Degradation of phenol by Pseudomonas putida ATCC 11172 in continuous culture at different ratios of biofilm surface to culture volume.

Pseudomonas putida ATCC 11172 was grown in continuous culture with phenol as the only carbon and energy source; a culture practically without biofilm was compared with biofilm cultures of differing surface area/volume ratios. The biofilm did not significantly affect the maximal suspended cell concentration in the effluent, but it increased the maximal phenol reduction rate from 0.23 g/liter per h (without biofilm) to 0.72 g/liter per h at the highest biofilm level (5.5 cm2 of biofilm surface per ml of reactor volume). The increase in phenol reduction rate was linear up to the surface area/volume ratio of 1.4 cm2/ml. The continuous cultures with biofilms could tolerate a higher phenol concentration of the medium (3.0 g/liter) than the nonbiofilm system (2.5 g/liter). At higher dilution rates an intermediate product, 2-hydroxymuconic semialdehyde, accumulated in the culture. When the biomass of the effluent started to decrease, the concentration of 2-hydroxymuconic semialdehyde reached a peak value. We conclude that biofilms in continuous culture have the potential to enhance the aerobic degradation of aromatic compounds.     

Phenol degradation by Ralstonia eutropha: colorimetric determination of 2-hydroxymuconate semialdehyde accumulation to control feed strategy in fed-batch fermentations.

Phenol biodegradation by Ralstonia eutropha was modeled in different culture modes to assess phenol feeding in biotechnological depollution processes. The substrate-inhibited growth of R. eutropha was described by the Haldane equation with a Ks of 2 mg/L, a Ki of 350 mg/L and a mumax of 0.41 h(-1). Furthermore, growth in several culture modes was characterized by the appearance of a yellow color, due to production of a metabolic intermediate of the phenol catabolic pathway, 2-hydroxymuconic semialdehyde (2-hms) which was directly correlated to the growth rate and/or the phenol-degradation rate, because these two parameters are coupled (as seen by the constant growth yield of 0.68 g biomass/g phenol whatever the phenol concentration). This correlation between color appearance and metabolic activity was used to develop a control procedure for optimal phenol degradation. A mass-balance equation modeling approach combined with a filtering step using an extended Kalman filter enabled state variables of the biological system to be simulated. A PI controller, using the estimation of the phenol concentration provided by the modeling step, was then built to maintain the phenol concentration at a constant set-point of 0.1 g/L which corresponded to a constant specific growth rate of 0.3 h(-1), close to the maximal specific growth value of the strain. This monitoring strategy, validated for two fed-batch cultures, could lead, in self-cycling fermentation systems, to a productivity of more than 19 kg of phenol consumed/m(3)/d which is the highest value reported to date in the literature. This system of monitoring metabolic activity also protected the bacterial culture against toxicity problems due to the transient accumulation of phenol.     

Cell growth optimization in microcarrier culture

Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephandex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm2 of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per milliliter of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for scale up into large volume production units.     

Characteristics of draft tube gas-liquid-solid fluidized-bed bioreactor with immobilized living cells for phenol degradation

Biological phenol degradation in a draft tube gas-liquid-solid fluidized bed (DTFB) bioreactor containing a mixed culture immobilized on spherical activated carbon particles was investigated. The characteristics of biofilms including the biofilm dry density and thickness, the volumetric oxygen mass transfer coefficient, and the phenol removal rates under different operating conditions in the DTFB were evaluated. A phenol degradation rate as high as 18 kg/m(3)-day with an effluent phenol concentration less than 1 g/m(3) was achieved, signifying the high treatment efficiency of using a DTFB.     

Cell growth optimization in microcarrier culture

Summary Three monkey kidney cell lines and primary chicken embryo cells were grown in microcarrier culture. The carrier support was DEAE-Sephadex gel beads at low anion exchange capacity prepared according to a protocol developed at the Massachusetts Institute of Technology. The growth rate of the cells and the final cell density in microcarrier culture was dependent on the concentration of the beads in culture and on the size of the initial cell inoculum. A bead concentration of 1.0 to 2.0 mg of beads/ml of tissue culture medium and a cell inoculum of 20,000 cells/cm² of bead surface appeared to be optimal. The efficiency of the microcarrier culture system was compared to that of stationary and roller bottle cultures. Stationary flasks gave cell densities about twofold higher than maximal densities in roller bottles and about threefold and twofold higher than cell densities in microcarrier culture at a bead concentration of 2.5 and 1.0 mg/ml, respectively. In terms of cell yield per millitier of tissue culture medium, the microcarrier culture was superior to roller bottle and stationary cultures. An advantage of the microcarrier culture system is its suitability for a scale up into large volume production units.     

Estimation of the biofilm formation ability of Pseudomonas putida in discrete samples from continuous culture

Summary Test systems were set up in order to evaluate the ability of biomass from a continuous culture to form biofilms. A film-forming strain of Pseudomonas putida was used as the test organism. The adsorption of resting cells onto glass surfaces was measured in specially designed chambers containing 1 ml of cell suspension. Both the quantity and the physiological activity of the adsorbed cells, in terms of optical density after detachment and pH change of a substrate exposed to the adsorbed cells, were measured. The analysis of biomass from continuous cultures of Pseudomonas putida verified the suitability of the methods. Furthermore, other properties of importance to biofilm formation such as hydrophobicity and flocculation capacity of the cells were investigated.It was shown for samples deriving from different dilution rates that the cell adsorption rate drastically increased at dilution rates higher than the _max of the culture. Simultaneously, higher values of hydrophobicity and flocculation capacity were observed.It was also shown that the age and thickness of the biofilm subsequently produced in the continuous culture influenced the metabolic activity per unit of biomass attached to the surface. The methods described in this investigation may facilitate the study of parameters important to biofilm formation as well as the metabolic activity of the attached biomass.     

Characterization of anaerobic microbial culture with high acidogenic activity

The mixed cultures which were used were isolated from municipal sludge digesters, and the production of organic acids (acetic, propionic, butyric, etc.) from carbohydrates was tested. The behavior of the reference population (culture R) obtained directly from the sewage treatment plant, is compared to that obtained after three months in a plug-flow reactor (Gradostat fermentor) without pH control (culture A) and after six months with pH control (culture B). For culture B, the specific rate of acid production is related to the cell growth rate by (1/X)r_p= 17 µ + 1.6 with a maximal acid concentration of 40 g/liter. The batch culture yields are improved from 0.36g/g for the initial culture (R) to 0.72 g/g for culture B after six months in continuous culture, and 0.8 g/g in plug-flow continuous culture. The productivity of organic acids reaches 1.7 g/liter·hr. It is suggested that the acidogenic fermentation, the first step of methanogenesis, is a potential process to produce acetic, propionic, and butyric acids.     

Growth yields of Desulfotomaculum orientis with hydrogen in chemostat culture

Desulfotomaculum orientis (strain Singapore 1) was grown autotrophically with H₂+CO₂ and sulfate, thiosulfate or sulfite as electron acceptor in sulfide- and pH-controlled continuous culture. Under sulfate-limiting conditions real growth yields of up to 9.7 g cell dry mass per mol sulfate were obtained. Electron acceptor limitation resulted in the excretion of up to 14.5 mmol acetate per liter, formed by reduction of CO₂ with H₂. Acetate production was not coupled to an increase of growth yields: under hydrogen-limiting conditions only 1.6 mmol acetate per liter was produced, and even higher growth yields of up to 12,4 g cell dry mass per mol sulfate were obtained. With thiosulfate or sulfite as electron acceptor growth yields increased up to 17.9 g cell dry mass per mol electron acceptor. Growth yields were not simply correlated with the growth rate, and did not allow the determination of maintenance coefficients and the extrapolation to maximal yields at infinite growth rate (Y ^max). The maximal growth rates (_max) with sulfate and thiosulfate were 0.090 and 0.109 h^-1, respectively, if cells were grown continuously in sulfidostat culture under nonlimiting conditions.The net energy yield of sulfate reduction and the energy requirement for the activation of sulfate by Desulfotomaculum orientis are discussed.     

Growth and enzyme synthesis during continuous culture of Trichosporon cutaneum on phenol

The soil yeast Trichosporon cutaneum was grown in continuous culture with phenol as the sole carbon source. The cultures were operated as carbon-limited chemostats or as steady-state continuous cultures without carbon limitation. Selected comparative runs were also conducted on glucose or acetate as carbon source. In addition to growth parameters, the activities of several intracellular enzymes were determined, comprising those directly involved in the degradation of phenol as well as auxiliary enzymes required for the generation of reducing power. All enzymes were assayed in detergent-permea-bilized cells. Phenol was found to serve as an excellent carbon source, comparable to glucose or acetate. The utilization of phenol in T. cutaneum is very efficient as indicated by a low maintenance requirement (0.01 g phenol/g cells.h). The cell yields obtained were on the order of 0.8 g cells/g phenol. Although the phenol-limited chemostats were run with fully phenol-induced cells, a further increase in the activities of isocitrate DH(NADP(+)), maleate DH and the phenol-degrading enzymes occurred after transition to nonlimiting condition. Enzyme activities increased in parallel with increasing phenol levels in the effluent, as well as with increasing toxicity. The significance of this phenomenon is discussed. The significance of this phenomenon is discussed. This elevation in enzyme activities in not related to an increase in specific growth rate.     

Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start-up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start-up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start-up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.The Pirt-maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 +/- 0.08 C-mol/C-mol, the maintenance value 0.019 +/- 0.012 C-mol/(C-mol h). The lowest actual biomass yield measured in this study was 0.15 C-mol/C-mol. (c) 1994 John Wiley & Sons, Inc.     

220D-F2 from Rubus ulmifolius Kills Streptococcus pneumoniae Planktonic Cells and Pneumococcal Biofilms

Streptococcus pneumoniae (pneumococcus) forms organized biofilms to persist in the human nasopharynx. This persistence allows the pneumococcus to produce severe diseases such as pneumonia, otitis media, bacteremia and meningitis that kill nearly a million children every year. While bacteremia and meningitis are mediated by planktonic pneumococci, biofilm structures are present during pneumonia and otitis media. The global emergence of S. pneumoniae strains resistant to most commonly prescribed antibiotics warrants further discovery of alternative therapeutics. The present study assessed the antimicrobial potential of a plant extract, 220D-F2, rich in ellagic acid, and ellagic acid derivatives, against S. pneumoniae planktonic cells and biofilm structures. Our studies first demonstrate that, when inoculated together with planktonic cultures, 220D-F2 inhibited the formation of pneumococcal biofilms in a dose-dependent manner. As measured by bacterial counts and a LIVE/DEAD bacterial viability assay, 100 and 200 µg/ml of 220D-F2 had significant bactericidal activity against pneumococcal planktonic cultures as early as 3 h post-inoculation. Quantitative MIC’s, whether quantified by qPCR or dilution and plating, showed that 80 µg/ml of 220D-F2 completely eradicated overnight cultures of planktonic pneumococci, including antibiotic resistant strains. When preformed pneumococcal biofilms were challenged with 220D-F2, it significantly reduced the population of biofilms 3 h post-inoculation. Minimum biofilm inhibitory concentration (MBIC)₅₀ was obtained incubating biofilms with 100 µg/ml of 220D-F2 for 3 h and 6 h of incubation. 220D-F2 also significantly reduced the population of pneumococcal biofilms formed on human pharyngeal cells. Our results demonstrate potential therapeutic applications of 220D-F2 to both kill planktonic pneumococcal cells and disrupt pneumococcal biofilms.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Pseudomonas putida Mutants Defective in the Metabolism of the Products of meta Fission of Catechol and Its Methyl Analogues

A selection procedure is described which was used to isolate mutants of Pseudomonas putida strain U in the following enzymes of the meta-fission pathway of phenol and cresols: hydrolase, tautomerase, and decarboxylase. Strains deficient in the hydrolase are unable to use either o- or m-cresol as a sole carbon source and were shown to accumulate 2-hydroxy-6-keto-2,4-heptadienoate when incubated in the presence of o- or m-cresol. When 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form) was metabolized by phenol-induced extracts of tautomerase-deficient strains, the enol tautomer of 4-oxalocrotonate accumulated and was then converted slowly to the keto tautomer by a nonenzymatic reaction. Phenol-induced extracts of decarboxylase-deficient strains accumulated the keto tautomer of 4-oxalocrotonate from 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form). Strains with an inactive decarboxylase are unable to completely metabolize either phenol or p-cresol. Tautomerase-defective strains are unable to grow with p-cresol as the sole carbon source and grow only very slowly on phenol.     

Production of fumonisin B1 by Fusarium moniliforme NRRL 13616 in submerged culture.

Fumonisin B1, a recently discovered mycotoxin, was synthesized by submerged cultures of Fusarium moniliforme NRRL 13616 grown for 29 days at 28 degrees C and 220 rpm in a basal salts medium (pH 5.0) supplemented with 90 g of glucose per liter and 3.5 g of ammonium sulfate per liter. Under these culture conditions, 74 +/- 23 micrograms of fumonisin B1 per ml was produced by 29-day-old F. moniliforme NRRL 13616 cultures. Fumonisin B1 was detected in liquid culture extracts by high-performance thin-layer chromatography. Fumonisin B1 was confirmed and quantitated by gas chromatography and gas chromatography-mass spectral analysis of the trimethylsilyl derivative. The use of a defined medium for producing fumonisin B1 in a submerged culture facilitates its isolation and provides an excellent method for conducting biosynthetic studies.     

Perfusion culture of hybridoma cells for hyperproduction of IgG(2a) monoclonal antibody in a wave bioreactor-perfusion culture system

A novel wave bioreactor-perfusion culture system was developed for highly efficient production of monoclonal antibody IgG2a (mAb) by hybridoma cells. The system consists of a wave bioreactor, a floating membrane cell-retention filter, and a weight-based perfusion controller. A polyethylene membrane filter with a pore size of 7 microm was floating on the surface of the culture broth for cell retention, eliminating the need for traditional pump around flow loops and external cell separators. A weight-based perfusion controller was designed to balance the medium renewal rate and the harvest rate during perfusion culture. BD Cell mAb Medium (BD Biosciences, CA) was identified to be the optimal basal medium for mAb production during batch culture. A control strategy for perfusion rate (volume of fresh medium/working volume of reactor/day, vvd) was identified as a key factor affecting cell growth and mAb accumulation during perfusion culture, and the optimal control strategy was increasing perfusion rate by 0.15 vvd per day. Average specific mAb production rate was linearly corrected with increasing perfusion rate within the range of investigation. The maximum viable cell density reached 22.3 x 105 and 200.5 x 105 cells/mL in the batch and perfusion culture, respectively, while the corresponding maximum mAb concentration reached 182.4 and 463.6 mg/L and the corresponding maximum total mAb amount was 182.4 and 1406.5 mg, respectively. Not only the yield of viable cell per liter of medium (32.9 x 105 cells/mL per liter medium) and the mAb yield per liter of medium (230.6 mg/L medium) but also the mAb volumetric productivity (33.1 mg/L.day) in perfusion culture were much higher than those (i.e., 22.3 x 105 cells/mL per liter medium, 182.4 mg/L medium, and 20.3 mg/L.day) in batch culture. Relatively fast cell growth and the perfusion culture approach warrant that high biomass and mAb productivity may be obtained in such a novel perfusion culture system (1 L working volume), which offers an alternative approach for producing gram quantity of proteins from industrial cell lines in a liter-size cell culture. The fundamental information obtained in this study may be useful for perfusion culture of hybridoma cells on a large scale.     

Formation of Short-Chain Fatty Acids from H(2) and CO(2) by a Mixed Culture of Bacteria

The biological utilization of CO(2) and H(2) for the formation of short-chain fatty acids was studied by using a mixed culture of bacteria. Optimization of a medium was carried out in continuous culture to identify limiting factors which controlled growth and production of organic acids. The optimal pH for growth and acid production was 7.0 at 37 degrees C; the maximal cell concentration obtained was 5.9 g of cells per liter (dry weight), and the maximal amount of volatile acids formed was 4.7 g/liter, with acetic acid as the predominant acid. With the optimized medium, it was found that the rate of transfer of hydrogen or carbon dioxide, or both, from gas to liquid was the limiting factor which controlled growth and production of acids.     

Degradation of 4-Chlorophenol via the meta Cleavage Pathway by Comamonas testosteroni JH5

Comamonas testosteroni JH5 used 4-chlorophenol (4-CP) as its sole source of energy and carbon up to a concentration of 1.8 mM, accompanied by the stoichiometric release of chloride. The degradation of 4-CP mixed with the isomeric 2-CP by resting cells led to the accumulation of 3-chlorocatechol (3-CC), which inactivated the catechol 2,3-dioxygenase. As a result, further 4-CP breakdown was inhibited and 4-CC accumulated as a metabolite. In the crude extract of 4-CP-grown cells, catechol 1,2-dioxygenase and muconate cycloisomerase activities were not detected, whereas the activities of catechol 2,3-dioxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, 2-hydroxymuconic semialdehyde hydrolase, and 2-oxopent-4-enoate hydratase were detected. These enzymes of the meta cleavage pathway showed activity with 4-CC and with 5-chloro-2-hydroxymuconic semialdehyde. The activities of the dioxygenase and semialdehyde dehydrogenase were constitutive. Two key metabolites of the meta cleavage pathway, the meta cleavage product (5-chloro-2-hydroxymuconic semialdehyde) and 5-chloro-2-hydroxymuconic acid, were detected. Thus, our previous postulation that C. testosteroni JH5 uses the meta cleavage pathway for the complete mineralization of 4-CP was confirmed.     

Kinetic parameters of continuous cultures of Bacillus thermoleovorans sp. A2 degrading phenol at 65 degrees C

In this paper, we report on the kinetics of phenol degradation and cell growth in continuous cultures of suspended cells of Bacillus thermoleovorans sp. A2 at 65 degrees C. A high yield coefficient of Y(x/s)=0.84 g cell dry weight g(-1) phenol was measured at a dilution rate of 0.5 h(-1). At the same dilution rate the coefficient for maintenance metabolism (m(s)) was determined to be 0.045 g phenol g(-1) cell dry weight h(-1). The maximal growth rate (wash-out) determined at a phenol inlet concentration of 188 mg l(-1) was 0.9 h(-1). Up to 7 g phenol l(-1) per day were degraded in a continuously operated 2-l stirred tank reactor with suspended cells (feed concentration 660 mg l(-1)). Additionally, yield coefficients for oxygen and ammonium are reported.     

Formation of glycosidases in batch and continuous culture of Bacteroides fragilis.

Nine strains of bacteroides fragilis were cultivated in stirred fermentors and tested for their ability to produce glycosidases. B. fragilis subsp. vulgatus B70 was used for optimizing the production of glycosidases. The highest bacterial yield was obtained in proteose peptone-yeast extract medium. The optimum pH for maximal bacterial yield was 7.0, and the optimum temperature for growth was 37 degrees C. The formation of glycosidases was optimal between pH 6.5 and 7.5, and the optimum temperature for synthesis of glycosidases was between 33 and 37 degrees C. Culture under controlled conditions in fermentors gave more reproducible production of glycosidases than static cultures in bottles. The strain was also grown in continuous culture at a dilution rate of 0.1 liter/h at pH 7.0 and 37 degrees C with a yield of 2.0 mg of dry weight per ml in the complex medium. The formation of glycosidases remained constant during the entire continuous process.     

Factors Affecting Oxidation of Thiosalts by Thiobacilli

The effects of temperature, initial pH, and the concentrations of ammonium, phosphate, and heavy metals on the oxidation of thiosalts by an authentic strain of Thiobacillus thiooxidans (ATCC 8085) and by a mixed culture isolated from a base metal-processing mill effluent pond were studied. The optimum temperature was 30°C and the optimum initial pH was 3.75 for both cultures using thiosulfate and for the mixed culture using tetrathionate. T. thiooxidans ATCC 8085 did not oxidize tetrathionate. For a thiosalt concentration of 2,000 ppm (2,000 mg/liter), maximal rates of destruction occurred at concentrations of ammonium ion above 2 mg/liter and in the presence of 1 mg of phosphate per liter. Under optimal conditions, the rate of thiosulfate oxidation by the pure culture was 55 ± 3 mg/liter per h; the mixed culture oxidized thiosulfate at the rate of 40 ± 1 mg/liter per h and tetrathionate at the rate of 50 ± 2 mg/liter per h. Metal ions caused normal inhibition kinetics in the oxidation of thiosulfate by T. thiooxidans ATCC 8085. K_i values were calculated for cadmium (16 mg/liter), copper (0.46 mg/liter), lead (2 mg/liter), silver (3.1 mg/liter), and zinc (33 mg/liter). Only a slight additive effect was apparent in the presence of all of these metal ions. The mixed culture of thiosalt-oxidizing bacteria was less sensitive to heavy metal inhibition; the order of inhibition of thiosulfate oxidation was Cd < Zn < Pb < Ag < Cu, and that of tetrathionate oxidation was Zn < Cd < Pb < Ag < Cu.