pH control for fed-batch culture of Bacillus thuringiensis

Summary Fed-batch culture of Bacillus thuringiensis for thuringiensin (-exotoxin) production was carried out in a modified airlift reactor. The feeding policy was based on the pH value in the broth. The feed rate of the substrate was regulated by an appropriate pH value. The yield of thuringiensin based on the fed-batch culture increased 30% in comparison with that of batch culture.     

The secretory activity of algal cells in buffered media

Summary The effect of the activity of algal cells on the buffering characteristics of suspending fluid was studied onChlorella 7-11-05 cells suspended in Tris buffer or in sodium citrate-citric acid buffer. It was observed that changes in the buffering capacity of the suspending fluid depended on the concentration of the original buffer, pH, and the nature of the buffer.In Tris buffer, which is not consumed byChlorella cells, the buffering capacity, measured in terms of the van Slyke's buffer index (), increased with time. In sodium citrate-citric acid buffer, which is used byChlorella cells in the course of their metabolism, not only the buffer index () changed, but also the pH at which the suspending fluid had maximum buffering capacity shifted to a new value. In both Tris and citrate buffers the newly formed buffer systems had a maximum buffering capacity at pH 6.4 corresponding to the pK₁ of carbonic acid.This work was supported by funds from the National Aeronautics and Space Administration.Scientific Article A 1237, Contribution No. 3742 of the University of Maryland Agricultural Experiment Station.     

Non-bicarbonate intracellular pH buffering of reptilian muscle

Summary Non-bicarbonate intracellular pH buffering values of skeletal and cardiac muscles were measured for 16 species of Australian reptiles from four orders (snakes, skelctal 19–36 slykes, cardiac 9–17 slykes; lizards, skeletal 25–54 slykes, cardiac 17–19 slykes; turtles, skeletal 25–43 slykes, cardiac 11–24 slykes; crocodile, skeletal 43 slykes). Although a positive correlation between pH buffering capacity and dependence on anaerobic muscle work was found, even the highest reptilian pH buffering values were low relative to equivalent white anaerobic muscles of fish, birds, and mammals. The low non-bicarbonate intracellular pH buffering capacity of reptilian muscle arises through lower contributions from proteins (10–14 slykes), non-protein histidine (7–18 slykes) and phosphate (5–15 slykes). It is concluded that while other vertebrates depend on these intracellular buffers for regulating muscle pH during anaerobic muscle work, reptiles rely less on buffering and instead may tolerate greater pH fluctuations.Abbreviations intracellular pH buffering capacity - EDTA ethylenediaminetetra-acetic acid - HPLC high performance liquid chromatography - I.D. internal diameter - LDH lactate dehydrogenase     

Production of thuringiensin from Bacillus thuringiensis using a net-draft-tube,modified airlift reactor

A net-draft-tube, modified airlift reactor and a stirred-tank reactor were used for thuringiensin production by Bacillus thuringiensis subsp. darmstadiensis growing with various concentrations of molasses. The optimum concentration of molasses for thuringiensin production in both reactors was 15 g/l. There was a 6 h delay in sporulation in the modified airlift reactor compared with that in the stirred-tank reactor. Thuringiensin yield in the modified airlift reactor (2.2 g/l) was consequently higher than that in the stirred-tank reactor (1.1 g/l).     

Optimization of agitation for production of swainsonine from Metarhizium anisopliae in stirred tank and airlift reactors

The optimal agitation rate for production of swainsonine from Metarhizium anisopliae grown in batch stirred tank reactors (2 to 20 l) was 400 rpm with a mixed hyphal and pelleted morphology where the specific swainsonine production rate was 9×10^–2 mg g^–1 cell dry wt h from 87 to 142 h. Culture of the fungus in a 6-l airlift reactor produced loose pellets and the production of swainsonine started at least 24 h earlier than in the stirred tank reactor. The final yield (5.9 mg swainsonine g^–1 cell dry wt) after 168 h in the airlift reactor was 18% less than those obtained in the stirred tank reactor with an agitation rate of 400 rpm.     

Bacterial cellulose production by Acetobacter xylinum in a 50-L internal-loop airlift reactor

Bacterial cellulose (BC) production was realized in a batch cultivation of Acetobacter xylinum subsp. sucrofermentans BPR2001 in a 50-L internal-loop airlift reactor. When the bacterium was cultivated with air supply, 3.8 g/L of BC was produced after 67 hours. When oxygen-enriched gas was supplied, the concentration of BC was doubled and the production rate of BC was 0.116 g/L. h, which was two times higher than that of air-supplied culture and comparable to that in a mechanically agitated stirred-tank fermentor. Bacterial cellulose produced by the airlift reactor formed a unique ellipse pellet (BC pellet), different from the fibrous form which was produced in an agitated stirred-tank fermentor. The BC-pellet suspension was demonstrated to have a higher volumetric oxygen transfer coefficient than the fibrous BC suspension in a 50-L internal-loop airlift reactor. The mixing time of BC-pellet suspension in the airlift reactor was also shorter than that in water.     

Comparison of photobioreactors for cultivation of Undaria pinnatifida gametophytes

Undaria pinnatifida gametophytes were grown in 2.5 l bubble column and airlift reactor at 25 °C and light intensity of 40 mol m^–2 s^–1 for 6 days. With aeration at 1 l min^–1, the airlift reactor yielded higher growth rate (0.12 mg DW ml^–1 d^–1) than a bubble column (0.08 mg DW ml^–1 d^–1). The advantages were related to the more homogeneous fluid dynamic characteristics of the airlift reactor.     

Growth and alkaloid production of a cell suspension culture of Thalictrum rugosum in shake flasks and membrane-stirrer reactors with bubble free aeration

Summary A novel membrane-stirrer reactor allowing bubble-free aeration was used for the cultivation of a cell suspension culture of Thalictrum rugosum. The immediate advantages were the absence of any foaming or flotation and the gentle mixing of the suspension by the tumbling movement of a coil or basket of membranes. In a 211 reactor under these conditions the suspensions were grown to high cell densities of 550 g fresh mass or 50 g dry mass/l. A pO₂ of 30% was maintained up to 400 g/l without air bubble formation at the gas-exchange membranes. Provided that the physiological states of the initial suspensions were comparable, growth rates and alkaloid production of shake-flask and fermenter-grown cells were rather similar.Abbreviations NAA -naphthylacetic acid - 2,4-d 2,4-dichlorophenoxyacetic acid     

A method for the regulation of microbial population density during continuous culture at high growth rates

A method for the continuous culture of microorganisms is described which employs growth-dependent pH changes to control the rate of addition of fresh medium to a culture vessel. The apparatus (the phauxostat) supports, at constant pH, longterm continuous culture at rates near or at the maximum of which the organisms are capable. The buffering capacity of the inflowing medium determines the steady-state population density of the culture, but the rate of growth is independent of the buffering capacity. The fundamental theory of operation is tested and some basic parameters of growth are estimated using Escherichia coli B growing continuously in media containing glucose, glycerol or DL-lactate.Joseph C. Wilson Scholar.     

An airlift loop reactor for the transformation of steroids by immobilized cells

Summary The flow behaviour of calcium alginate beads in an airlift reactor (ALR) with external loop was dependent on the airflow rate into and the amount of beads in the reactor. The performance of immobilizedArthrobacter simplex for the ¹-dehydrogenation of hydrocortisone in the ALR compared favourably to that in a stirred tank reactor. The physical stability of the calcium alginate beads was significantly greater in the ALR.     

Performance of airlift bioreactors in the cultivation of some antibiotic producing microorganisms

The specific aspects of airlift reactors emphasizing their function relevance to particular application as bioreactors are presented. The two main groups of airlift reactors – external-loop and concentric-tube reactors – were investigated on a pilot-plant scale with regard to their performance during the cultivation of unicellular and filamentous microorganisms which produce Bacitracin, Cephalosporin C and Nystatin. Some results were compared to those obtained in conventional stirred tank bioreactors. The comparison was carried out based on physical properties (oxygen transfer rate (OTR), volumetric mass transfer coefficient (k_La) and efficiency of oxygen transfer (E)), cell mass, productivity and substrate consumption, secondary metabolite production, and efficiency of the product formation with regard to the specific power input. It was shown that B. licheniformis, C. acremonium and S. noursei fermentations occurred similarly to those performed in stirred vessels, proving that the capacity of the airlift bioreactors surpassed the problems which arise from the morphology and rheology of the broths. From the chemical engineering point of view, it was obvious that the primary tasks of a bioreactor (uniform distribution of microorganisms and nutrients over the entire fermenter volume, appropriate supply of biomass with nutrients and oxygen) were fulfilled by the airlift bioreactors tested. In addition, the efficiency of oxygen transfer (OTR referred to power input) in the airlift fermenters proved to be about 38% higher than in the stirred tank bioreactors (expressed as average values), while the sorption efficiency (OTR referred to antibiotic production) was found to be 22% greater in the airlift system than in an STR. Therefore, the biosyntheses were performed with about a 30–40% increase in energy efficiency and energy savings compared to the conventional system. Moreover, the lack of mechanical devices in the airlift system provides greater safety and a gentler environment for the cultivation of microorganisms.     

Repeated elicitation enhances taxane production in suspension cultures of Taxus chinensis in bioreactors

Methyl jasmonate added at 100 M on days 8, 14 and 20 to cultures of Taxus chinensis growing in flasks and 1 l airlift bioreactors improved taxuyunnanine C (TC) production by 50–60% in flasks and by about 80% in airlift bioreactors compared with single addition of 100 M methyl jasmonate on day 8. The final TC production in the airlift bioreactors reached 565±47 mg l^–1, which is the highest reported for taxoid production in a bioreactor.     

Production of ginsenoside saponins by culturing ginseng (Panax ginseng) embryogenic tissues in bioreactors

Summary Ginseng (Panax ginseng) embryogenic tissues were cultured in three types of reactors and the ginsenoside productivities in these tissues were compared. As a result, the saponin productivity was the best when an airlift reactor was used, and more than twice of that when a paddle or internal turbine reactor was used. The tissues grew 9 fold during 42 days, and the ginsenoside pattern resembled that of ginseng leaves.Part 98 in the series Studies on Plant Tissue Cultures For Part 97 see Orihara, Y., and Furuya, T., (1993) submitted for publication.     

The high intracellular ph buffering of hagfish (Eptatretus cirrhatus) dental plate retractor muscle cannot be attributed to the known histidine-related compounds present in other vertebrates

• 1.1. Intracellular pH buffering capacity of hagfish (Eplatretus cirrhatus) dental plate retractor muscles is among the highest reported for any vertebrate muscle.
• 2.2. Over 80% of the pH buffering capacity of hagfish retractor and myotome muscle is due to components other than proteins and phosphate.
• 3.3. The muscles have less than 0.5 μmol/g wet weight of l-histidine, and lack l-l-methyl histidine, l-3-methyl histidine and the histidine-containing dipeptides anserine, carnosine and ophidine.
• 4.4. Instead, they contain an unidentified low molecular weight acid-soluble compound to which the high pH buffering capacity can be attributed.

     

CaCO<Subscript>3</Subscript> supplementation alleviates the inhibition of formic acid on acetone/butanol/ethanol fermentation by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis>

Objective

To investigate the inhibiting effect of formic acid on acetone/butanol/ethanol (ABE) fermentation and explain the mechanism of the alleviation in the inhibiting effect under CaCO₃ supplementation condition.

Results

From the medium containing 50 g sugars l^?1 and 0.5 g formic acid l^?1, only 0.75 g ABE l^?1 was produced when pH was adjusted by KOH and fermentation ended prematurely before the transformation from acidogenesis to solventogenesis. In contrast, 11.4 g ABE l^?1 was produced when pH was adjusted by 4 g CaCO₃ l^?1. The beneficial effect can be ascribed to the buffering capacity of CaCO₃. Comparative analysis results showed that the undissociated formic acid concentration and acid production coupled with ATP and NADH was affected by the pH buffering capacity of CaCO₃. Four millimole undissociated formic acid was the threshold at which the transformation to solventogenesis occurred.

Conclusion

The inhibiting effect of formic acid on ABE fermentation can be alleviated by CaCO₃ supplementation due to its buffering capacity.

     

Induced Production of Acidic Polysaccharide by Benzalkonium Chloride-resistant Bacterium (Enterobacter species) in the Presence of Hexachlorophen and Chlorhexidine

The bacterium, which was isolated from soil and identified as Enterobacter sp., was induced by hexachlorophen (HCP) and chlorhexidine (CH), as well as benzalkonium chloride (BC), to produce acidic polysaccharide. HCP is a bisphenol and CH is a bisbiguanido, while BC is a quarternary ammonium compound. The cells produced the maximum amount of the polysaccharide (0.3 ~ 0.9 mg as total sugar/mg dry weight cells) in a 0.07m potassium phosphate buffer (pH 7.2) containing 0.22 m glucose and approximately 0.1 mm BC or HCP, or 0.06 mm CH. There was no growth of the cells in these conditions. The polysaccharides produced in the presence of each drug were all composed of fucose, glucose, galactose and glucuronic acid. At the optimum concentration for polysaccharide production, a large amount of UV-absorbing material was released from the cells.     

Production of d-lactic acid by Lactobacillus delbrueckii ssp. delbrueckii from orange peel waste: techno-economical assessment of nitrogen sources

In this study, the effect of several organic nitrogen sources (namely peptone, meat extract—ME, yeast extract—YE, and corn steep liquor—CSL) on d-lactic acid production by Lactobacillus delbrueckii ssp. delbrueckii has been studied. While lactic acid bacteria (LAB) are well-known for their complex nutritional requirements, organic nitrogen source-related cost can be as high as 38% of total operational costs (OPEX), being its nature and concentration critical factors in the growth and productivity of the selected strain. Corn steep liquor (CSL) has been chosen for its adequacy, on the grounds of the d-lactic acid yield, productivity, and its cost per kilogram of product. Finally, orange peel waste hydrolysate supplemented with 37 g/l CSL has been employed for d-lactic acid production, reaching a final yield of 88% and a productivity of 2.35 g/l h. CSL cost has been estimated at 90.78$/ton of d-lactate.

     

Oxygen transfer in an airlift reactor with multiple net draft tubes

A pilot scale airlift reactor with multiple net draft tubes was developed to improve oxygen transfer in the reactor. The reactor was 0.29 m in diameter and 2 m height. A steadystate sulfite oxidation method was applied to determine an overall volumetric mass transfer coefficient. Oxygen transfer of the proposed airlift reactor can be 60–100% higher than that of bubble columns under the same operating conditions.List of Symbols C ^* mol·dm^–3 saturated concentration of dissolved oxygen - C _L mol·dm^–3 bulk concentration of dissolved oxygen - G mol/min nitrogen flow rate - k _L a hr^–1 the volumetric gas-liquid mass transfer coefficient - Mo ₂ g/mol molecular weight of oxygen - OTR g/min the oxygen transfer rate - U _g cm/s superficial air velocity - V _L dm³ volume of the liquid phase - _in oxygen mole ratio in the inlet gas - _out oxygen mole ratio in the outlet gas     

Penicillin acylase production by E. coli

Enzyme production with E. coli ATCC 11105, in a complex medium using phenylacetic acid as inducer is carried out in a stirred-tank reactor of 10 dm³ and an airlift tower-loop reactor of 60 dm³ with outer loop at a temperature of 27 °C. The optimum inducer concentration was 0.8 kg/m³, which was kept constant by fed-batch operation. The optimum of the relative dissolved O₂-concentration with regard to saturation is below 10% in a stirred-tank reactor and at 35% in a tower-loop reactor. It was kept constant by parameter-adaptive control of the aeration rate. In a stirred-tank enzyme productivity is slightly higher than in a tower-loop reactor, and much higher than in a bubble column reactor.List of Symbols CPR kg/(m³ h) CO₂-production rate - OTR kg/(m³ h) O₂-transfer rate - OUR kg/(m³ h) O₂-utilization rate - PAA phenylacetic acid (inducer) - RQ = CPR/OUR respiratory quotient - X kg/m³ cell mass concentration - _m h^–1 maximum specific growth rate     

Effect of pH on exocellular riboflavin production byEremothecium ashbyii

Summary The production of riboflavin byEremothecium ashbyii grown in a chemically defined medium in batch culture was affected by pH of the medium. Highest yields were obtained at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5. The medium pH also affected cell morphology. When the organism was grown in a stirred tank reactor and an airlift vessel at pH 4.5 very similar levels of riboflavin were obtained.