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.

Selenium-Enriched Probiotic Saccharomyces boulardii CCT 4308 Biomass Production Using Low-Cost Sugarcane Molasses Medium

Abstract Food supplements have been increasingly investigated. Probiotics have several benefits for human and animal health and selenium (Se) is widely recommended against oxidative stress. In this context, the aim of this study was to develop a low-cost bioprocess to produce a functional food product comprising both probiotic and Se accumulation. Yeast cells of Saccharomyces boulardii CCT 4308 were cultivated using sugarcane molasses as substrate. Optimization studies were performed to evaluate the best medium composition for biomass production and Se-accumulation in batch and fed-batch systems. Optimized conditions were defined with a medium composed of 150 g L-1 sugarcane molasses and 12 g L-1 yeast extract, with feeding of 100 g L-1 sugarcane molasses and 100 μg mL-1 of Se incorporation after 4 h and 10 h of fermentation, respectively, during 48 h in STR (stirred tank reactor). Best biomass production reached 14.52 g L-1 with 3.20 mg Se g-1 biomass at 12 h. Process optimization led to 4.82-fold increase in biomass production compared to initial condition. A final Se-enriched S. boulardii CCT 4308 biomass was obtained, which is comparable to commercial products. An alternative probiotic yeast biomass was efficiently produced as a new food-form of Se supplement in a sustainable process using an inexpensive agro-industrial residue.

Control de las tasas de aireación y agitación en la producción de surfactina en un cultivo alimentado (fed-batch) en espuma desbordante con fermentación industrial / Control of agitation and aeration rates in the production of surfactin in foam overflowing fed-batch culture with industrial fermentation

Bacillus amyloliquefaciens fmb50 produces a high yield of surfactin, a lipopeptide-type biosurfactant that has been widely studied and has potential applications in many fields. A foam overflowing culture has been successfully used in the combined production-enrichment fermentation of surfactin. In this study, the agitation and aeration rates were found to have relationships with foam formation and surfactin enrichment. A maximum surfactin concentration of 4.7 g/l of foam was obtained after 21 h of culture with an agitation rate of 150 rpm and an aeration rate of 1 vvm in fed-batch culture. By controlling the foam overflow rate (f out) of a fed-batch culture, surfactin concentration in the foam was continuously maintained above 4 g/l.

Bacillus amyloliquefaciens fmb50 produce gran cantidad de surfactina, un biosurfactante de tipo lipopeptídico que ha sido objeto de estudios pormenorizados y tiene aplicaciones en muchos campos. El cultivo en espuma desbordante se ha utilizado con éxito en la fermentación combinada de producción-enriquecimiento de surfactina. En este estudio, se halló que las tasas de aireación y agitación tienen relación con la formación de espuma y el enriquecimiento de la surfactina. Se obtuvo una concentración máxima de surfactina de 4,7 g/l de espuma después de 21 h de cultivo con una tasa de agitación de 150 rpm y una tasa de aireación de 1 vvm en un cultivo alimentado (fed-batch). Al controlar la tasa de espuma desbordante (f out) de un cultivo fed-batch, la concentración de surfactina en la espuma se mantuvo continua por encima de 4 g/l.

The use of date waste for lactic acid production by a fed-batch culture using Lactobacillus casei subsp. rhamnosus

The production of lactic acid from date juice by Lactobacillus caseisubsp. rhamnosus in batch and fed-batch cultures has been investigated. The fed-batch culture system gave better results for lactic acid production and volumetric productivity. The aim of this work is to determine the effects of the feeding rate and the concentration of the feeding medium containing date juice glucose on the cell growth, the consumption of glucose and the lactic acid production by Lactobacillus casei subsp. rhamnosus in fed-batch cultures. For this study, two concentrations of the feeding medium (62 and 100 g/L of date juice glucose) were tested at different feeding rates (18, 22, 33, 75 and 150 mL/h). The highest volumetric productivity (1.3 g/L.h) and lactic acid yield (1.7 g/g) were obtained at a feeding rate of 33 mL/h and a date juice glucose concentration of 62 g/L in the feeding medium. As a result, most of the date juice glucose was completely utilised (residual glucose 1 g/L), and a maximum lactic acid production level (89.2 g/L) was obtained.

The effect of high concentrations of glycerol on the growth, metabolism and adaptation capacity of Clostridium butyricum DSP1

Background The production of biofuels from renewable energy sources is one of the most important issues in biotechnology today. The process is known to generate various by-products, for example glycerol that is obtained in the making of biodiesel from rapeseed oil. Crude glycerol may be utilized in many ways, including microbial conversion to 1,3-propanediol. The main drawback of that technology is the use of high concentrations of glycerol, which inhibits the growth of bacterial cells. Results This study investigated the impact of crude glycerol on Clostridium butyricum DSP1 and its ability to adapt to an environment of high osmotic pressure. It was found that a crude glycerol concentration of up to 70 g/L did not have an inhibitory effect on C. butyricum DSP1. Adaptation procedures involving the passage of metabolically active biomass from a fermentation medium with a lower concentration of crude glycerol to one with a greater substrate concentration allowed breaking the barrier of high osmotic pressure (150 g/L crude glycerol) and receiving a 1,3-PD concentration of 74 g/L in a batch culture operation. The work looked into intracellular modifications shown by proteomic profiling in order to explain the mechanisms underlying the response and adaptation of bacterial cells exposed to unfavorable environmental conditions. Conclusions This study of the effect of glycerol on the growth and metabolism of C. butyricum DSP1 demonstrated that the maximum substrate concentrations that do not inhibit the metabolic activity of bacterial cells are 90 g/L and 70 g/L for pure and crude glycerol, respectively.

Cultivo por lote de Wickerhamomyces anomaìus en un biorreactor a escala laboratorio para la producción de una poligalacturonasa / Batch culture of Wickerhamomyces anomaìus in a lab scale bioreactor for poligalacturonase production

Wickerhamomyces anomalus, una levadura aislada de frutas cítricas en la provincia de Misiones, Argentina, produce una poligalacturonasa (endo-PG) con capacidad macerante de tejidos vegetales. El objetivo del presente trabajo fue determinai los parámetros cinéticos y estequiométricos del crecimiento de W. anomalus y la producción de la enzima poligalacturonasa en un medio de cultivo sintético, operado en sistema por lote, en un biorreactor a escala laboratorio. Los cultivos se realizaron en un biorreactor de 4 l que contenía 3 l de un medio sintético compuesto por glucosa, pectina de citrus, vitaminas, aminoácidos, sulfato de amonio y sales, y se incubaron con agitación y aireación, a 30 °C durante 12 h. El transcurso de proceso fermentativo se siguió por medidas de biomasa, glucosa residual, actividad poligalacturonasa y contenido de O2 y CO2 de los gases a la salida del reactor. La velocidad específica de crecimiento máxima (-im) de W. anomalus fue de 0,337 h-1 y el rendimiento de biomasa producida (Yx/s) de 0,401 gx/gs. Al finalizar el cultivo, la actividad PG en el sobrenadante fue de PG de ~ 83,7 UE/ml. La actividad específica y la productividad obtenidas fueron de ~ 1,91. 10(4) UE/gx y ~ 9.301 UE/l.h, respectivamente. El cociente respiratorio fue cercano a 1 durante el proceso fermentativo. No se formó ningún otro producto, además de biomasa y CO 2 . El cultivo por lote resultó ser una buena alternativa para la producción de PG a partir de W. anomalus, obteniéndose un extracto con elevada actividad enzimática, en un medio de cultivo sintético y de bajo costo.

Wickerhamomyces anomalus, a yeast isolated from citrus fruit peels in the province of Misiones, Argentina, produces a polygalacturonase (endo-PG) with maceration activity of vegetable tissues. The objective of the present work was to determine kinetic and stoichiometric parameters of W. anomalus growth and polygalacturonase production in a synthetic culture medium, operating in a batch-type bioreactor at laboratory scale. Cultures were performed in a bioreactor of 4 l, containing 3 l of a synthetic medium composed of glucose, citrus pectin, vitamins, amino acids, ammonium sulfate and salts, and were incubated with agitation (450 rpm) and aeration at 30 °C, during 12 h. The course of the fermentation process was followed by measuring biomass, residual glucose, polygalacturonase activity and O2 and CO2 content of outlet gases from the reactor. The maximum specific growth rate (Um) of W. anomalus was 0.337 h-1 and the biomass yield (Yx/s) was 0.40 gx/gs. At the end of the culture, PG activity in the supernatant was ~84 UE/ml. The specific activity and the productivity obtained were ~1.91 104 UE/gx and ~9,301 UE/l.h, respectively. Respiratory quotient was approximately 1.0 throughout the fermentation process. No other product different from biomass and CO2 was detected. Batch culture could be an adequate alternative for the production of polygalacturonase from W. anomalus and an extract with high enzymatic activity using a synthetic and economic culture medium could be obtained.

Effect of Aflatoxin B1 on in vitro Rumen Microbial Fermentation Responses Using Batch Culture.

Aims: To evaluate the effect of aflatoxin B1 (AFB1) on in vitro dry matter disappearance (IVDMD), gas production and ammonia-N formation of an alfalfa hay based diet using batch culture system. Place and Duration of Study: Department of Animal Science, between July 2011 and August 2012. Methodology: In an anaerobic batch culture system, 50 ml of buffered rumen fluid was dispensed into a 125-ml serum bottle containing 0.5 g dry matter (DM) of the experimental diet. Experimental treatments included four dose levels of AFB1 (0, 300, 600 and 900 ng/ml). All bottles were purged with anaerobic CO2, sealed and placed in a shaking water bath for 72 h at 38.6ºC. Gas production of each bottle was recorded at 2, 4, 8, 12, 16, 24, 48 and 72 h of the incubation and then gas released. The batch cultures were repeated in three incubation runs. After 72 h incubation, bottles were opened and 2-ml sample of each bottle were taken for ammonia-N analysis. The biomass residues were centrifuged and the pellet was dried at 65°C for the determination of t he residual DM and IVDMD. Results: Addition of AFB1 affected the rate and cumulative gas production (P<0.05), so, by increasing the level of AFB1 from 0 to 900 ng/ml, the gas production rate decreased from 0.071 to 0.051 and cumulative gas production decreased from 196.4 to 166.0 ml/g DM, respectively. In addition, IVDMD decreased significantly with inclusion of AFB1 in culture medium, so that the lowest and the highest IVDMD values were observed in treatments with 900 and 0 ng/ml AFB1, respectively (0.54 vs. 0.68). The results indicated that addition of AFB1 significantly (P<0.05) decreased ammonia-N concentrations, so the lowest value was observed at 900 ng/ml AFB1. Conclusion: The addition of different levels of AFB1 affected in vitro fermentation characteristic, as represented in reduced gas production, dry matter digestibility and ammonia-N concentrations. Therefore it is necessary to control and manage aflatoxin contaminations in ruminants.

A cost effective fermentative production of glutathione by Saccharomyces cerevisiae with cane molasses and glycerol

This work aimed to evaluate the effect of sugar cane molasses and glycerol on glutathione (GSH) fermentation by Saccharomyces cerevisiae ATCC 7754 in flask culture using response surface methodology. Under optimized conditions (80 g/L of molasses and 60 g/L of glycerol), the highest GSH and biomass concentration achieved were 119.6 mg/L and 25.3 g/L, respectively. Further studies done in 5 L bioreactor resulted 241.3 mg/L GSH after 96 h in batch fermentation without amino acids addition and the concentration of biomass was 12.1 g/L. In batch fermentation with the addition of the three amino acids (4 mM cysteine, glycine and glutamic acid at 32 h), biomass reached to 25 g/L and GSH, 236.1 mg/L at 96 h of fermentation. The strategy of precursor amino acids addition is a key aspect in increasing the synthesis of GSH.

PARÁMETROS FISICOQUÍMICOS PARA LA SÍNTESIS DE ÁCIDO LÁCTICO O ETANOL DE LA BACTERIA (Corynebacterium glutamicum) / Physico-Chemical Parameter for Production of Lactic Acid or Ethanol of (Corynebacterium glutamicum) Bacteria

El interés por obtener productos para la industria de biocombustibles a partir de desechos agrícolas, conduce a la búsqueda de nuevos sistemas biotecnológicos resistentes y costo-efectivos. Corynebacterium glutamicum, es un microorganismo usado para producir amino-ácidos que crece en gran variedad de sustratos y es resistente durante la fermentación, a variaciones de pH, temperatura, presión osmótica y acumulación de alcohol, características que lo hacen candidato a ser mejorado para la síntesis de ácido láctico y etanol. Aún se desconocen aspectos de su fisiología que aumenten su eficiencia en convertir azúcares (C5 y C6) en estos dos metabolitos. Por tanto, este trabajo buscó identificar los parámetros fisicoquímicos que tuvieron un mayor efecto sobre crecimiento bacteriano y síntesis de ácido láctico o etanol en un sistema por lotes. Para lograr este objetivo, ocho variables fueron evaluadas en un modelo estadístico producido en erlenmeyer; con los resultados obtenidos, se hallaron las mejores condiciones que fueron puestas a prueba en un cultivo en biorreactor. La temperatura, concentración de biotina y azúcar fueron las variables de mayor impacto (p< 0,05). Usando las mejores condiciones, 36 °C; 6,1 mg/L de biotina y 50 g/L de glucosa, se obtiene una µmax de 0,394 h-1, 16 g/L de ácido láctico a las 15 h del proceso con un rendimiento del 32%; observándose un mayor consumo de sustrato durante el crecimiento y poca disponibilidad para la fermentación, sugiriendo una alimentación del cultivo al final de la fase exponencial que aumente los rendimientos de producción.

The interest to obtain products for the bio-fuel industry from renewable resources has directed research to find resistant and costs-effective biotechnological systems. Corynebacterium glutamicum, is a microorganism used to produce amino acids, that grows in wide variety of substrates and its resistance during fermentation to pH, temperature, osmotic pressure variations and alcohol aggregate, renders this organism a suitable candidate to improve by genetic modifications lactic acid and ethanol synthesis. However, some aspects of its physiology remain unknown, such us increase lactic acid and ethanol production from C5 and C6 sugars. For this reason, the main aim in our work was to identify the most important variables with impact on culture and the best culture conditions to produce lactic acid or ethanol in batch culture. To achieve this objective, eight variables were tested in culture using a statistical model. The best culture conditions were obtained and tested in a bacth biorreactor system. Temperature, biotin and glucose concentration were the variables with most impact (p< 0.05) in culture. Using optimal conditions, 36 °C; 6.1 mg/L of biotin and 50 g/L of glucose; a µmax of 0.394 h-1, 16 g/L of lactic acid was obtained after 15 h of culture with an efficiency of 32%. High glucose consumption was observed during bacterial growth, which leads to low concentration of substrate for the production process; this suggests a culture feeding at the end of exponential growth phase, which can increase the production yield.

Valorization of raw glycerol for citric acid production by Yarrowia lipolytica yeast

In the present report, citric acid production from raw glycerol in two fed-batch systems by acetate negative-mutants of Yarrowia lipolytica: Wratislavia 1.31 and Wratislavia AWG7 was compared. In the system, in which the total glycerol concentration was 200 g∙L-1, the substrate was added by pulsed additions, and in the other, in which the total glycerol concentration was 300 g∙L-1, the substrate was added at a constant feeding rate of 1.4 g∙h-1. Despite high citric acid concentrations (155.2 and 157.5 g∙L-1 with Y. lipolytica Wratislavia 1.31 and Y. lipolytica Wratislavia AWG7, respectively) obtained from 300 g∙L-1 of glycerol, the yield of citric acid was similar, i.e. about 0.6 g∙g-1. The volumetric citric acid productivity was markedly higher (1.05 and 0.94 g∙L-1h-1 with Y. lipolytica Wratislavia 1.31 and Y. lipolytica Wratislavia AWG7 strains, respectively) in the cultures containing 200 g∙L-1 of carbon source.

Sequential use of ammonium and leucine as nitrogen sources during growth of Geotrichum candidum on a glucose based medium / Uso secuencial del amonio y leucina como fuentes de nitrógeno durante el crecimiento de Geotrichum candidum en un medio basado en glucosa

Geotrichum candidum growth on ammonium and leucine as nitrogen sources and glucose as a carbon source was examined. A clear preference of G. candidum for ammonium over leucine as a nitrogen source was shown. Indeed, ammonium was completely exhausted at the end of exponential growth after less than 35 hrs of culture; in contrast only 5 percent of leucine was concomitantly assimilated. Growth continued at slower rates on glucose and leucine as carbon and nitrogen sources respectively, and at the end of culture (185 hrs), leucine was completely exhausted.

Fermentation technologies for the production of exopolysaccharide-synthesizing Lactobacillus rhamnosus concentrated cultures

The exopolysaccharide (EPS)-producing cultures such as Lactobacillus rhamnosus RW-9595M present a challenge for the culture producers because the high viscosity of the fermented growth medium makes it difficult to recover the cells by centrifugation or filtration. This study examined four approaches to reduce viscosity of the medium while producing high cell densities: incubation temperature, extended incubation in the stationary growth phase, production in alginate gel beads and fed-batch fermentation technology. Automated spectrophotometry (AS) was used to study the effects of temperature, pH and lactate level on growth of the strain. In AS assays, there was no significant difference in final maximal biomass production at temperatures ranging between 34 ºC to 44 ºC, but lower yields were noted at 46 C. A pH below 6.0 and a lactate concentration higher than 4 percent almost completely prevented growth. Under batch fermentation conditions, the viscosity of the medium obtained at 37 C was two fold higher than for 44 ºC. For cultures produced at 37 ºC, centrifugation at 10000 g during 5 min did not allow complete recovery of cells, in contrast to cultures grown at 44 ºC. An extended period of incubation (5 hrs) in the stationary growth phase did not reduce the final viscosity of the growth medium. For similar biomass levels, the glucose-based fed-batch fermentation allowed a 40 percent reduction in viscosity of the fermented medium in comparison to traditional batch cultures. High-density cell populations (3 x 10(10) CFU/g) were obtained when L. rhamnosus RW-9595M was grown in alginate beads. However, overall biomass yields in the immobilized cell bioreactor were half of those obtained in free-cell fermentations. Therefore three methods of producing concentrated EPS-producing cultures are proposed.

The Current State and Trend of in Fed-Batch Mammalian Cell Culture / 中国生物工程杂志

Fed-batch culture is the predominant mode of the current animal cell culture process for many recombinant protein production. Fed-batch culture operation is mainly based on the nutrient continuous consumption and demand of cells to design continuous or semi-continuous concentration feed medium to maintain or support high-density cell growth and improve volumetric productivity of target protein in the reactor. The main methods to improve production efficiency of fed-batch cells culture include the optimization of medium design, selection and optimization of feed strategy and regulation of cell metabolism.

ULTRASTRUCTURE AND BATCH CULTURE OF MAGNETOTACTIC BACTERIA STRAIN WD-1 / 微生物学通报

The ultrastructure of magnetotactic bacteria strain WD\|1 was reported.A thin sectioned profile of it clearly showed its cell wall,cell membrane,poly\|?\|hydroxybutyrates and magnetosomes.Meanwhile,the method of batch culture of strain WD\|1 was also reported,135mg/L dry cells could be gotten with this method.The energy profiles of X\|ray collected after electron excitation in SEM indicated that both dry cells and magnetosomes contained such mineral elements as P,S,Ca,Si,Al and Fe with different content.The iron content of its cells and magnetosomes was 3 07% and 84 57% respectively.