Scale-up and fed-batch cultivation strategy for the enhanced co-production of microbial lipids and carotenoids using renewable waste feedstock.

Agro-industrial by-product valorization as a feedstock for the bioproduction of high-value products has demonstrated a feasible alternative to handle the environmental impact of waste. Oleaginous yeasts are promising cell factories for the industrial production of lipids and carotenoids. Since oleaginous yeasts are aerobic microorganisms, studying the volumetric mass transfer (kLa) could facilitate the scale-up and operation of bioreactors to grant the industrial availability of biocompounds. Scale-up experiments were performed to assess the simultaneous production of lipids and carotenoids using the yeast Sporobolomyces roseus CFGU-S005 and comparing the yields in batch and fed-batch mode cultivation using agro-waste hydrolysate in a 7 L bench-top bioreactor. The results indicate that oxygen availability in the fermentation affected the simultaneous production of metabolites. The highest production of lipids (3.4 g/L) was attained using the kLa value of 22.44 h-1, while higher carotenoid accumulation of 2.58 mg/L resulted when agitation speed was increased to 350 rpm (kLa 32.16 h-1). The adapted fed-batch mode in the fermentation increased the production yields two times. The fatty acid profile was affected according to supplied aeration and after the fed-batch cultivation mode. This study showed the scale-up potential of the bioprocess using the strain S. roseus in the obtention of microbial oil and carotenoids by the valorization of agro-industrial byproducts as a carbon source.

Cost Reduction in the Production of Spirulina Biomass and Biomolecules from Indole-3-Acetic Acid Supplementation in Different Growth Phases.

Despite the great potential for the industrial application of microalgae, production costs are still too high to make them a competitive raw material for commodities. Therefore, studying more efficient cultivation strategies in biomass production and economic viability is necessary. In this sense, this work aimed to reduce the production costs of biomass and biomolecules using phytohormone indole-3-acetic acid in different phases of Spirulina sp. LEB 18 cultivation. The experiments were conducted on bench scale indoor for 30 days. In each couple of experiments, the phytohormone was added on different days. The supplementation of indole-3-acetic acid on half of the growth deceleration phase of the microalga showed a cost reduction of 27%, 34%, and 75% for biomass, proteins, and carbohydrates, respectively. In addition, the strategy increased the final biomass concentration and carbohydrate content at 31.2 and 33.8%, respectively, compared to the condition without phytohormone. This study is the starting point for implementing phytohormone supplementation in industrial microalgal cultures.

A Modeled High-Density Fed-Batch Culture Improves Biomass Growth and ß-Glucans Accumulation in Microchloropsis salina.

Algae and microalgae are used as a source of different biomolecules, such as lipids and carbohydrates. Among carbohydrates, polysaccharides, such as ß-glucans, are important for their application as antioxidants, antisepsis, and immunomodulators. In the present work, the ß-glucans production potential of Microchloropsis salina was assessed using two different culture conditions: a high-density batch and a modeled high-density fed-batch. From the biochemical parameters determined from these two cultures conditions, it was possible to establish that the modeled high-density fed-batch culture improves the biomass growth. It was possible to obtain a biomass productivity equal to 8.00 × 10-2 ± 2.00 × 10-3 g/(L × day), while the batch condition reached 5.13 × 10-2 ± 4.00 × 10-4 g/(L × day). The same phenomenon was observed when analyzing the ß-glucans accumulation, reaching volumetric productivity equal to 5.96 × 10-3 ± 2.00 × 10-4 g of product/(L × day) against the 4.10 × 10-3 ± 2.00 × 10-4 g of product/(L × day) obtained in batch conditions. These data establish a baseline condition to optimize and significantly increase ß-glucan productivity, as well as biomass, adding a new and productive source of this polymer, and integrating its use in potential applications in the human and animal nutraceutical industry.

Continuous Production of Biogenic Magnetite Nanoparticles by the Marine Bacterium Magnetovibrio blakemorei Strain MV-1T with a Nitrous Oxide Injection Strategy.

Magnetotactic bacteria (MTB) produce magnetosomes, which are membrane-embedded magnetic nanoparticles. Despite their technological applicability, the production of magnetite magnetosomes depends on the cultivation of MTB, which results in low yields. Thus, strategies for the large-scale cultivation of MTB need to be improved. Here, we describe a new approach for bioreactor cultivation of Magnetovibrio blakemorei strain MV-1T. Firstly, a fed-batch with a supplementation of iron source and N2O injection in 24-h pulses was established. After 120 h of cultivation, the production of magnetite reached 24.5 mg∙L-1. The maximum productivity (16.8 mg∙L-1∙day-1) was reached between 48 and 72 h. However, the productivity and mean number of magnetosomes per cell decreased after 72 h. Therefore, continuous culture in the chemostat was established. In the continuous process, magnetite production and productivity were 27.1 mg∙L-1 and 22.7 mg∙L-1∙day-1, respectively, at 120 h. This new approach prevented a decrease in magnetite production in comparison to the fed-batch strategy.

Improving 3-hydroxypropionic acid production in E. coli by in silico prediction of new metabolic targets.

3-Hydroxypropionic acid (3-HP) production from renewable feedstocks is of great interest in efforts to develop greener processes for obtaining this chemical platform. Here we report an engineered E. coli strain for 3-HP production through the ß-alanine pathway. To obtain a new strain capable of producing 3-HP, the pathway was established by overexpressing the enzymes pyruvate aminotransferase, 3-hydroxyacid dehydrogenase, and L-aspartate-1-decarboxylase. Further increase of the 3-HP titer was achieved using evolutionary optimizations of a genome-scale metabolic model of E. coli containing the adopted pathway. From these optimizations, three non-intuitive targets for in vivo assessment were identified: L-alanine aminotransferase and alanine racemase overexpression, and L-valine transaminase knock-out. The implementation of these targets in the production strain resulted in a 40% increase in 3-HP titer. The strain was further engineered to overexpress phosphoenolpyruvate carboxylase, reaching 0.79 ± 0.02 g/L of 3-HP when grown using glucose. Surprisingly, this strain produced 63% more of the desired product when grown using a mixture of glucose and xylose (1:1, C-mol), and gene expression analysis showed that the cellular adjustment to consume xylose had a positive impact on 3-HP accumulation. Fed-batch culture with xylose feeding led to a final titer of 29.1 g/L. These results reinforce the value of computational methods in strain engineering, enabling the design of more efficient strategies to be assessed. Moreover, higher production of 3-HP under a sugar mixture condition points towards the development of bioprocesses based on renewable resources, such as hemicellulose hydrolysates.

The Chemical, Microbiological and Volatile Composition of Kefir-like Beverages Produced from Red Table Grape Juice in Repeated 24-h Fed-Batch Subcultures.

The aim of this work was to study the production of kefir-like beverages via the fed-batch fermentation of red table grape juice at initial pHs of 3.99 (fermentation A) and 5.99 (fermentation B) with kefir grains during 4 repeated 24-h fed-batch subcultures. All kefir-like beverages (KLB) were characterized by low alcoholic grade (≤3.6%, v/v) and lactic and acetic acid concentrations. The beverages obtained from fermentation B had lower concentrations of sugars and higher microbial counts than the KLB obtained in fermentation A. Additionally, the KLB samples from fermentation B were the most aromatic and had the highest contents of alcohols, esters, aldehydes and organic acids, in contrast with the nonfermented juice and KLB from fermentation A. These results indicate the possibility of obtaining red table grape KLB with their own distinctive aromatic characteristics and high content in probiotic viable cells, contributing to the valorization of this fruit.

Scale-Up of Capsular Polysaccharide Production Process by Haemophilus influenzae Type b Using kLa Criterion.

Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h-1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h-1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains.

BACKGROUND: Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. RESULTS: The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h-1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h-1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h-1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. CONCLUSION: The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

Saturated constructed wetland-microbial fuel cell system and effect on dissolved oxygen gradient, electricity generation and ammonium removal.

The aim of this work was to assess effect of saturated constructed wetland-Microbial fuel cell system on dissolved oxygen gradient, electricity generation and ammonium removal. Two laboratory-scale systems, one planted with Schoenoplectus californicus (SCW1-MFC) and other without plant (SCW2-MFC), were fed discontinuously with synthetic wastewater over 90 days. Both systems were operated at different organic loading rate (12 and 28 g COD/m2d) and ammonium loading rate (1.6 and 3.0 g NH4+- N/m2 d) under open circuit and close circuit mode. The results indicate that between lower and upper zones of wetlands the average values were in the range of 1.22 ± 0.32 to 1.39 ± 0.27 mg O2/L in SCW1-MFC and 1.28 ± 0.24 to 1.56 ± 0.31 mg O2/L in SCW2-MFC. The effect of operating mode (closed and open circuit) and vegetation on DO was not significant (p > 0.05). Chemical oxygen demand (COD) removal efficiencies, fluctuated between 90 and 95% in the SCW1-MFC and 82 and 94% in the SCW2-MFC system. Regarding NH4+- N, removal efficiencies were above 85% in both systems reaching values maximus 98%. The maximum power density generated was 4 and 10 mW/m2 in SCW1-MFC, while SCW2-MFC recorded the highest values (12 and 22 mW/m2).

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.

Escherichia coli coculture for de novo production of esters derived of methyl-branched alcohols and multi-methyl branched fatty acids.

BACKGROUND: A broad diversity of natural and non-natural esters have now been made in bacteria, and in other microorganisms, as a result of original metabolic engineering approaches. However, the fact that the properties of these molecules, and therefore their applications, are largely defined by the structural features of the fatty acid and alcohol moieties, has driven a persistent interest in generating novel structures of these chemicals. RESULTS: In this research, we engineered Escherichia coli to synthesize de novo esters composed of multi-methyl-branched-chain fatty acids and short branched-chain alcohols (BCA), from glucose and propionate. A coculture engineering strategy was developed to avoid metabolic burden generated by the reconstitution of long heterologous biosynthetic pathways. The cocultures were composed of two independently optimized E. coli strains, one dedicated to efficiently achieve the biosynthesis and release of the BCA, and the other to synthesize the multi methyl-branched fatty acid and the corresponding multi-methyl-branched esters (MBE) as the final products. Response surface methodology, a cost-efficient multivariate statistical technique, was used to empirical model the BCA-derived MBE production landscape of the coculture and to optimize its productivity. Compared with the monoculture strategy, the utilization of the designed coculture improved the BCA-derived MBE production in 45%. Finally, the coculture was scaled up in a high-cell density fed-batch fermentation in a 2 L bioreactor by fine-tuning the inoculation ratio between the two engineered E. coli strains. CONCLUSION: Previous work revealed that esters containing multiple methyl branches in their molecule present favorable physicochemical properties which are superior to those of linear esters. Here, we have successfully engineered an E. coli strain to broaden the diversity of these molecules by incorporating methyl branches also in the alcohol moiety. The limited production of these esters by a monoculture was considerable improved by a design of a coculture system and its optimization using response surface methodology. The possibility to scale-up this process was confirmed in high-cell density fed-batch fermentations.

Scale-up of capsular polysaccharide production process by Haemophilus influenzae type b using kLa criterion

Polyribosyl-ribitol-phosphate (PRP) from Haemophilus influenzae type b (Hib) is an active immunizing molecule used in the production of the vaccine against H. influenzae, and industrial production could contribute to satisfying a world demand especially in developing countries. In this sense, the aim of this study was to establish a scale-up process using the constant oxygen mass transfer coefficient (kLa) such as the criterion for production of PRP in three different sizes of bioreactor systems. Three different kLa values (24, 52 and 80 h−1) were evaluated in which the biological influence in a 1.5 L bioreactor and 52 h−1 was selected to scale-up the production process until a 75 L pilot-scale bioreactor was achieved. Finally, the fed-batch phase was started under a dissolved oxygen concentration (pO2) at 30% of the saturation in the 75 L bioreactor to avoid oxygen limitation; the performance of production presented high efficiency (9.0 g/L DCW-dry cell weight and 1.4 g/L PRP) in comparison with previous scale-up studies. The yields, productivity and kinetic behavior were similar in the three-size bioreactor systems in the batch mode indicating that kLa is possible to use for PRP production at large scales. This process operated under two stages and successfully produced DCW and PRP in the pilot scale and could be beneficial for future bioprocess operations that may lead to higher production and less operative cost.

Development of a chemically defined medium for Planctopirus limnophila to increase biomass production

BACKGROUND Planctomycetes is a phylum of biofilm-forming bacteria with numerous biosynthetic gene clusters, offering a promising source of new bioactive secondary metabolites. However, the current generation of chemically defined media achieves only low biomass yields, hindering research on these species. We therefore developed a chemically defined medium for the model organism Planctopirus limnophila to increase biomass production. RESULTS We found that P. limnophila grows best with a 10 mM sodium phosphate buffer. The replacement of complex nitrogen sources with defined amino acid solutions did not inhibit growth. Screening for vitamin requirements revealed that only cyanocobalamin (B12) is needed for growth. We used response surface methodology to optimize the medium, resulting in concentrations of 10 g/L glucose, 34 mL/L Hutner's basal salts, 23.18 mM KNO3, 2.318 mM NH4Cl and 0.02 mg/L cyanocobalamin. The analysis of amino acid consumption allowed us to develop a customized amino acid solution lacking six of the amino acids present in Aminoplasmal 10%. Fed-batch cultivation in a bioreactor using the optimized medium achieved a final DOD600 of 46.8 ± 0.5 after 108 h, corresponding to a cell dry weight of 13.6 ± 0.7 g/L. CONCLUSIONS The optimized chemically defined medium allowed us to produce larger amounts of biomass more quickly than reported in earlier studies. Further research should focus on triggering P. limnophila biofilm formation to activate the gene clusters responsible for secondary metabolism

TCA Cycle and Its Relationship with Clavulanic Acid Production: A Further Interpretation by Using a Reduced Genome-Scale Metabolic Model of Streptomyces clavuligerus.

Streptomyces clavuligerus (S. clavuligerus) has been widely studied for its ability to produce clavulanic acid (CA), a potent inhibitor of ß-lactamase enzymes. In this study, S. clavuligerus cultivated in 2D rocking bioreactor in fed-batch operation produced CA at comparable rates to those observed in stirred tank bioreactors. A reduced model of S. clavuligerus metabolism was constructed by using a bottom-up approach and validated using experimental data. The reduced model was implemented for in silico studies of the metabolic scenarios arisen during the cultivations. Constraint-based analysis confirmed the interrelations between succinate, oxaloacetate, malate, pyruvate, and acetate accumulations at high CA synthesis rates in submerged cultures of S. clavuligerus. Further analysis using shadow prices provided a first view of the metabolites positive and negatively associated with the scenarios of low and high CA production.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min-1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g-1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein-1, 10.6 times higher than in fed-batch.

State estimation and nonlinear tracking control simulation approach. Application to a bioethanol production system.

Tracking control of specific variables is key to achieve a proper fermentation. This paper analyzes a fed-batch bioethanol production process. For this system, a controller design based on linear algebra is proposed. Moreover, to achieve a reliable control, on-line monitoring of certain variables is needed. In this sense, for unmeasurable variables, state estimators based on Gaussian processes are designed. Cell, ethanol and glycerol concentrations are predicted with only substrates measurement. Simulation results when the controller and estimators are coupled, are shown. Furthermore, the algorithms were tested with parametric uncertainties and disturbances in the control action, and are compared, in all cases, with neural networks estimators (previous work). Bayesian estimators show a performance improvement, which is reflected in a decrease of the total error. Proposed techniques give reliable monitoring and control tools, with a low computational and economic cost, and less mathematical complexity than neural network estimators.

Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Buried Kex2 Sites in Glargine Precursor Aggregates Prevent Its Intracellular Processing in Pichia pastoris Muts Strains and the Effect of Methanol-Feeding Strategy and Induction Temperature on Glargine Precursor Production Parameters.

Glargine is a long-acting insulin analog with less hypoglycemia risk. Like human insulin, glargine is a globular protein composed of two polypeptide chains linked by two disulfide bonds. Pichia pastoris KM71 Muts strains were engineered to produce and secrete insulin glargine through the cleavage of two Kex2 sites. Nevertheless, the recombinant product was the single-chain insulin glargine (glargine precursor) instead of the expected double-chain glargine. Molecular model analysis of the dimeric and hexameric forms of the single-chain glargine showed buried Kex2 sites that prevent intracellular glargine precursor processing. The effect of the methanol-feeding strategy (methanol limited fed-batch vs. methanol non-limited fed-batch) and the induction temperature (28 °C vs. 24 °C) on the cell growth and production parameters in bioreactor cultures was also evaluated. Exponential growth at a constant specific growth rate was observed in all the cultures. The volumetric productivities and specific substrate consumption rates were directly proportional to the specific growth rate. The lower temperature led to increased metabolic activity of the yeast cells, which increased the specific growth rate. The methanol non-limited fed-batch culture at 24 °C showed the highest values for the process parameters. After 75 h of induction, 0.122 g/L of glargine precursor was obtained from the culture medium.

Establishment of a production process for a novel vaccine candidate against Lawsonia intracellularis

BACKGROUND: Lawsonia intracellularis remains a problem for the swine industry worldwide. Previously, we designed and obtained a vaccine candidate against this pathogen based on the chimeric proteins: OMP1c, OMP2c, and INVASc. These proteins formed inclusion bodies when expressed in E. coli, which induced humoral and cellular immune responses in vaccinated pigs. Also, protection was demonstrated after the challenge. In this study, we established a production process to increase the yields of the three antigens as a vaccine candidate. RESULTS: Batch and fed-batch fermentations were evaluated in different culture conditions using a 2 L bioreactor. A fed-batch culture with a modified Terrific broth medium containing glucose instead of glycerol, and induced with 0.75 mM IPTG at 8 h of culture (11 g/L of biomass) raised the volumetric yield to 627.1 mg/L. Under these culture conditions, plasmid-bearing cells increased by 10% at the induction time. High efficiency in cell disruption was obtained at passage six using a high-pressure homogenizer and a bead mill. The total antigen recovery was 64% (400 mg/L), with a purity degree of 70%. The antigens retained their immunogenicity in pigs, inducing high antibody titers. CONCLUSIONS: Considering that the antigen production process allowed an increment of more than 70-fold, this methodology constitutes a crucial step in the production of this vaccine candidate against L. intracellularis.

High Gravity and Very High Gravity Fermentation of Sugarcane Molasses by Flocculating Saccharomyces cerevisiae: Experimental Investigation and Kinetic Modeling.

Substantial progress has been made in ethanol fermentation technology under high gravity (HG) and very high gravity (VHG), which offer environmental and economic benefits. HG and VHG processes increase the productivity of ethanol, reduce distillation costs, and enable higher yields. The aim of the present study was to evaluate the use of sugarcane molasses as the medium component along with flocculating yeasts for fermentation in a fed-batch process employing this promising technology. We evaluated fed-batch fermentation, HG, and VHG involving a molasses-based medium with high concentrations of reducing sugars (209, 222, and 250 g/L). Fermentation of 222 g/L of total reducing sugars achieved 89.45% efficiency, with a final ethanol concentration of 104.4 g/L, whereas the highest productivity (2.98 g/(L.h)) was achieved with the fermentation of 209 g/L of total reducing sugars. The ethanol concentration achieved with the fermentation of 222 g/L of total reducing sugars was close to the value obtained for P'max (105.35 g/L). The kinetic model provided a good fit to the experimental data regarding the fermentation of 222 g/L. The results revealed that sugarcane molasses and flocculating yeasts can be efficiently used in HG fermentation to reduce the costs of the process and achieve high ethanol titers.