained between the CFD-based average shear rates and the values obtained from universal velocity profiles, indicating that CFD simulation is a valuable tool for [Formula: see text] prediction.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus

ABSTRACT Clavulanic acid is a β-lactam compound with potent inhibitory activity against β-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20 g L-1), threonine (0.0-1.44 g L-1), ornithine (0.0-4.08 g L-1), and glutamate (0.0-8.16 g L-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437 mg L-1, while a formulation without this salt produced only 41 mg L-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

On-Site Production of Cellulolytic Enzymes by the Sequential Cultivation Method.

The conversion of renewable lignocellulosic biomass into fuels, chemicals, and high-value materials using the biochemical platform has been considered the most sustainable alternative for the implementation of future biorefineries. However, the high cost of the cellulolytic enzymatic cocktails used in the saccharification step significantly affects the economics of industrial large-scale conversion processes. The on-site production of enzymes, integrated to the biorefinery plant, is being considered as a potential strategy that could be used to reduce costs. In such approach, the microbial production of enzymes can be carried out using the same lignocellulosic biomass as feedstock for fungal development and biofuels production. Most of the microbial cultivation processes for the production of industrial enzymes have been developed using the conventional submerged fermentation. Recently, a sequential solid-state followed by submerged fermentation has been described as a potential alternative cultivation method for cellulolytic enzymes production. This chapter presents the detailed procedure of the sequential cultivation method, which could be employed for the on-site production of the cellulolytic enzymes required to convert lignocellulosic biomass into simple sugars.

Screening of medium constituents for clavulanic acid production by Streptomyces clavuligerus.

Clavulanic acid is a ß-lactam compound with potent inhibitory activity against ß-lactamases. Studies have shown that certain amino acids play essential roles in CA biosynthesis. However, quantitative evaluations of the effects of these amino acids are still needed in order to improve CA production. Here, we report a study of the nutritional requirements of Streptomyces clavuligerus for CA production. Firstly, the influence of the primary nitrogen source and the salts composition was investigated. Subsequently, soybean protein isolate was supplemented with arginine (0.0-3.20gL-1), threonine (0.0-1.44gL-1), ornithine (0.0-4.08gL-1), and glutamate (0.0-8.16gL-1), according to a two-level central composite rotatable design. A medium containing ferrous sulfate yielded CA production of 437mgL-1, while a formulation without this salt produced only 41mgL-1 of CA. This substantial difference suggested that Fe2+ is important for CA biosynthesis. The experimental design showed that glutamate and ornithine negatively influenced CA production while arginine and threonine had no influence. The soybean protein isolate provided sufficient C5 precursor for CA biosynthesis, so that supplementation was unnecessary. Screening of medium components, together with experimental design tools, could be a valuable way of enhancing CA titers and reducing the process costs.

A closed-loop strategy for endoglucanase production using sugarcane bagasse liquefied by a home-made enzymatic cocktail.

Use of the same lignocellulosic biomass as feedstock for enzymes and ethanol production has been suggested as a lower cost option in future biorefineries. Here, we propose a closed-loop strategy to produce the cellulolytic enzymes required for biomass hydrolysis using sugarcane bagasse liquefied by a home-made enzymatic cocktail as carbon source and inducer. The fed-batch liquefaction conditions were firstly evaluated using commercial enzymes. Subsequently, the effects of different liquefied materials and solids loadings on endoglucanase production by Aspergillus niger cultivated in submerged fermentation were investigated. The liquefied bagasse produced using the home-made cocktail was more favorable for endoglucanase production, resulting in improvement up to 17%, compared to bagasse liquefied by commercial enzymes. The results indicated that liquefied bagasse produced by home-made enzymatic cocktail could provide a cost-effective carbon source and inducer for cellulolytic enzyme production, and could contribute to closing loops within the biorefinery, thus reducing costs and minimizing waste.

Soybean protein as a cost-effective lignin-blocking additive for the saccharification of sugarcane bagasse.

Addition of surfactants, polymers, and non-catalytic proteins can improve the enzymatic hydrolysis of lignocellulosic materials by blocking the exposed lignin surfaces, but involves extra expense. Here, soybean protein, one of the cheapest proteins available, was evaluated as an alternative additive for the enzymatic hydrolysis of pretreated sugarcane bagasse. The effect of the enzyme source was investigated using enzymatic cocktails from A. niger and T. reesei cultivated under solid-state, submerged, and sequential fermentation. The use of soybean protein led to approximately 2-fold increases in hydrolysis, relative to the control, for both A. niger and T. reesei enzymatic cocktails from solid-state fermentation. The effect was comparable to that of BSA. Moreover, the use of soybean protein and a 1:1 combination of A. niger and T. reesei enzymatic cocktails resulted in 54% higher glucose release, compared to the control. Soybean protein is a potential cost-effective additive for use in the biomass conversion process.

Secretome data from Trichoderma reesei and Aspergillus niger cultivated in submerged and sequential fermentation methods.

The cultivation procedure and the fungal strain applied for enzyme production may influence levels and profile of the proteins produced. The proteomic analysis data presented here provide critical information to compare proteins secreted by Trichoderma reesei and Aspergillus niger when cultivated through submerged and sequential fermentation processes, using steam-explosion sugarcane bagasse as inducer for enzyme production. The proteins were organized according to the families described in CAZy database as cellulases, hemicellulases, proteases/peptidases, cell-wall-protein, lipases, others (catalase, esterase, etc.), glycoside hydrolases families, predicted and hypothetical proteins. Further detailed analysis of this data is provided in "Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation process: enzyme production for sugarcane bagasse hydrolysis" C. Florencio, F.M. Cunha, A.C Badino, C.S. Farinas, E. Ximenes, M.R. Ladisch (2016) [1].

Secretome analysis of Trichoderma reesei and Aspergillus niger cultivated by submerged and sequential fermentation processes: Enzyme production for sugarcane bagasse hydrolysis.

Cellulases and hemicellulases from Trichoderma reesei and Aspergillus niger have been shown to be powerful enzymes for biomass conversion to sugars, but the production costs are still relatively high for commercial application. The choice of an effective microbial cultivation process employed for enzyme production is important, since it may affect titers and the profile of protein secretion. We used proteomic analysis to characterize the secretome of T. reesei and A. niger cultivated in submerged and sequential fermentation processes. The information gained was key to understand differences in hydrolysis of steam exploded sugarcane bagasse for enzyme cocktails obtained from two different cultivation processes. The sequential process for cultivating A. niger gave xylanase and ß-glucosidase activities 3- and 8-fold higher, respectively, than corresponding activities from the submerged process. A greater protein diversity of critical cellulolytic and hemicellulolytic enzymes were also observed through secretome analyses. These results helped to explain the 3-fold higher yield for hydrolysis of non-washed pretreated bagasse when combined T. reesei and A. niger enzyme extracts from sequential fermentation were used in place of enzymes obtained from submerged fermentation. An enzyme loading of 0.7 FPU cellulase activity/g glucan was surprisingly effective when compared to the 5-15 times more enzyme loadings commonly reported for other cellulose hydrolysis studies. Analyses showed that more than 80% consisted of proteins other than cellulases whose role is important to the hydrolysis of a lignocellulose substrate. Our work combined proteomic analyses and enzymology studies to show that sequential and submerged cultivation methods differently influence both titers and secretion profile of key enzymes required for the hydrolysis of sugarcane bagasse. The higher diversity of feruloyl esterases, xylanases and other auxiliary hemicellulolytic enzymes observed in the enzyme mixtures from the sequential fermentation could be one major reason for the more efficient enzyme hydrolysis that results when using the combined secretomes from A. niger and T. reesei.

Power consumption evaluation of different fed-batch strategies for enzymatic hydrolysis of sugarcane bagasse.

The minimization of costs in the distillation step of lignocellulosic ethanol production requires the use of a high solids loading during the enzymatic hydrolysis to obtain a more concentrated glucose liquor. However, this increase in biomass can lead to problems including increased mass and heat transfer resistance, decreased cellulose conversion, and increased apparent viscosity with the associated increase in power consumption. The use of fed-batch operation offers a promising way to circumvent these problems. In this study, one batch and four fed-batch strategies for solids and/or enzyme feeding during the enzymatic hydrolysis of sugarcane bagasse were evaluated. Determinations of glucose concentration, power consumption, and apparent viscosity were made throughout the experiments, and the different strategies were compared in terms of energy efficiency (mass of glucose produced according to the energy consumed). The best energy efficiency was obtained for the strategy in which substrate and enzyme were added simultaneously (0.35 kg(glucose) kWh⁻¹). This value was 52% higher than obtained in batch operation.

Mixing design for enzymatic hydrolysis of sugarcane bagasse: methodology for selection of impeller configuration.

One of the major process bottlenecks for viable industrial production of second generation ethanol is related with technical-economic difficulties in the hydrolysis step. The development of a methodology to choose the best configuration of impellers towards improving mass transfer and hydrolysis yield together with a low power consumption is important to make the process cost-effective. In this work, four dual impeller configurations (DICs) were evaluated during hydrolysis of sugarcane bagasse (SCB) experiments in a stirred tank reactor (3 L). The systems tested were dual Rushton turbine impellers (DIC1), Rushton and elephant ear (down-pumping) turbines (DIC2), Rushton and elephant ear (up-pumping) turbines (DIC3), and down-pumping and up-pumping elephant ear turbines (DIC4). The experiments were conducted during 96 h, using 10 % (m/v) SCB, pH 4.8, 50 °C, 10 FPU/g biomass, 470 rpm. The mixing time was successfully used as the characteristic parameter to select the best impeller configuration. Rheological parameters were determined using a rotational rheometer, and the power consumptions of the four DICs were on-line measured with a dynamometer. The values obtained for the energetic efficiency (the ratio between the cellulose to glucose conversion and the total energy) showed that the proposed methodology was successful in choosing a suitable configuration of impellers, wherein the DIC4 obtained approximately three times higher energetic efficiency than DIC1. Furthermore a scale-up protocol (factor scale-up 1000) for the enzymatic hydrolysis reactor was proposed.