Production of cephalosporin C in a fluidized-bed bioreactor

Production of cephalosporin C was investigated in a fluidized-bed bioreactor using bioparticles of Cephalosporium acremonium. Bioparticles were developed by forming a biofilm of growing hyphae around celite particles which contained spores of the microorganism. Production of the antibiotic was significantly improved by using bioparticles over the free mycelial culture, possibly due to the enhanced mass transfer capacity of the bioreactor system and successive generation of highly productive morphological forms of the microorganism. The maximum attainable titer of cephalosporin C from the bioreactor system was almost double that from a jar fermentor operation with a free mycelial culture of the same strain. The biofilm of the bioparticles became unstable as the fermentation proceeded. Morphological differentiation of the microorganism caused a gradual loss of biofilm and an increase of free cells in the culture broth. Additional feeding of a limited amount of methionine to the fermentation broth was not as effective as expected for improving the bioparticle stability. However, repeated use of the bioparticles revealed a strong possibility to improved the overall reactor performance since it allowed an enhanced production of the antibiotic with fewer free cells.     

Effects of by-products of fermentation of banana pseudostem on ethanol separation by pervaporation

In this work, we performed recovery of ethanol from a fermentation broth of banana pseudostem by pervaporation (PV) as a lower-energy-cost alternative to traditional separation processes such as distillation. As real fermentation systems generally contain by-products, it was investigated the effects of different components from the fermentation broth of banana pseudostem on PV performance for ethanol recovery through commercial flat sheet polydimethylsiloxane (PDMS) membrane. The experiments were compared to a binary solution (ethanol/water) to determine differences in the results due to the presence of fermentation by-products. A real fermented broth of banana pseudostem was also used as feed for the PV experiments. Seven by-products from fermented broth were identified: propanol, isobutanol, methanol, isoamyl alcohol, 1-pentanol, acetic acid, and succinic acid. Moreover, the residual sugar content of 3.02 g/L¹ was obtained. The presence of methanol showed the best results for total permeate flux (0.1626 kg·m⁻²·h⁻¹) and ethanol permeate flux (0.0391 kg·m⁻²·h⁻¹) during PV at 25°C and 3 wt% ethanol, also demonstrated by the selectivity and enrichment factor. The lowest total fluxes of permeate were observed in the experiments containing the acids. Better permeance of 0.1171 from 0.0796 kg·m⁻²·h⁻¹ and membrane selectivity of 9.77 from 9.30 were obtained with real fermentation broth than with synthetic solutions, possibly due to the presence of by-products in the multicomponent mixtures, which contributed to ethanol permeation. The results of this work indicate that by-products influence pervaporation of ethanol with hydrophobic flat sheet membrane produced from the fermented broth of banana pseudostem.     

A comparative evaluation of oxygen mass transfer and broth viscosity using Cephalosporin-C production as a case strategy

The production of Cephalosporin-C (CPC) a secondary metabolite, using a mold Acremonium chrysogenum was studied in a lab scale Internal loop air lift reactor. Cephalosporin-C production process is a highly aerobic fermentation process. Volumetric gas–liquid mass transfer coefficient (k_La) and viscosity (η) were evaluated, during the growth and production phases of the microbial physiology. An attempt has been made to correlate the broth viscosity, η and volumetric oxygen transfer coefficient, k_La during the Cephalosporin-C production in an air lift reactor. The impact of biomass concentration and mycelial morphology on broth viscosity has been also evaluated. The broth exhibits a typical non-Newtonian fermentation broth. Rheology parameters like consistency index and fluidity index are also studied.     

Continuous ethanol production by yeast immobilized on to channeled alumina beads

Vertical and near-horizontal (15° angle) packed-bed columns were compared for continuous ethanol fermentation using an alcohol- and glucose-tolerant Saccharomyces cerevisiae strain immobilized on to channeled alumina beads (5·0 × 10⁹ cells g⁻¹ beads). Spaces between beads (1·0–6·5 mm) and angle (15°) of near-horizontal reactor columns (with six ports in each) efficiently removed CO₂ and increased ethanol productivity. Malt-glucose-yeast-extract broth containing 16·7% glucose at 35°C fed at a dilution rate of 3· h⁻¹ to thw two horizontal columns (in series) yielded maximum ethanol productivity of 40·0 g liter⁻¹ h⁻¹. Feedstock flow rate and other factors (temperature, pH, nutrients, and glucose levels) affected productivities. The immobilized-cell system showed operational stability for >3 months without plugging, and could be stored for at least one year with no loss of bioreactor performance. Scanning electron micrographs of the beads revealed large numbers of yeast-cells attached on to internal and external surfaces of beads.     

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.     

Continuous production of an antibiotic polypeptide (nisin) by Lactococcus lactis using a bioreactor coupled to a microfiltration module

The continuous production of nisin, an antibiotic polypeptide, by Lactococcus lactis in a bioreactor system coupled to a microfiltration module is described. Nisin productivity with respect to both cultivation time (ND) and the quantity of glucose consumed (ND/S_f) in continuous production was enhanced by maintaining a low concentration of lactic acid in the broth. A maximum ND of 7.80 × 10⁴ U·l⁻¹·h⁻¹ and ND/S_f of 5.20 × 10³ U·g⁻¹·h⁻¹ were obtained when the glucose concentration in the feed medium was 15 g/l. These values represent about 4.1- and 4.5-fold increases, respectively, over those obtained in batch culture.     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

Diacetyl fermentation coupled with pervaporation using oleyl alcohol supported liquid membrane

Pervaporation using oleyl alcohol supported liquid membrane was successfully applied to diacetyl fermentation by immobilized lactic acid bacteria. Diacetyl productivity was about 10 g·m⁻³·h⁻¹, while productivity during batch fermentation was about 6 g·m⁻³·h⁻¹. Diacetyl yield from consumed glucose was about 0.04 g·g⁻¹ which was 4 times as large as that of batch fermentation. The pervaporation functioned favorably on actual fermentation broth. The flux of the permeate and the diacetyl separation factor for the pervaporation were about 9 g·m⁻²·h⁻¹ and 36, respectively, and these values were maintained at almost constant levels during fermentation. Diacetyl concentration in the permeate was about 2 kg·m⁻³, which is sufficiently high for commercial use.     

The study of renin–angiotensin–aldosterone in experimental diabetes mellitus

It is generally accepted that hypertension and other vascular pathologies increase in diabetes mellitus (DM) patients as a result of the renin–angiotensin–aldosterone (RAA) system. In this study, changes in the renin‐angiotensin‐aldosterone (RAA) system level was determined in Streptozotocin (STZ)‐injected rats. A total of 46 female Wistar albino rats (180–220 g body weight) was utilized in these experiments. STZ was given intraperitoneally to induce diabetes in rats. Streptozotocin (60 mg kg⁻¹ body weight) was dissolved in 0·1 m citrate–‐phosphate buffer (pH 4–5). The non‐diabetic rats were injected with sterilized buffer alone to act as a control group. Blood glucose levels were 398±8·2 mg dl⁻¹, 488±11·75 mg dl⁻¹ and 658±29·6 mg dl⁻¹ at days 3, 12 and 30 respectively. The level of plasma renin activity (PRA) was measured as 7·69±1·07 ng ml⁻¹ h⁻¹; 1·82±0·22 ng ml⁻¹ h⁻¹ and 0·67±0·12 ng ml⁻¹ h⁻¹ at days 3, 12 and 30, respectively. These values showed that the PRA levels are decreased with increased time period. Serum angiotensin converting enzyme (ACE, E.C. 3.4.15.1) levels were increased at days 12 and 30 (p<0·05 and p<0·005), whereas serum aldosterone levels were increased at days 3 and 12 (p<0·05). The level of urea and creatinine increased at days 12 and 30 (p<0·05 and p<0·005, respectively) when compared to the control group. The data from these experiments indicate that the PRA level decreased whereas ACE activity level increased in diabetic rats compared with the control. Aldosterone levels increased at the first stage of the experiment, but then decreased by the end of the experiment as a result of changes in renin and ACE levels. Copyright © 1999 John Wiley & Sons, Ltd.     

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).     

Poly(β-hydroxybutyric acid) thermoplastic production by Alcaligenes latus: Behavior of fed-batch cultures

Fed-batch culture of Alcaligenes latus, ATCC 29713, was investigated for producing the intracellular bioplastic poly(β–hydroxybutyric acid), PHB. Constant rate feeding, exponentially increasing feeding rate, and pH-stat fed batch methods were evaluated. pH-stat fed batch culture reduced or delayed accumulation of the substrate in the broth and led to significantly enhanced PHB productivity relative to the other modes of feeding. Presence of excessive substrate appeared to inhibit PHB synthesis, but not the production of cells. In fed-batch culture, the maximum specific growth rate (0.265?h^?1) greatly exceeded the value (0.075?h^?1) previously observed in batch culture of the same strain. Similarly, the maximum PHB production rate (up to 1.15?g?·?l^?1?·?h^?1) was nearly 8-fold greater than values observed in batch operations. Fed-batch operation was clearly superior to batch fermentation for producing PHB. A low growth rate was not a prerequisite for PHB accumulation, but a reduced or delayed accumulation of substrate appeared to enhance PHB accumulation. Under the best conditions, PHB constituted up to 63% of dry cell mass after 12?h of culture. The average biomass yield coefficient on sucrose was about 0.35, or a little less than in batch fermentations. The highest PHB concentrations attained were about 18?g?·?l^?1.     

Interactions of cell morphology and transport processes in the lovastatin fermentation

The cholesterol lowering drug, Lovastatin (Mevacor), acts as an inhibitor of HMGCoA reductase, and is produced from an Aspergillus terreus fermentation.Pilot scale studies were carried out in 800 liter fermenters to determine the effects of cell morphology on the oxygen transport properties of this fermentation. Specifically, parallel fermentations giving (i) filamentous mycelial cells, and (ii) discrete mycelial pellets, were quantitatively characterized in terms of broth viscosity, availability of dissolved oxygen, oxygen uptake rates and the oxygen transfer coefficient under identical operating conditions.The growth phase of the fermentation, was operated using a cascade control strategy which automatically changed the agitation speed with the goal of maintaining dissolved oxygen at 50% saturation. Subsequently stepwise changes were made in agitation speed and aeration rate to evaluate the response of the mass transfer parameters (DO, OUR, and k _L a). The results of these experiments indicate considerable potential advantages to the pellet morphology from the standpoint of oxygen transport processes.List of Symbols DO % sat. Dissolved oxygen concentration - k _L a h^–1 Gas-liquid mass transfer coefficient - OUR mmol/dm³h Oxygen uptake rate - P/V KW/m³ Agitator power per unit volume - V _s m/s Superficial air velocity - _app cP Apparent viscosity     

Lipid and biomass production by the halotolerant microalga Nannochloropsis salina

The effect of environmental factors on cell-lipid content, on the growth rate and on the overall productivity of Nannochloropsis salina was tested in the laboratory and in outdoor cultures. Under optimum conditions in the laboratory, the maximum growth rate (μ_max) was 0·030 h⁻¹, which corresponds to a doubling time of 23 h. Cellular lipid content was affected by the phase of growth and the temperature, but not by nitrogen starvation, pH or the source of sea water. The most important factor affecting the output rate of biomass was the cell concentration. The maximum biomass productivity obtained in outdoor ponds was 24·5 g·m⁻²·day⁻¹, and the lipid production rate was 4·0 g m⁻²·day⁻¹.     

Does midazolam inhibit the development of acute tolerance to the analgesic effect of alfentanil?

Alfentanil-midazolam analgesic interactions were studied in rats with continuous infusions or bolus injections of the drugs. Analgesia was determined by measuring the threshold of motor response to noxious pressure. The continous constant-rate infusion of alfentanil demonstrated that after an initial peak, the analgesia profoundly declined due to the development of acute tolerance. When alfentanil (250 μg·kg⁻¹·h⁻¹) was given together with midazolam (3 mg·kg⁻¹·h⁻¹), the decline in the analgesic effect of alfentanil was attenuated. Following the 4 h period of the constant-rate (250 μg·kg⁻¹·h⁻¹) infusion of alfentanil, when acute tolerance was already developed, midazolam (3 mg·kg⁻¹) given as a bolus injection enhanced the alfentanil-induced anesthesia. At the same time, when alfentanil was given as a bolus injection (30 μg·kg⁻¹) with or without midazolam (3 mg·kg⁻¹) also by bolus injection, no changes were seen to indicate an enhancement of the analgesic effect of alfentanil by midazolam. The results suggest that midazolam attenuates the development of acute tolerance to the analgesic effect of alfentanil.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

Glutamine synthetase activity and free amino acid pools of eelgrass (Zostera marina L.) roots

Activity of the enzyme glutamine synthetase (GS, EC 6.3.1.2) was determined in vitro for roots of the marine angiosperm Zostera marina L. (eelgrass) collected from a population in Great Harbor, Woods Hole, Massachusetts, U.S.A. The GS synthetase activity was lowest in roots of plants collected from the shallow region of the eelgrass bed (12.0 μmol·g⁻¹ (fresh wt)· h⁻¹) and increased in the mid (3.0 m, 40.3 μmol·g⁻¹ (fresh wt)·h⁻¹) and deep (5.0 m, 72.3 μmol·g⁻¹ (fresh wt)·h⁻¹) plant collection depths. GS transferase activity increased with collection depth in a similar manner: shallow, 28.6 μmol·g⁻¹ (fresh wt)·h⁻¹; mid, 52.0 μmol·g⁻¹ (fresh wt)·h⁻¹; deep, 92.8 μmol·g⁻¹ (fresh wt)·h⁻¹. When sediment-embedded plants were held in continuous darkness for 2 days to create extended root anoxia, root GS activities nearly doubled. In contrast, in vivo incorporation of ¹⁴C-glutamate into glutamine and protein residue remained constant or declined under short-term hypoxia and anoxia. During aerobic recovery from anoxia, root labelling of glutamine and protein increased markedly. Free amino acid patterns of eelgrass roots growing in situ were determined over a diurnal cycle. Total free amino acid content was maximal at dawn and decreased 50% by noon. In contrast, the proportion of glutamine was lowest at dawn and maximal at noon for both shallow and deep-growing plants. Despite differences in depth-specific plant sizes, root/rhizome/shoot ratios, and relative growth rates, the daily whole plant nitrogen demand of shallow and deep growing plants were equivalent. When corrected for assay temperature response, the enzyme synthetase activities measured in vitro suggest that all of the plant nitrogen assimilation requirements can be met within daylight hours during the period of peak summer biomass.     

Effect of light intensity and eye development on prey capture by larval striped bass Morone saxatilis

The efficacy of visual and non-visual feeding among pelagic striped bass Morone saxatilis larvae adapted to a turbid estuary was determined in the laboratory in clear water. Capture of Artemia salina (density 100 l⁻¹) was significantly affected by the interaction between age of larvae (range: 8–25 days post-hatch, dph) and light intensity (range: 0–10·6 μmol s⁻¹ m⁻² at the water surface). Visual feeding by larvae aged 9–11 dph was highest in dim light (0·086–0·79 μmol s⁻¹ m⁻²), with fish capturing up to 5 prey larva⁻¹ h⁻¹. As the larvae grew, prey capture in brighter light improved, associated with an increasing proportion of twin cone photoreceptors and improving ability of the retina to light- and dark-adapt. By age >22 dph, mean prey capture was greatest at highest light intensities (0·79 and 10·6 μmol s⁻¹ m⁻²) exceeding 100 prey larva⁻¹ h⁻¹. Incidence of feeding larvae generally improved as the larvae grew, reaching >80% in all light intensities from 16 dph onwards. The lower threshold for visual feeding, between 0·0084 and 0·03 μmol s⁻¹ m⁻², remained constant as the larvae grew, despite an increasing density of rod photoreceptors. Below this threshold, non-visual feeding was evident at a low rate (<6 prey larva⁻¹ h⁻¹) that was independent of larval age.     

Enhanced production of peroxidase in continuous culture of Arthromyces ramosus by selective bleeding of mycelium

A new method of continuous culture with selective bleeding of mycelia using 9-mesh screen was developed to improve the production rate of peroxidase (POD) by Arthromyces ramosus. At the dilution rate of 0.05 h⁻¹ with the mycelium leakage rate of 60%, a high production rate (average value was 1.67 U·ml⁻¹·h⁻¹) was maintained for over 100 h: the rate was 3.2 times that in a glucose-fed batch culture. At the same dilution rate, the volumetric and specific production rates of POD in the continuous culture without the screen were lower than those in the first continuous culture and decreased gradually in the later phase of the culture. In the continuous culture with low mycelium leakage rate of 1.6%, the POD production rate was not improved further, although the mycelial concentration (43 g·l⁻¹) increased 2.9 times. It is suggested that the high agitation rate required to meet the oxygen demand is unfavorable for the POD production.     

Submerged citric acid fermentation: rheological properties ofAspergillus niger broth in a stirred tank reactor

Summary A large number of submerged citric acid fermentations in a beet molasses substrate was studied. The development of Aspergillus niger from conidia to pellets was followed. Rheological characteristics of the fermentation broth including the pellets were determined. The results obtained confirm the fact that the non-Newtonian pseudoplastic behaviour of the fermentation broth was due to the presence of mycelial pellets. The most significant changes in rheological properties occurred during the period of maximal citric acid production and increase in biomass. Offprint requests to: M. Berovi     

Zooplankton and bacteria contribution to phosphorus and nitrogen internal cycling in a tropical and eutrophic reservoir: Pampulha Lake,Brazil

Nutrient regeneration and respiration rates of natural zooplankton from a tropical reservoir were experimentally measured. Excretion rates of ammonia (E_a), orthophosphate (E_p) and community respiration rates (R) were estimated considering the variations in the concentrations of ammonia, orthophosphate and dissolved oxygen between control and experimental units. The ranges obtained for these rates from the 2 h assays were E_a = 1.95–4.95 μg N-NH₄ · mg · DW⁻¹ · h⁻¹; E_p = 0.12–0.76 μg P-PO₄ mg DW⁻¹ · h⁻¹. Respiratory rates were quite constant (R = 0.01–0.02 mg O₂ · mg DW⁻¹ · h⁻¹). The uptake of nutrients due to bacteria can affect the experimental determination of excretion rates of zooplankton. Orthophosphate release increased from 0.28 to 0.82 μg P-PO₄ · mg DW⁻¹ · h⁻¹ when bacterial activity was depleted by antibiotic addition in experimental vessels (Exp IV). This demonstrates that free living bacteria are able to consume promptly most phosphorus excreted by zooplankton. Ammonia excretion rates were lower in experimental units containing antibiotics. Lower excretion rates were also obtained with longer exposure times and higher biomass levels in the experimental units. Finally, this study also showed that zooplankton excretion can affect significantly turn over rates of total phosphorus in Pampulha Reservoir. In some periods, specially during the dry season when zooplankton biomass was very high, phosphorus release by zooplankton, during one single day, can be as high as 40% of the total phosphorus content in lake water (Turn over time = 2.5 days).