High Cell Density Cultivation of Escherichia coli DH5α in Shake Flasks with a New Formulated Medium

High cell density cultivation necessitates cell division and biomass formation, the mechanisms of which remain poorly understood, especially from the cellular energetics perspective. Specifically, the sensing of energy abundance and the channelling of nutritional energy into biomass formation and cell maintenance remains enigmatic at the sensory, effector and decision levels. Thus, optimization of cell growth remains an iterative trial and error process where the principal parameters are growth medium composition and incubation temperature. In this study, a new semidefined formulated medium was shown to be useful for high cell density cultivation of Escherichia coli DH5α (ATCC 53868). Comprising K2HPO4, 12.54; KH2PO4, 2.31; D-Glucose, 4.0; NH4Cl, 1.0; Yeast extract, 12.0; NaCl, 5.0; MgSO4, 0.24; the medium possessed a high capacity phosphate buffer able to moderate pH fluctuations during cell growth known to be detrimental to biomass formation. With glucose and NH4Cl providing the nutrients for initial growth, followed by a lag phase of 3 hours, a maximal optical density of 12.0 was obtained after 27 hours of cultivation at 37 oC and 230 rpm. Yeast extract provides a secondary source of carbon and nitrogen. Maximal optical density obtained in formulated medium was higher than the 10.1, 4.2, and 3.4 obtained in Tryptic Soy Broth, M9 with 1 g/L of yeast extract, and LB Lennox, respectively. Cultivation of E. coli DH5α in formulated medium with 6 g/L of glucose resulted in a longer lag phase of 8 hours and a longer time (68 hours) to attainment of maximal optical density, which marked the upper limit of glucose concentration beyond which biomass formation would be reduced. Specifically, glucose concentration above 6 g/L markedly reduced biomass formation possibly due to the environmental stress arising from low pH in the culture broth. Glucose concentration below 4 g/L, on the other hand, reduced biomass formation through a smaller pool of nutrients serving as biomass building blocks. Deviation from 1:1 molar ratio between glucose and NH4Cl was not detrimental to biomass formation and growth rates. Collectively, a semi-defined formulated medium could increase optical density of E. coli DH5α beyond that of LB Lennox and Tryptic Soy Broth, and may find use in cultivation of cells for applied microbiology research.

Antifungal Activity of the Bark Extract of Michelia Alba Against Curvularia Verruculosa Fungal the Cause of Leaf Spot Disease on Rice

The purpose of this study was observe the antifungal activity of the bark extract of Michelia alba against Curvularia verruculosa fungal of the cause of leaf spot disease in rice. The antifungal activities was carried out using the diffusion well, colony, biomass formation methods. The bark extract of Michelia alba has showed the antifungal activity against Curvularia verruculosa fungal with a minimum inhibition concentratiotn value to be 0.5%. The bark extract of Michelia alba with 2.0% concentration can strongly inhibit the growth of C. Verruculosa with inhibiting capabality is 33.17 mm. This extract at 0.6% concentration was able to completely inhibit the growth of fungal colony and at 2.0% concentration has been able to inhibit completely the biomass formation of C. Verruculosa fungal for a 14-day period of incubation.

A novel strain of Stenotrophomonas acidaminiphila produces super-active alkaline protease during cassava effluent fermentation: process optimization, kinetic modeling and scale-up

Microbial fermentations for value-added metabolites production are exploited for efficient bioconversion of agro-industrial wastes for the dual purposes of pollution abatement and cost-effectiveness. In the present study, the regular 2-level factorial design was employed to screen fermentation parameters that enhance production of a novel alkaline protease by a strain of Stenotrophomonas acidaminiphila using cassava processing effluent as substrate. Data from randomized experiments of central composite rotatable design for improved enzyme activity, guided by path of steepest ascent experiments, were modeled and optimized by response surface methodology (RSM). Shake flask kinetics of production under optimized conditions was modeled by logistic and modified Gompertz models and determinations of maximum specific growth rate, µmax, maximum volumetric rate of substrate consumption, rsm, maximum volumetric rate of biomass formation, rxm and specific yield of product, Yp/x were made. Logistic model poorly fitted RSM-generated product formation and substrate consumption data. However, biomass formation was accurately fitted (adjusted r2 >99%), with µmax of 0.471 h-1. The modified Gompertz model, on the contrary, more accurately fitted all three major response data with minimal mean squared error. Potential for scale-up of bioprocess evaluated in 5-L bioreactor satisfactorily revealed 8.5-fold more substrate consumption in bioreactor than in shake flask. The 86.76-fold aqueous two-phase system-purified protease had a specific activity of 1416.73 Umg-1 which improved with increasing surfactant concentration. These results suggest significant bioprocess potential for sustainable cassava effluent management and concomitant commercial production of alkaline protease for industrial detergent application.

A Novel Strain of Stenotrophomonas Acidaminiphila Produces Super-active Alkaline Protease During Cassava Effluent Fermentation: Process Optimization, Kinetic Modeling and Scale-up

PurposeMicrobial fermentations for value-added metabolites production are exploited for efficient bioconversion of agro-industrial wastes for the dual purposes of pollution abatement and cost-effectiveness.MethodsIn the present study, the regular 2-level factorial design was employed to screen fermentation parameters that enhance production of a novel alkaline protease by a strain of Stenotrophomonas acidaminiphila using cassava processing effluent as substrate. Data from randomized experiments of central composite rotatable design for improved enzyme activity, guided by path of steepest ascent experiments, were modeled and optimized by response surface methodology (RSM). Shake flask kinetics of production under optimized conditions was modeled by logistic and modified Gompertz models and determinations of maximum specific growth rate, µmax, maximum volumetric rate of substrate consumption, rsm, maximum volumetric rate of biomass formation, rxm and specific yield of product, Yp/x were made.ResultsLogistic model poorly fitted RSM-generated product formation and substrate consumption data. However, biomass formation was accurately fitted (adjusted r2 >99%), with µmax of 0.471 h-1. The modified Gompertz model, on the contrary, more accurately fitted all three major response data with minimal mean squared error. Potential for scale-up of bioprocess evaluated in 5-L bioreactor satisfactorily revealed 8.5-fold more substrate consumption in bioreactor than in shake flask. The 86.76-fold aqueous two-phase system-purified protease had a specific activity of 1416.73 Umg-1 which improved with increasing surfactant concentration.ConclusionThese results suggest significant bioprocess potential for sustainable cassava effluent management and concomitant commercial production of alkaline protease for industrial detergent application.

Monod model is insufficient to explain biomass growth in nitrogen-limited yeast fermentation

The yeast Saccharomyces cerevisiae is an essential microorganism in food biotechnology; particularly, in wine and beer making. During wine fermentation, yeasts transform sugars present in the grape juice into ethanol and carbon dioxide. The process occurs in batch conditions and is, for the most part, an anaerobic process. Previous studies linked limited-nitrogen conditions with problematic fermentations, with negative consequences for the performance of the process and the quality of the final product. It is, therefore, of the highest interest to anticipate such problems through mathematical models. Here we propose a model to explain fermentations under nitrogen-limited anaerobic conditions. We separated the biomass formation into two phases: growth and carbohydrate accumulation. Growth was modelled using the well-known Monod equation while carbohydrate accumulation was modelled by an empirical function, analogous to a proportional controller activated by the limitation of available nitrogen. We also proposed to formulate the fermentation rate as a function of the total protein content when relevant data are available. The final model was used to successfully explain experiments taken from the literature, performed under normal and nitrogen-limited conditions. Our results revealed that Monod model is insufficient to explain biomass formation kinetics in nitrogen-limited fermentations of S. cerevisiae . The goodness-of-fit of the herewith proposed model is superior to that of previously published models, offering the means to predict, and thus control fermentations. Importance: Problematic fermentations still occur in the winemaking industrial practise. Problems include sluggish rates of fermentation, which have been linked to insufficient levels of assimilable nitrogen. Data and relevant models can help anticipate poor fermentation performance. In this work, we proposed a model to predict biomass growth and fermentation rate under nitrogen-limited conditions and tested its performance with previously published experimental data. Our results show that the well-known Monod equation does not suffice to explain biomass formation.

Analysis and Reconstitution of the Menaquinone Biosynthesis Pathway in Lactiplantibacillus plantarum and Lentilactibacillus buchneri

In Lactococcus lactis and some other lactic acid bacteria, respiratory metabolism has been reported upon supplementation with only heme, leading to enhanced biomass formation, reduced acidification, resistance to oxygen, and improved long-term storage. Genes encoding a complete respiratory chain with all components were found in genomes of L. lactis and Leuconostoc mesenteroides, but menaquinone biosynthesis was found to be incomplete in Lactobacillaceae (except L. mesenteroides). Lactiplantibacillus plantarum has only two genes (menA, menG) encoding enzymes in the biosynthetic pathway (out of eight), and Lentilactobacillus buchneri has only four (menA, menB, menE, and menG). We constructed knock-out strains of L. lactis defective in menA, menB, menE, and menG (encoding the last steps in the pathway) and complemented these by expression of the extant genes from Lactipl. plantarum and Lent. buchneri to verify their functionality. Three of the Lactipl. plantarum biosynthesis genes, lpmenA1, lpmenG1, and lpmenG2, as well as lbmenB and lbmenG from Lent. buchneri, reconstituted menaquinone production and respiratory growth in the deficient L. lactis strains when supplemented with heme. We then reconstituted the incomplete menaquinone biosynthesis pathway in Lactipl. plantarum by expressing six genes from L. lactis homologous to the missing genes in a synthetic operon with two inducible promoters. Higher biomass formation was observed in Lactipl. plantarum carrying this operon, with an OD600 increase from 3.0 to 5.0 upon induction.

Monod model is insufficient to explain biomass growth in nitrogen-limited yeast fermentation

The yeast Saccharomyces cerevisiae is an essential microorganism in food biotechnology; particularly, in wine and beer making. During wine fermentation, yeasts transform sugars present in the grape juice into ethanol and carbon dioxide. The process occurs in batch conditions and is, for the most part, an anaerobic process. Previous studies linked limited-nitrogen conditions with problematic fermentations, with negative consequences for the performance of the process and the quality of the final product. It is, therefore, of the highest interest to anticipate such problems through mathematical models. Here we propose a model to explain fermentations under nitrogen-limited anaerobic conditions. We separated the biomass formation into two phases: growth and carbohydrate accumulation. Growth was modelled using the well-known Monod equation while carbohydrate accumulation was modelled by an empirical function, analogous to a proportional controller activated by the limitation of available nitrogen. We also proposed to formulate the fermentation rate as a function of the total protein content when relevant data are available. The final model was used to successfully explain experiments taken from the literature, performed under normal and nitrogen-limited conditions. Our results revealed that Monod model is insufficient to explain biomass formation kinetics in nitrogen-limited fermentations of S. cerevisiae. The goodness-of-fit of the herewith proposed model is superior to that of previously published models, offering the means to predict, and thus control fermentations.

Carbon-Source Dependent Interplay of Copper and Manganese Ions Modulates the Morphology and Itaconic Acid Production in Aspergillus terreus

The effects of the interplay of copper(II) and manganese(II) ions on growth, morphology and itaconic acid formation was investigated in a high-producing strain of Aspergillus terreus (NRRL1960), using carbon sources metabolized either mainly via glycolysis (D-glucose, D-fructose) or primarily via the pentose phosphate shunt (D-xylose, L-arabinose). Limiting Mn2+ concentration in the culture broth is indispensable to obtain high itaconic acid yields, while in the presence of higher Mn2+ concentrations yield decreases and biomass formation is favored. However, this low yield in the presence of high Mn2+ ion concentrations can be mitigated by increasing the Cu2+ concentration in the medium when D-glucose or D-fructose is the growth substrate, whereas this effect was at best modest during growth on D-xylose or L-arabinose. A. terreus displays a high tolerance to Cu2+ which decreased when Mn2+ availability became increasingly limiting. Under such conditions biomass formation on D-glucose or D-fructose could be sustained at concentrations up to 250 mg L–1 Cu2+, while on D-xylose- or L-arabinose biomass formation was completely inhibited at 100 mg L–1. High (>75%) specific molar itaconic acid yields always coincided with an “overflow-associated” morphology, characterized by small compact pellets (<250 μm diameter) and short chains of “yeast-like” cells that exhibit increased diameters relative to the elongated cells in growing filamentous hyphae. At low concentrations (≤1 mg L–1) of Cu2+ ions, manganese deficiency did not prevent filamentous growth. Mycelial- and cellular morphology progressively transformed into the typical overflow-associated one when external Cu2+ concentrations increased, irrespective of the available Mn2+. Our results indicate that copper ions are relevant for overflow metabolism and should be considered when optimizing itaconic acid fermentation in A. terreus.