Biochemical and physiological characterization of the efrotomycin fermentation

Summary An efrotomycin fermentation was characterized through physical, chemical and biochemical studies. Growth of the actinomycete,Nocardia lactamdurans occurred during the first 50 h of the fermentation cycle at the expense of glucose, protein, and triglycerides. The initiation of efrotomycin biosynthesis was observed when glucose dropped to a low concentration. Upon glucose depletion, cell growth ceased and a switch in the respiratory quotient occurred. Efrotomycin biosynthesis was supported by the utilization of soybean oil and starch. Analysis of triglyceride metabolism showed that no diglycerides or monoglycerides accumulated during the fermentation. The activity of extracellular enzymes (lipase, protease, and amylase) increase during the cell growth phase and decreased significantly after 150 h. The concentrations of DNA, tetrahydro-vitamin K₂ (a membrane component), and free amino acids in the supernatant increased dramatically late in the fermentation cycle (225 h), indicating massive cell lysis. During this same time period, a reduction in cellular respiratory activity and efrotomycin biosynthesis were observed.     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

Continuous acetone-butanol fermentation: a global approach for the improvement in the solvent productivity in synthetic medium

Summary The present chemostat culture studies were performed in a partial gas recycle system using Clostridium acetobutylicum ATCC 824. Initiation of chemostat cultures at two different levels of vitamins has demonstrated a more than 3 fold improvement in the solvent productivity. The production of extracellular autobacteriocin was increased markedly, when strict anaerobic conditions of the feed vessel were not maintained. When pH was regulated at 4.4, very little variation was seen in the concentration of acids. The duration of solvent production was reduced significantly when NH₄OH was replaced by NaOH for pH regulation, whereas a marked increase in the production of extracellular autobacteriocin was observed. In the optimised medium conditions, a solvent productivity of 2.09 g l^-1 h^-1 (highest ever) could be obtained in the synthetic medium.     

Ouabain-resistant cells cultured in a synthetic medium

Several strains of kidney and liver cells cultured in a synthetic medium were found to be resistant to ouabain. These cell strains were characterized because this resistance may serve as a good marker in genetic studies on somatic cells in chemically defined conditions in the absence of Na+ related growth factors and hormones. The phenotype was stable in the absence of selection for at least two years, and the original strains before adaptation to the synthetic medium were found to have ouabain sensitivity equal to the corresponding cells in the synthetic medium. The resting membrane potential, Na+,K+-ATPase activity, and growth rate of the resistant cells were similar to those of ouabain-sensitive cells. The resistance of the cells was not affected by serum or antibodies against some cytoskeletal proteins and the sensitivity of the Na+,K+-ATPase was not restored by partial purification of the membranes. Western blotting of the Na+,K+-ATPase of the ouabain-resistant cells showed that the molecular weights of its two subunits and its immunoreactivity were similar to those of the enzyme from the ouabain-sensitive strain. Thus the ouabain resistance is caused not by ouabain-like hormone produced by the cells or change in the cytoskeletal system, but by a mutation resulting in expression of an ouabain-resistant ATPase gene.     

Kinetics of fermentation processes

Kinetic studies on fermentation processes were made and a general equation of production rate was newly presented applying the kinetic theory on mierobial cell growth which was reported previously by the authors.^l,2 Equations for product concentration in fermentation time courses were derived by developing mathematically the general equation of production rate, and characteristic properties of fermentation processes were clarified. Some examples of fermentations were analyzed kinetically using the new kinetic theory. The calculated values of product, and cell concentrations were in good agreement with the observed values.     

Characteristics of granular methanogenic sludge grown on phenol synthetic medium and methanogenic fermentation of phenolic wastewater in a UASB reactor

The growth of granules on a phenol synthetic medium and the methanogenic fermentation of industrial phenolic wastewater from a steel factory in an upflow anaerobic sludge blanket (UASB) reactor were investigated. Total granular sludge concentration retained in the UASB reactor was 6.7 g MLSS/l (6.0 g MLVSS/l) during the 10 months' operation on the phenol synthetic medium. This realized a maximum phenol removal rate of 2.2 g/l·d (phenol concentration of influent = 500 mg/l), which corresponded to 5.2 g COD/l·d at space velocity (SV) of 4.4 d⁻¹. The granules formed were of relatively small size ranging from 0.61 to 0.77 mm, and had a relatively low density of 0.013–0.023 g MLVSS/cm³ and low specific gravity (1.11) due to very low ash content (8.7–11.9%). Electron microscopic analysis showed that Methanothrix spp. appeared dominantly on the granule surface as well as within it. The specific metabolic activities of bacterial trophic groups were the highest for H₂ followed by acetate, benzoate, phenol, and propionate. In the case of industrial phenolic wastewater, although phenol efficiency was only 50% at SV of 0.4 d⁻¹, when the wastewater was diluted twofold and the treated wastewater was recycled at SV of 7.3 d⁻¹, the removal efficiencies of phenol and CODcr were restored to 90% (influent=400 mg/l) and 80% (influent=5,000 mg/l), respectively. It was suggested that recycling of the treated wastewater might be improved by partly degrading unknown toxic compounds contained in phenolic wastewater.     

Evaluation of ethanol production by a new isolate of yeast during fermentation in synthetic medium and sugarcane bagasse hemicellulosic hydrolysate

A new xylose fermenting yeast was isolated from over-ripe banana by enrichment in xylose-containing medium. The phylogenetic analysis of ITS1-5.8S-ITS2 region sequences of ribosomal RNA of isolate BY2 revealed that it shows affiliation to genus Pichia and clades with Pichia caribbica. In batch fermentation, Pichia strain BY2 fermented xylose, producing 15 g l^?1 ethanol from 30 g l^?1 xylose under shaking conditions at 28°C, with ethanol yield of 0.5 g g^?1 and volumetric productivity of 0.31 g l^?1 h^?1. The optimum pH range for ethanol production from xylose by Pichia strain BY2 was 5–7. Pichia strain BY2 also produced 6.08 g l^?1 ethanol from 30 g l^?1 arabinose. Pichia strain BY2 can utilize sugarcane bagasse hemicellulose acid hydrolysate for alcohol production, efficiency of fermentation was improved by neutralization, and sequential use of activated charcoal adsorption method. Percent total sugar utilized and ethanol yield for the untreated hydrolysate was 17.14% w/v and 0.33 g g^?1, respectively, compared with 66.79% w/v and 0.45 g g^?1, respectively, for treated hemicellulose acid hydrolysate. This new yeast isolate showed ethanol yield of 0.45 g g^?1 and volumetric productivity of 0.33 g l^?1 h^?1 from sugarcane bagasse hemicellulose hydrolysate detoxified by neutralization and activated charcoal treatment, and has potential application in practical process of ethanol production from lignocellulosic hydrolysate.     

Kinetics of the xanthan fermentation

Xanthan gum, a heteropolysaccharide with unusual and useful properties, is now produced commercially by fermentation with Xanthomonas compestris NRRL B–1459 in a medium containing glucose, minerals, and a complex nitrogen source—distillers' dried solubles (DDS). Understanding the kinetics of the fermentation should contribute to process improvements and increase the market potential for the gum. Earlier studies showed that although DDS determined initial growth rate, growth was stopped by some mechanism other than substrate exhaustion, probably an effect related to product formation. Product formation did not require active growth, but its rate increased with cell concentration. Specific product formation rate declined at high viscosities. Varying glucose concentration from 0.5 to 5.0% and dissolved O₂ tension between 20 and 90% air saturated had no effect on the rates, but pH had to be maintained near 7 and temperature near 28°C to permit continued product formation. Xanthan yield could be explained by the energy required for growth and polymerization, that energy coming from dissimilation of the part of the carbohydrate substrate not converted to polymer.     

Improvement of a synthetic medium for Dictyostelium discoideum

Dictyostelium discoideum is of considerable interest as an expression system for the production of proteins of high value. The cultivation of this social amoeba is not as easy as that of other common microbial expression systems. Wildtype strains grow on bacteria. Mutant strains growing on axenic media reach cell densities of 1–2×10⁷ ml⁻¹ when cultivated in commonly used complex media. A totally synthetic medium formulated by Franke and Kessin (Proc. Natl. Acad. Sci. USA 74 (1977) 2157) has become popular and allows cell densities of about 3×10⁷ ml⁻¹ to be obtained. This medium (FM) is being improved mainly on the basis of the analysis of limitations with respect to amino acids. With this improved synthetic medium (SIH) cell densities in the order of 5–6×10⁷ ml⁻¹ have been achieved.     

Triiodothyronine accelerates the synthesis of Synapsin I in developing neurons from fetal rat brain cultured in a synthetic medium

The effect of Triiodothyronine (T3) on Synapsin I appearance in rat cortical neurons has been investigated in vitro. Neuronal cultures from 16-day-old fetal rat brain grown in the absence of T3, express immunohystochemically detectable Synapsin I at the 14th day in vitro. The addition of the hormone to the culture medium determines an early (at the 7th day in vitro) appearance of fluorescent dots specific for Synapsin I.     

Kinetics of ethanol inhibition in alcohol fermentation

The inhibitory effect of ethanol on yeast growth and fermentation has been studied for the strain Saccharomyces cerevisiae ATCC No. 4126 under anaerobic batch conditions. The results obtained reveal that there is no striking difference between the response of growth and ethanol fermentation. Two kinetic models are also proposed to describe the kinetic pattern of ethanol inhibition on the specific rates of growth and ethanol fermentation: \documentclass{article}\pagestyle{empty}\begin{document}$$\begin{array}{*{20}c} {\frac{{\mu _i }}{{\mu _0 }} = 1{\rm } - {\rm }\left( {\frac{P}{{P_m }}} \right);\alpha } \hfill & {\left( {{\rm for}\ {\rm growth}} \right)} \hfill \\ {\frac{{\nu _i }}{{\nu _0 }} = 1{\rm } - {\rm }\left( {\frac{P}{{P'_m }}} \right);\beta } \hfill & {\left( {{\rm for}\ {\rm ethanol}\ {\rm production}} \right)} \hfill \\ \end{array}$$\end{document} The maximum allowable ethanol concentration above which cells do not grow was predicted to be 112 g/L. The ethanol-producing capability of the cells was completely inhibited at 115 g/L ethanol. The proposed models appear to accurately represent the experimental data obtained in this study and the literature data.     

Kinetics of inhibition in propionic acid fermentation

The inhibitory effect of propionic acid P and biomass concentration X is studied in batch and continuous fermentations with cell recycle.In batch fermentations, the specific growth rate decreases and cancels out at a critical propionic acid concentration Pc ₁; the formerly decreasing specific production rate becomes constant after Pc ₁ and cancels out when a second critical propionic acid concentration Pc ₂ is reached.In continuous fermentation with cell recycle, a similar inhibition is observed with biomass. The specific rates decrease and become constant at a critical biomass concentration Xc. They cancel out at different high biomass concentrations.In both cases, the specific production rate can be related to the specific growth rate by the Luedeking and Piret expression: =+, [1], where the constants and are determined by the fermentation parameters.List of Symbols t h time - X kg/m³ biomass concentration - P kg/m³ propionic acid concentration - A kg/m³ acetic acid concentration - S kg/m³ lactose concentration - dX/dt kg/(m³h) instantaneous rate of cell growth - dP/dt kg/(m³h) instantaneous rate of propionic acid production - h^–1 specific growth rate - h^–1 specific propionic acid production rate - D h^–1 dilution rate     

Development of Thermoactinomyces vulgaris on a synthetic medium]

A synthetic medium was used to obtain the dormant spores of Thermoactinomyces vulgaris. The fraction of the dormant spores depended on the amino acid composition of the growth medium. The rate of growth and development of the organism on the synthetic medium is lower as compared to the routinely employed complex medium.     

Development of a synthetic minimal medium for Listeria monocytogenes

A defined solid and liquid minimal medium, HTM, which contained methionine and cysteine as the sole amino acids, was developed for Listeria monocytogenes. Complex broth-grown L. monocytogenes had to adapt to HTM by inducing amino acid biosyntheis. HTM is the simplest minimal medium available for growth of L. monocytogenes.     

Kinetics of polysaccharide B-1459 fermentation

Polysaccharide gum was made by fermentation with Xanthomonas campestris NRRL B-1459 in a medium of glucose, minerals, and distillers' solubles. The effect of distillers' solubles on growth rate can be described by the familiar saturation equation. Although a quasistoichiometric relationship was observed between nitrogen utilization and growth, total nitrogen supply was not growth limiting, nor was polymer formation growth associated. Cell growth primarily took place in the early part of the fermentation; polysaccharide biosynthesis occurred throughout the fermentation. Glucose was converted to polysaccharide at a fairly constant yield, which was 70–80% of glucose consumed, under optimum conditions. The kinetic patterns observed indicate that multistage continuous fermentation will be suitable for polysaccharide production.