Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Lipid accumulation in <Emphasis Type="Italic">Schizochytrium</Emphasis> G13/2S produced in continuous culture

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h⁻¹, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h⁻¹, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.     

Cell yields of Escherichia coli during anaerobic growth on fumarate and molecular hydrogen

Escherichia coli was grown anaerobically on sodium fumarate and molecular hydrogen or sodium formate in continuous culture. The maximal growth yield and the maintenance coefficient were determined. In a mineral medium a Y _fum ^max value of 6.6 g dry weight per mol fumarate was found. This value increased to 7.5 when casamino acids were present in the medium. From these data and the corresponding Y _ATP ^max values it could be calculated that per mol of fumarate reduced, 0.4 mol of ATP became available for growth. In batch culture a Y_fum value of 4.8 g dry weight per mol fumarate was determined.     

Inhibitor effect of products of metabolism on growth of Clostridium acetobutylicum

Summary The growth of Clostridium acetobutylicum was studied by three ways. 1. In batch fermentation, referred to as the control. 2. Fermentation in dialysis which permits elimination of all the products of metabolism: acids, solvents and gases. In order to test the toxic effect of acids, cultures were dialysed against 2 g l^-1 acetic acid or 2 g l^-1 butyric acid. 3. To test the toxic effect of gases only, batch fermentations were carried out under vacuum or with a continuous bubbling of nitrogen. The first method resulted in a productivity of 1.2 g l^-1 dry cell weight and a maximal specific growth rate of 0.2 h^-1; the second, 20 g l^-1 dry cell weight and a constant maximum specific growth rate (μ=0.39 h^-1) between 14 and 20 h. The toxic effect of acetic and butyric acids, starts at low concentrations and about 4 g l^-1 of both acids results in a decrease of 50% of maximal specific growth rate. The third series of experiments showed that gases produced by the bacteria have a high toxic effect, comparable to that of 5 g l^-1 of acid.     

Production of l-Methionine-enriched cells of a mutant derived from a methylotrophic yeast,Candida boidinii

l-Methionine-enriched cells production of an ethionine-resistant mutant of Candida boidinii no. 2201 was greatly improved by the control of pH and by feeding of methanol and other medium components during cultivation in a jar fermentor. Under the optimal conditions, 38.5 g (as dry weight)_of cells abd 282 mg of pool methionine (intracellular pool of free l-methionine) per l of culture broth were obtained after 11 d of cultivation.The culture conditions for production of l-methionine-enriched cells in continuous culture were investigated. With limited methanol in continuous cultivation, pool methionine productivity reached a maximum value of 1.14 mg·l⁻¹·h⁻¹ at a dilution rate of 0.05·h⁻¹. During methanol-limited growth in continuous cultivation, the pool methionine content of the mutant was about 20–35% higher than that in batch cultivation.     

Drosophila cells can be grown to high cell densities in a bioreactor

Summary Cell cultures of the fruitfly, Drosophila melanogaster, were grown in a bioreactor to high cell densities: 5.7×10⁷ cells/ml, in batch, semi-batch and continuous flow modes. Using a semi-batch culture mode 500g wet weight of cell mass was produced in 16 days, using 5 l of cheap, commercially available, serum-free medium.     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Isolation and Characterization of a Pseudomonas putida Strain able to Grow with Trimethyl-1,2-dihydroxy-propyl-ammonium as Sole Source of Carbon, Energy and Nitrogen

Trimethyl-1,2-dihydroxypropyl-ammonium (TM) originates from the hydrolysis of the parent esterquat surfactant, which is widely used as softener in fabric care. Based on test procedures mimicking complex biological systems, TM is supposed to degrade completely when reaching the environment. However, no organisms able to degrade TM were isolated nor has the degradation pathway been elucidated so far. We isolated a Gram-negative rod able to grow with TM as sole source of carbon, energy and nitrogen. The strain reached a maximum specific growth rate of 0.4 h^–1 when growing with TM as the sole source of carbon, energy and nitrogen. TM was degraded to completion and surplus nitrogen was excreted as ammonium into the growth medium. A high percentage of the carbon in TM (68% in continuous culture and 60% in batch culture) was combusted to CO₂ resulting in a low yield of 0.54 mg cell dry weight per mg carbon during continuous cultivation and 0.73 mg cell dry weight per mg carbon in batch cultures. Choline, a natural structurally related compound, served as a growth substrate, whereas a couple of similar other quaternary aminoalcohols also used in softeners did not. The isolated bacterium was identified by 16S-rDNA sequencing as a strain of Pseudomonas putida with a difference of only one base pair to P. putida DSM 291^T. Despite their high identity, the reference strain P. putida DSM 291^T was not able to grow with TM and the two strains differed even in shape when growing on the same medium. This is the first microbial isolate able to degrade a quaternary ammonium softener head group to completion. Previously described strains growing on quaternary ammonium surfactants (decyltrimethylammonium, hexadecyltrimethylammonium and didecyldimethylammonium) either excreted metabolites or a consortium of bacteria was required for complete degradation.     

Enhancement of growth rate and β-galactosidase activity, and variation in organic acid profile of Bifidobacterium animalis subsp. lactis Bb 12

The behavior of Bifidobacterium animalis subsp. lactis Bb 12 under batch cultivation, after continuous culturing for up to 12 d, was monitored in skim milk-based media. Previous continuous culture for longer than 6 d affected the physiology of said microorganism. The minimum inhibitory concentrations of lactic and acetic acids increased from 18 to 26 g/l, whereas the molar ratio of acetic to lactic acid increased from 0.8 to 1.55, when the previous continuous culture increased its duration from 1 to 12 d. The specific lactose consumption rate decreased from 0.94 to 0.77 g_lactose/g_{cell dry mass}/h within the batch culture timeframe; this was concomitant with greater amounts of acetic and formic acids, and lower amounts of lactic acid produced. The β-galactosidase activity increased as continuous culturing time increased, and reached 446 units/ml by 12 d; however, the rate of enzyme synthesis decreased concomitantly. Succinic acid was produced during the exponential growth and stationary phases of the batch culture, but the former at exponential growth phase was higher as the continuous culturing time was longer. For comparison purposes, batch cultivation of samples taken from continuous cultures by 1 and 12 d was done using a semi-synthetic medium with glucose as carbon source; a pattern similar to that observed when using skim milk-based media was observed.     

Production of NO and N2O by the Heterotrophic Nitrifier Alcaligenes faecalis parafaecalis under Varying Conditions of Oxygen Saturation

Production of NO and N ₂ O by the heterotrophic nitrifier Alcaligenes faecalis subsp. parafaecalis was studied during growth in batch and continuous culture on peptone-meat extract medium. Depending on oxygen saturation level, medium redox status and amount of substrate supplied, the microorganisms produced 0.002–0.25 mg NO-N h^{- 1} (g protein)^{- 1} and 0.16–2.4 mg N ₂ O-N h^{- 1} (g protein)^{- 1} . Maximum rates of nitrogen oxides production were observed during peak events initiated by sudden changes of oxygen supply in the medium and were due to combined nitrification/denitrification taking place simultaneously within the cells. Based on model simulations of enzymatic kinetics of denitrification, possible mechanisms of increased nitrogen oxides production during periods of changes in oxygen supply are suggested.     

Invertase and phosphatase of yeast in a phosphate-limited continuous culture

Summary Deficiency of inorganic phosphate caused the hyper production of invertase and the derepression of acid phosphatase in a continuous culture ofSaccharomyces carlsbergensis. The specific invertase activity was 40,000 enzyme units per g dry cell weight at a dilution rate lower than 0.05 h^–1 with a synthetic glucose medium of which the molecular ratio of KH₂PO₄ to glucose was less than 0.006. This activity is eight fold higher than in a batch growth and 1.5 fold as much as the highest enzyme activity observed so far in a glucose-limited continuous culture.For the hyper production of invertase, it is necessary to culture the yeast continuously by keeping the Nyholm's conservative inorganic phosphate concentration at less than 0.2 m mole per g dry weight cell. The derepression of acid phosphatase brought about by phosphate deficiency, was similar in both batch and continuous cultures.Nomenclature D dilution rate of continuous culture (h^–1) - E_i invertase concentration in culture (enzyme unit l^–1) - E_p acid phosphatase concentration in culture (enzyme unit l^–1) - P inorganic phosphate concentration in culture (mM) - S glucose concentration in culture (mM) - X cell concentration in culture (g dry weight cell l^–1) Greek Letter specific rate of growth (h^–1) Suffix f feed - 0 initial value     

Energetic aspects of anaerobic growth of Aerobacter aerogenes in complex medium

Molar growth yields for anaerobic growth of Aerobacter aerogenes in complex medium were much higher than for growth in minimal medium. In batch cultures the molar growth yield for glucose varied from 44 to 50 and Y _ATP from 17.1 to 18.8. For glucose-limited chemostat cultures a value of 17.5 g/mole was found for Y _ATP ^max and a value of 2.3 mmoles ATP/g dry weight h for the maintenance coeficient. Growth dependent pH changes were used to control the addition of fresh medium, containing excess of glucose to a continuous culture. The specific growth rate and the population density were dependent on the pH difference between the inflowing medium and the culture. At a value of 1.44 h^-1 the molar growth yield for glucose was about 70 and Y _ATP about 28.5. An-equation is presented, which gives the relation between theoretical and experimental Y _ATP ^max values.     

Growth of Thermoanaerobium brockii in batch and continuous culture at supraoptimal temperatures

Summary Thermoanaerobium brockii was grown in batch and continuous culture at supraoptimal temperatures (>65° C). Specific growth rates were lower in batch (_max>1.0 h^-1) than in continuous cultures (_max1.2–1.4 h^-1). Acetone addition to the medium did not increase critical dilution rate significantly. The media used contained significantly less organic material and sulfide than previously reported media; however, yeast extract requirements were shown to be exceptionally high (60% of the glucose concentration used). Organic substrates inhibited growth and product formation in chemostat cultures whereas the slow formation of acetic acid was observed in batch cultures, but also with virtually no growth. The inhibiting concentration was found to be approximately 15 g organic carbon·l^-1. The maintenance requirements of T. brockii were in the same range as expected of aerobic extreme thermophiles (m_s0.5 g·g^-1·h^-1) and could be met only by glucose and not by yeast extract. Maintenance was obviously not independent of specific growth rate. Production of the stereospecific alcohol-aldehyde/ketone oxidore-ductase was strictly growth associated and its formation was not affected by acetone added to medium.     

Optimization of lipid production in the oleaginous yeastApiotrichum curvatum in wheypermeate

Summary Lipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h^-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h^-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.Nomenclature C/N-ratio carbon to nitrogen ratio of the growth medium (g/g) - C/N_crit C/N-ratio at which there is just enough nitrogen to allow all carbon source to be converted to biomass - D dilution rate=volume of incoming medium per unit time/volume of medium in the culture vessel (h^-1) - D_max maximum dilution rate (h^-1) - DW cell dry weight - L lipid yield (g storage lipid/g carbon source) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - Q_L lipid production rate (g/l/h) - Y_i molecular fraction of carbon substrate that is converted to storage carbohydrate (C-mol/C-mol) - Y_ls maximal amount of storage lipid that can be produced per mol carbon source (C-mol/C-mol)     

Effect of malic acid on the growth kinetics of<Emphasis Type="Italic"> Lactobacillus plantarum</Emphasis>

The fermentation kinetics of Lactobacillus plantarum were studied in a specially designed broth formulated from commercially available, dehydrated components (yeast extract, trypticase, ammonium sulfate) in batch and continuous culture. During batch growth in the absence of malic acid, the specific growth rate was 0.20 h^–1. Malic acid in the medium, at 2 mM or 10 mM, increased the specific growth rate of L. plantarum to 0.34 h^–1. An increase in the maximum cell yield due to malic acid also was observed. Malic acid in the medium (12 mM) reduced the non-growth-associated (maintenance energy) coefficient and increased the biomass yield in continuous culture, based on calculations from the Luedeking and Piret model. The biomass yield coefficient was estimated as 27.4 mg or 34.3 mg cells mmol^–1 hexose in the absence or presence of malic acid, respectively. The maintenance coefficient was estimated as 3.5 mmol or 1.5 mmol hexose mg^–1 cell h^–1 in the absence or presence of malic acid. These results clearly demonstrate the energy-sparing effect of malic acid on the growth- and non-growth-associated energy requirements for L. plantarum. The quantitative energy-sparing effect of malic acid on L. plantarum has heretofore not been reported, to our knowledge.     

Modeling of yeast <Emphasis Type="Italic">Brettanomyces bruxellensis</Emphasis> growth at different acetic acid concentrations under aerobic and anaerobic conditions

Glucose utilization by Brettanomyces bruxellensis at different acetic acid concentrations under aerobic and anaerobic conditions was investigated. The presence of the organic acid disturbs the growth and fermentative activity of the yeast when its concentration exceeds 2 g l⁻¹. A mathematical model is proposed for the kinetic behavior analysis of yeast growing in batch culture. A Matlab algorithm was used for estimation of model parameters, whose confidence intervals were also calculated at a 0.95 probability level using a t-Student distribution for f degrees of freedom. The model successfully simulated the batch kinetics observed at different concentrations of acetic acid under both oxygen conditions.     

Optimization of culture conditions and continuous production of chitosan by the fungi,Absidia coerulea

The production of chitosan from the mycelia ofAbsidia coerulea was studied to improve cell growth and chitosan productivity. Culture conditions were optimized in batch cultivation (pH 4.5 agitator speed of 250 rpm, and aeration rate of, 2 vvm) and the maximum chitosan concentration achieved was 2.3 g/L under optimized conditions. Continuous culture was carried out successfully by the formation of new growth spots under optimized conditions, with a chitosan productivity of 0.052 gL⁻¹ h⁻¹, which is the highest value to date, and was obtained at a dilution rate of 0.05 h⁻¹. Cell chitosan concentrations reached about 14% in the steady state, which is similar to that achieved in batch culture. This study shows that for the continuous culture ofAbsidia coerulea it is vital to control the medium composition.     

Recombinant protein production in cultures of an Escherichia coli trp − strain

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp ^– derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-l batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l. Correspondence to: F. Bolivar     

Growth of Nitrosomonas europaea in batch and in continuous culture

Summary Techniques are described in this paper for growing Nitrosomonas europea in batch and in continuous culture with apparatus constructed from readily available laboratory materials.The methods employed in batch culture have enabled the collection of cells concentrated in small volumes. Nitrosomonas europaea has been grown successfully in continuous culture and yields of 23 g wet weight were obtained, an average of 0.16 g/l with a flow rate of 300 ml/h over twenty days. Results show that high levels of nitrite do not materially affect the growth of the bacterium. The continuous culture equipment has also been used to grow Azotobacter vinelandii and Thiobacillus concretivorus.     

Relationships between nucleic acid,nitrogen, and growth rate of tobacco cells in suspension culture

Summary Changes in the amount of nucleic acid and nitrogen, and the relationships between these amounts and the growth rate of tobacco cells (Nicotiana tabacum L. cv. Bright Yellow-2) at different initial nitrogen concentrations in the medium, were examined in batch cultures. During culture in basal medium, the amount of intracellular nucleic acid expressed per unit of dry biomass was 36.3 mg RNA g^–1 cell and 8.1 mg DNA g^–1 cell at the beginning of batch culture. These values increased 2.5 fold for RNA and 1.5 fold for DNA during the exponential growth phase and then gradually decreased with the decline in the growth rate. Similar changes were also observed in the medium containing less nitrogen. The specific growth rate, (day^–1), of the culture corresponded to the magnitude of the intracellular RNA content (mg RNA g^–1 cell), and the linear relationship, RNA=38+23 was obtained. In addition, there were remarkable positive correlations between the total and protein nitrogen, and during the cultures. The mononucleotide composition of total RNA (AMP+UMP)/(GMP+CMP) which was suggested to be a convenient index of metabolic activity was nearly constant (0.78 to 0.80) during tobacco cell culture in the basal medium.