Scaling of the process of biosynthesis of surfactants by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> EK-1 on hexadecane

Peculiarities of synthesis of surface-active substances (SAS) are studied at periodical cultivation of Rhodococcus erythropolis EK-1 in the AK-210 fermenter on medium containing n-hexadecane. Maximum indicators of SAS synthesis (concentration of extra cellular SAS is 7.2 g/l; factor of emulsification of the cultural liquid 50%; SAS yield from the substrate 50%) have been observed at 60–70% concentration of dissolved oxygen from the saturation level with aerial oxygen (pH 8.0) fractional supply of the substrate by portions each being 0.3–0.4% every 5–6 h to a final volume concentration of 2.4% and with the use of 10% inoculate grown until mid-exponential phase on the medium with 1.0 vol % of n-hexadecane. Implementation of the process of SAS biosynthesis with the fermentation equipment provided the possibility to increase almost two-fold the amount of the synthesized SAS and reduce 3.5-fold the time of cultivation of the producer strain compared with the growth in flasks at shake-flask propagator.     

Intensification of surfactant synthesis in Rhodococcus erythropolis EK-1 cultivated on hexadecane

Activity of key enzymes of n-alkane metabolism was determined in cells of Rhodococcus erythropolis EK-1, a surfactant producer grown on n-hexadecane. Potassium cations were found to inhibit alkane hydroxylase and NADP(+)-dependent aldehyde dehydrogenase, while sodium cations were found to activate these enzymes. Decreased potassium concentration (to 1 mM), increased sodium concentration (to 35 mM), and addition of 36 micromol/l Fe(II), required for alkane hydroxylase activity, resulted in increased activity of the enzymes of n-hexadecane metabolism and in a fourfold increase of surfactant synthesis. A 1.5-1.7-fold increase in surfactant concentration after addition of 0.2% fumarate (gluconeogenesis precursor) and 0.1% citrate (lipid synthesis regulator) to the medium with n-hexadecane results from enhanced synthesis of trehalose mycolates, as evidenced by a 3-5-fold increase in phosphoenolpyruvate synthetase and trehalose phosphate synthase, respectively.     

Factors influencing the cultivability of lake water bacteria

Counting bacteria in natural water samples by cultivation yields only low recovery efficiencies (ca. 1%), compared to total counts obtained after 4,6-diamidino-2-phenylindol (DAPI) staining. In order to optimize the cultivation of heterotrophic planktonic bacteria from Lake Constance (Germany), selected parameters of the medium composition were modified. The most important factor was the concentration of organic substrate (nutrient broth plus yeast extract), which significantly influenced the "most probable number" obtained in liquid growth medium. Reduced oxygen concentrations (3-12%) lowered the "most probable number". Addition of N-acyl homoserine lactones to the medium increased the cultivability slightly. Low substrate concentrations [0.03-0.06% (w/v)], an incubation atmosphere of 21% oxygen at 16 degrees C for 4 weeks were optimal and increased the cultivability ("most probable number" related to total bacterial counts) to an average cultivability of 18+/-11%, (n=8). The results indicate that cultivabilities of heterotrophic bacteria from lakewater samples can be significantly increased by modifying the cultivation methods.     

Use of n-hexadecane as an oxygen vector to improve Phaffia rhodozyma growth and carotenoid production in shake-flask cultures

AIMS: To identify beneficial oxygen vectors for Phaffia rhodozyma in liquid cultures, and to evaluate their use to improve the oxygen transfer and carotenoid production in the yeast cultures. METHODS AND RESULTS: Several liquid hydrocarbons were tested as oxygen vectors for improving the yeast growth and carotenoid production in shake-flask cultures of P. rhodozyma. While all nontoxic organic liquids (Log P: > or =5.6) showed a positive effect, n-hexadecane was proved to be the most beneficial for the yeast growth and carotenoid production. The addition of 9% (v/v) n-hexadecane to the liquid medium at the time of inoculation was found to be optimal, increasing the carotenoid yield by 58% (14.5 mg l(-1) vs 9.2 g l(-1) in the control) and the oxygen transfer rate (OTR) by 90%. CONCLUSIONS: The addition of n-hexadecane to shake-flask cultures of P. rhodozyma significantly improved the oxygen transfer in culture, thus increasing the carotenoid production. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of organic oxygen vectors such as n-hexadecane may be a simple and useful means for enhancing oxygen transfer and carotenoid production in liquid fermentation of P. rhodozyma.     

Gordonia sp. LAM0048的分离鉴定及其降解正十六烷的研究

以正十六烷为唯一碳源,从长期受石油污染的土壤中筛选到一株高效降解正十六烷的菌株LAM0048。通过形态学观察、生理生化试验、细胞化学组分分析、16S rRNA基因序列分析、细胞脂肪酸和极性脂试验,确定其属于棒杆菌亚目(Corynebacterineae)、诺卡菌科(Nocardiaceae)、戈登氏属(Gordonia),且可能为戈登氏属的一株新种。采用单因素实验对菌株LAM0048在无机盐培养基中降解正十六烷的降解率进行初步探讨,发现该菌株能在以正十六烷为唯一碳源的培养基中生长,菌株LAM0048能够在36 h内完全降解0.05%(V/V)的正十六烷,当烷烃浓度达到1.0%(V/V)时,降解率达46.4%。结果表明LAM0048是一株具有耐受高浓度烷烃的石油降解菌,在石油污染环境的微生物修复中具有一定的应用潜力。     

Production of a Pseudomonas lipase in n-alkane substrate and its isolation using an improved ammonium sulfate precipitation technique

Among the various lipidic and non-lipidic substances, normal alkanes within the chain lengths of C-12 to C-20 served as the best carbon substrates for the production of extracellular lipase by Pseudomonas species G6. Maximum lipase production of 25 U/ml of the culture broth was obtained by using n-hexadecane as the sole carbon substrate. The optimum pH of 8 and temperature of 34 + 1 degrees C were demonstrated for the production of lipase in n-hexadecane substrate. The optimum concentration of iron, which played a critical role on the lipase production, was found to be 0.25 mg/l. Lipase production could be enhanced to nearly 2.4-fold by using tributyrin at a concentration of 0.05% (v/v) in the culture medium. High recovery of the lipase protein (83%) from the culture broth was achieved by treating the culture supernatant with Silicone 21 Defoamer followed by ammonium sulfate (60% saturation) fractionation.     

The influence of conditions of <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> K-4 strain cultivation on surface-active substances synthesis

It has been observed that the Acinetobacter calcoaceticus K-4 strain produces surface-active substances (SAS) while growing either on hydrophilic (ethanol) or on hydrophobic substrates (hexadecane). Maximal SAS synthesis (with a conditional SAS concentration of 3.6; emulsifying activity of culture liquid dissolved in 50 times equal to 96%) was detected with growth on an ethanol-containing medium with the addition of urea, yeast autolysate and microelements, C/N ratio 60:1 and 10% inoculate, cultivated on ethanol-containing medium by the end of the exponential phase of growth. With respect to its chemical nature, extracellular SAS synthesized by A. calcoaceticus K-4 growing on ethanol-containing medium under optimal cultivating conditions form a glycolipid-aminolipid complex.     

Production of biosurfactant using different hydrocarbons by Pseudomonas aeruginosa EBN-8 mutant

The present investigation dealt with the use of previously isolated and studied gamma-ray mutant strain Pseudomonas aeruginosa EBN-8 for the production of biosurfactant by using different hydrocarbon substrates viz. n-hexadecane, paraffin oil and kerosene oil, provided in minimal medium, as the sole carbon and energy sources. The batch experiments were conducted in 250 mL Erlenmeyer flasks, containing 50 mL minimal salt media supplemented with 1% (w/v) hydrocarbon substrate, inoculated by EBN-8 and incubated at 37 degrees C and 100 rpm in an orbital shaker. The sampling was done on 24 h basis for 10 d. The surface tension of cell-free culture broth decreased from 53 to 29 mN/m after 3 and 4 d of incubation when the carbon sources were paraffin oil and n-hexadecane, respectively. The largest reduction in interfacial tension from 26 to 0.4 mN/m was observed with n-hexadecane, while critical micelle dilution was obtained as 50 x CMC for paraffin oil as carbon source. When grown on n-hexadecane and paraffin oil, the EBN-8 mutant strain gave 4.1 and 6.3 g of the rhamnolipids/L, respectively. These surface-active substances subsequently allowed the hydrocarbon substrates to disperse readily as emulsion in aqueous phase.     

Effect of cultivation conditions on the adhesive activity of Rhodococcus cells towards n-Hexadecane

The effect of cultivation conditions (the composition, acidity, and salinity of the cultivation medium; temperature; and the hydrodynamic conditions of cultivation) on the adhesion of actinobacteria of the genus Rhodococcus to n-hexadecane has been investigated. A study performed showed that the adhesive activity of rhodococci depends on the composition of the cultivation medium and on the cultivation temperature. The possible mechanisms underlying the effect of growth conditions on the adhesion of rhodococci to liquid hydrocarbons and involving changes in the cell lipid content or the zeta potential of cells are addressed. Rhodococcal strains displaying high adhesive activity (80–90%) at a low temperature (18°C), high salinity (5.0% NaCl), and acidity (pH 6.0) of the cultivation medium have been selected as a result of the present work; these strains have a considerable potential for use in bioremediation of soil and water contaminated by hydrocarbons.     

Enhanced plasmid production in miniaturized high-cell-density cultures of Escherichia coli supported with perfluorinated oxygen carrier

A simple method for plasmid minipreps in closed 1.5 mL microcentrifuge tubes using a cultivation medium with internal substrate delivery (EnBase^®) in combination with a two-phase perfluorodecalin (PFD) system supplying additional oxygen to the E. coli culture is described. The procedure can simply be performed on a thermoshaker using only 50 μL cultivation volume. Twenty and twenty-five percent higher cell densities and plasmid concentration, respectively, were obtained with the additional oxygen delivery system when compared to cultures without PFD. Compared to standard 2 mL LB cultures ninefold higher cell densities and eightfold higher plasmid concentrations were achieved for the smaller culture volume. The μL-scale cultures can be directly utilized in further plasmid purification without any centrifugation step or the subsequent removal of the supernatant. This simplifies the routine procedure considerably. Furthermore, the new method is very robust considering the time of cultivation. Highest plasmid concentrations were already obtained after only 6 h of cultivation, but the plasmid concentration remained high (87 % of the maximum) even until 8 h of cultivation. Aside from the advantage of this method for the daily routine, we believe that it could also be applied to automated high-throughput processes.     

Utilization of Collagenous By-Products from the Meat Packing Industry: Production of Single-Cell Protein by the Continuous Cultivation of Bacillus megaterium

The conditions for continuous cultivation of Bacillus megaterium on a collagen-derived substrate (SP-100) were determined. The optimum conditions of temperature, pH, and dilution rate were 34 C, pH 7.0, and 0.25/hr, respectively. Increasing the substrate concentration in plain tap water resulted in proportional increases in the productivity of cell mass from 0.6 g per liter per hr at 1% substrate to 1.8 g per liter per hr at 10% substrate; however, the protein content of the biomass decreased from 60 to 36%, and the protein yield decreased from 91 to 50% at substrate concentrations of 1 and 10%, respectively. These effects (decreases) were reversed up to 7.5% substrate by mineral supplementation of the medium. The productivity of biomass increased from 0.6 to 1.9 per liter per hr; the protein content of the biomass, from 43 to 54%; and the protein yield, from 60 to 93%, respectively, as the substrate concentration (with mineral supplementation of the medium) was increased from 1 to 7.5%. Spent medium could be refortified and recycled as often as five times. The amino acids in the substrate protein appeared to be utilized for growth and metabolism more or less uniformly. Analysis of the B. megaterium biomass indicated considerable enrichment of the essential amino acids and reduction of proline, glycine, and hydroxyproline as compared to the collagen-derived substrate. The Protein Efficiency Ratios obtained on the collagen-derived substrate (SP-100) and on the B. megaterium biomass, expressed as percentages of the casein reference protein, were 14 and 74%, respectively. Thus, considerable improvement in nutritional value was effected by bacterial conversion of the collagen-derived substrate into single-cell protein.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

Growth and substrate consumption of Nitrobacter agilis cells immobilized in carrageenan: part 2. Model evaluation

A dynamic model that predicts substrate and biomass concentration profiles across gel beads and from that the overall substrate consumption rate by the gel beads containing growing cells was evaluated with immobilized Nitrobacter agilis cells in an airlift loop reactor with oxygen as the limiting substrate. The model predictions agreed well with the observed oxygen consumption rates at three different liquid phase oxygen concentrations. Image analysis showed that 90% of the immobilized cells after 42 days of cultivation was situated in the outer shells in a film of 140 mum, while the bead radius was about 1 mm. The maximum biomass concentration in the outmost film of 56 mum was 11 kg . m(-3) gel.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Optimum conditions for ursodeoxycholic acid production from lithocholic acid by Fusarium equiseti M41.

Ursodeoxycholic acid dissolves cholesterol gallstones in humans. In the present study optimum conditions for ursodeoxycholic acid production by Fusarium equiseti M41 were studied. Resting mycelia of F. equiseti M41 showed maximum conversion at 28 degrees C, pH 8.0, and dissolved oxygen tension of higher than 60% saturation. Monovalent cations, such as Na+, K+, and Rb+, stimulated the conversion rate more than twofold. In the presence of 0.5 M KCl, the initial uptake rate and equilibrium concentration of lithocholic acid (substrate) were enhanced by 5.7- and 1.7-fold, respectively. We confirmed that enzyme activity catalyzing 7 beta-hydroxylation of lithocholic acid was induced by substrate lithocholic acid. The activity in the mycelium was controlled by dissolved oxygen tension during cultivation: with a dissolved oxygen tension of 15% and over, the activity peak appeared at 25 h of cultivation, whereas the peak was delayed to 34 and 50 h with 5 and 0% dissolved oxygen tension, respectively. After reaching the maximum, the 7 beta-hydroxylation activity in the mycelium declined rapidly at pH 7.0, but the decline was retarded by increasing the pH to 8.0. Several combinations of operations, such as pH shift (from pH 7 to 8), addition of 0.5 M KCl, and dissolved oxygen control, were applied to the production of ursodeoxycholic acid in a jar fermentor, and a much larger amount of ursodeoxycholic acid (1.2 g/liter) was produced within 96 h of cultivation.     

Aerobic degradation of toluene in a closed chemostat with and without a head space outlet

The present paper describes the continuous aerobic cultivation of a Pseudomonas strain with toluene as the substrate in a closed chemostat with oxygen or air as the gas phase. Due to the constant supply of a nitrogen-saturated aqueous medium, nitrogen passes from the liquid phase of the chemostat into the gas phase (head space). This results in an increasing nitrogen content (asymptotic approach to 100%). The concomitant decrease in the partial pressure of the oxygen in the gas phase finally leads to an oxygen limitation for the bacteria in the medium and an incomplete toluene degradation. The critical nitrogen content of the gas phase at which oxygen limitation begins depends on the toluene concentration in the incoming medium. However, when the gas is continuously removed from the head space, the nitrogen content reaches a steady-state value of less than 100%, depending on the flow rate of the outgoing gas. The oxygen limitation and the associated incomplete toluene degradation can be prevented in this way. The method of gas removal from the head space to avoid oxygen limitation is also applicable when the reactor is supplied with air instead of oxygen. Waste waters contaminated with highly volatile pollutants can thus be biologically decontaminated under aerobic conditions, without shifting the pollution problem from the liquid to the gas phase.     

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.     

Biotransformation of 2,2-Dimethyl-1,3-Propanediol to 3-Hydroxypivalic Acid by Acetobacter Acetii DSMZ3508 and Related Bacteria

Acetobacter acetii DSMZ3508 and related bacteria converted 2,2-dimethyl-1,3-propanediol into 3-hydroxypivalic acid (2,2-dimethyl-3-hydroxypropionic acid; 3HP) during submerged cultivation in mineral salt medium. The maximum yield of 3-hydroxypivalic acid was 24.4% of the fed substrate after 18 days. Cultivation parameters, as pH, cell density, optimal substrate concentration, and oxygen supply for the bioconversion process were determined.     

Control of dissolved oxygen concentration in mammalian cell culture using a computer-coupled mass flow controller

Summary A simple proportional control system for dissolved oxygen (DO) concentration in cell culture medium was developed by using a computer-coupled mass flow controller. The DO levels were very stable during the cultivation of Vero-6, while flow rates of air and/or oxygen enriched air were gradually changed depending on the DO concentration and the preset DO level. Vero-6 cells could grow normally to the confluence in the range of 30% and 50% of DO. Growth of Vero-6 at 10% of DO was markedly retarded.     

Function and regulation of cytochrome P-450 in alkane-assimilating yeast

Transition of n-hexadecane utilizing cultures of Candida maltosa to oxygen-limited growth caused an up to 6-fold increase of the cellular cytochrome P-450 content. Enhanced cytochrome P-450 formation required protein de novo synthesis and was not due to a change of the apo/holo-enzyme ratio as demonstrated by cycloheximide inhibition and immunological quantitation. The effect of low oxygen concentration (pO₂=3–5%) was simulated by selective inhibition of alkane hydroxylation with carbon monoxide (at a pO₂ of 70–75%). Enhanced cytochrome P-450 formation occurred even when a constant growth rate was maintained through utilization of a second non-repressive growth substrate. However, the presence of n-alkanes was an essential precondition. It was concluded, that the cytochrome P-450 formation was mainly regulated by the intracellular inducer concentration which depends on the relative rates of alkane transport into the cell and the actual alkane hydroxylating activity of the enzyme system.Abbreviation cyt cytochrome