Production of the antitumoral retamycin during continuous fermentations of Streptomyces olindensis

Continuous fermentations were performed in order to correlate the production of retamycin, an anthracycline antibiotic produced by Streptomyces olindensis in submerged cultures, with the dilution rate. Maximum retamycin production was achieved at a dilution rate of 0.05 h⁻¹ (D=μ_x=0.05 h⁻¹), while higher dilution rates caused a decrease in antibiotic production, which ceased completely at a dilution rate of 0.30 h⁻¹. Otherwise, biomass productivity was favoured by high dilution rates, achieving a maximum at D=0.25 h⁻¹, whereas retamycin productivity reached a maximum at D=0.05 h⁻¹. Dilution rate influenced morphology, which was assessed by image analysis. The percentage of clumps decreased with an increase in dilution rate, with a correspondent increase in pellet percentage.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Continuous production of lactic acid from whey permeate by Lactobacillus helveticus in two chemostats in series

The effect of dilution rate on the production of lactic acid from whey permeate by Lactobacillus helveticus has been investigated. In the first chemostat of a two-stage system, total conversion (98.1%) and maximum lactic acid concentration (43.7 g l⁻¹) were obtained at a dilution rate (D_Itot) of 0.06 h⁻¹. Maximum volumetric productivities of lactic acid (8.27 g l⁻¹ h⁻¹) and biomass (1.90 g l⁻¹ h⁻¹) occurred at D_Itot of 0.40 h⁻¹. The fraction of -lactate in the product was found to increase with dilution rate and reached a maximum of 66% at the same dilution rate. The maximum specific growth rate (μ_max) on this medium was 0.7 h⁻¹. A Y_ATP (max) value of 22.4 g dry weight (mol ATP)⁻¹ and a maintenance coefficient of 8.0 mmol ATP (g dry weight h)⁻¹ were determined. The second stage, in series with the first, confirmed these results and further showed that the total residence time could be reduced by 50%, compared with a single chemostat for the same nearly complete level of substrate conversion.     

Ontogenetic photosynthetic changes, dispersal and survival of Thalassia testudinum (turtle grass) seedlings in a sub-tropical lagoon

Northward expansion of Thalassia testudinum (turtle grass) in Laguna Madre is occurring faster than can be explained by rhizome growth. We hypothesized that seedling establishment can account for the measured rates of meadow expansion and that seedling carbohydrate reserves are utilized until the plant is photosynthetically self-sufficient. To address seedling establishment, we estimated seed output, seedling dispersal and survival. Carbon dynamics were calculated from measurements of biomass allocation, non-structural carbohydrate carbon reserves and photosynthetic parameters in relation to T. testudinum seedling age. Potential seed production calculated for 1996 was consistent with field observations and was estimated at 66±14 seeds m⁻² bare area. Fruits can be positively buoyant for up to 10 days, while seeds were generally buoyant for <1 day. Water current measurements, made at about the time of seed release, indicate a positive net transport of 1.5 km d⁻¹ to the north. Seedling survival in laboratory culture after 6 months was 96% compared to 11% in the field after 1 year. The average root:rhizome+seed:leaf ratio changed from 0:11:1 for a 1 week old plant to 1:3:1 for a 15 month old plant. Seedlings used to determine whole plant photosynthesis ranged in age from about 1 week (0.25 months) to 15 months. Gross P_max increased from 80 to 220 μmol O₂ gdw sht⁻¹ h⁻¹, while whole plant respiration decreased from 170 to 60 μmol O₂ gdw sht⁻¹ h⁻¹. As the photosynthetic parameters changed, the average non-structural carbohydrate carbon (NSCC) reserves of the seeds decreased from 24 to 3.0 mg NSCC plant⁻¹. Subsequent increases in NSCC were the result of rhizome development. Daily carbon balance, assessed using H_sat periods of 8–18 h d⁻¹, predicts that T. testudinum seedlings become photosynthetically self-sufficient between 2 and 6 months. The unique characteristics of T. testudinum, including seed buoyancy, high seed production and survival rates, coupled with ontogenetic changes in carbon allocation and production imply that sexual reproduction can be important in the long distance dispersal and colonization for this species.     

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi

The effect of changing dilution rate (D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55 h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1 g L⁻¹ at D values of 0.15–0.35 h⁻¹. Maximal biomass productivity was found to be 0.39 g L⁻¹ h⁻¹, obtained at D = 0.35 h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45 mmol g⁻¹ h⁻¹ occurring at D = 0.4 h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4 h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.     

Aerobic biological treatment of black table olive washing wastewaters: effect of an ozonation stage

The present work is a study of oxidative degradation of the organic matter present in the washing waters from the black table olive industry. Pollutant organic matter reduction was studied by an aerobic biological process and by the combination of two successive steps: ozonation pretreatment followed by aerobic biological degradation. In the single aerobic biological process, the evolution of biomass and organic matter contents was followed during each experiment. Contaminant removal was followed by means of global parameters directly related to the concentration of organic compounds in those effluents: chemical oxygen demand (COD) and total phenolic content (TP). A kinetic study was performed using the Contois model, which applied to the experimental data, provides the specific kinetic parameters of this model: 4.81×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.279 g VSS g COD⁻¹ for the cellular yield coefficient and 1.92×10⁻² h⁻¹ for the kinetic constant for endogenous metabolism. In the combined process, an ozonation pretreatment is conducted with experiments where an important reduction in the phenolic compounds is achieved. The kinetic parameters of the following aerobic degradation stage are also evaluated, being 5.42×10⁻² h⁻¹ for the kinetic substrate removal rate constant, 0.280 g VSS g COD⁻¹ for the cellular yield coefficient and 9.1×10⁻³ h⁻¹ for the kinetic constant for the endogenous metabolism.     

Activity of oxyntomodulin on gastric acid secretion induced by histamine or a meal in the rat

Conscious rats with chronic gastric fistula were trained for drinking a 14-ml milk meal. The activity of an intestinal hormone, oxyntomodulin (OXM), was studied in this model and compared to that observed when histamine was the stimulus. Under histamine (0.25 mg·kg⁻¹·h⁻¹) stimulation, OXM at doses (60–120 pmol·kg⁻¹·h⁻¹) that induced physiological circulating levels inhibited gastric acid secretion up to 50%. Under meal stimulation, OXM reduced up to 29% acid secretion at doses (1–1.5 nmol·kg⁻¹·h⁻¹) inducing supraphysiological levels. We conclude that at physiological concentrations OXM cannot counteract the complex processes triggered by a meal. OXM would be a component of enterogastrone, a combination of several intestinal hormones acting in synergy. The OXM action is related to pathways recognizing the C-terminal 19–37 moiety of the molecule.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

The influence of hexoses addition on the fermentation of d-xylose in Debaryomyces hansenii under continuous cultivation

The effect of hexoses (glucose and galactose) addition to the feed xylose mineral medium of Debaryomyces hansenii chemostat cultures grown at a constant dilution rate of 0.055 h⁻¹ was studied. Xylitol was the major product detected amongst all tested conditions. The maximal values for xylitol yield and volumetric productivity (0.56 gg⁻¹ xylose and 0.21 gl⁻¹h⁻¹, respectively) were obtained for a glucose/xylose feeding ratio of 10%, showing that the addition of small amounts of glucose, but not galactose, enhanced the xylitol production. A xylitol yield increase of 30%, compared with the sole xylose-containing feed medium, was observed. It was found that the oxygen requirement for D. hansenii growth is lower under glucose compared with xylose. Ethanol and glycerol were only produced for glucose/xylose feeding ratio above 30%. The byproducts accumulation was correlated with glucose metabolism, because a direct relationship between the increase of ethanol (and glycerol) concentration and the increase of glucose in the feed medium was found.     

Influence of phosphorus concentration on the growth kinetics and stoichiometry of the microalga Scenedesmus obliquus

The growth of the freshwater microalga Scenedesmus obliquus was studied at 30°C in a mineral culture medium with phosphorus concentrations of between 0 and 372 μ . The values for the specific growth rates, between and , fitted a semistructured substrate-limitation model with μ_m1 = 0·0466 h⁻¹, μ_m2 = 0·0256 h⁻¹ and . The specific uptake rate of phosphorus reached a maximum value of q_Sm1 = 658·01 × 10⁻⁴ μmol P mg⁻¹ biomass h⁻¹.     

Continuous ethanol production and evaluation of yeast cell lysis and viability loss under very high gravity medium conditions

A combined bioreactor system, composed of a stirred tank and a three-stage tubular bioreactor in series and with a total working volume of 3260 ml, was established. Continuous ethanol production was carried out using Saccharomyces cerevisiae and a very high gravity (VHG) medium containing 280 g l⁻¹ glucose. An average ethanol concentration of 124.6 g l⁻¹ or 15.8% (v) was produced when the bioreactor system was operated at a dilution rate of 0.012 h⁻¹. The yield of ethanol to glucose consumed was calculated to be 0.484 or 94.7% of its theoretical value of 0.511 when ethanol entrapped in the exhaust gas was incorporated. Meanwhile, quasi-steady states and non-steady oscillations were observed for residual glucose, ethanol and biomass concentrations for all of these bioreactors during their operations. Models that can be used to predict yeast cell lysis and viability loss were developed.     

The thermal biology of three southern African elephant-shrews

1. 1. The thermal characteristics of Petrodromus tetradactylus, Elephantulus intufi and E. brachyrhynchus were investigated and compared with other elephant-shrews that occur in the southern African subregion.

2. 2. E. intufi and E. brachyrhynchus appear to have lower than expected basal metabolic rates (1.1185 ± 0.1623 and 0.9649 ± 0.1638 ml O₂ g⁻¹ h⁻¹, respectively) and high, narrow thermoneutral zones, similar to other elephant-shrews investigated previously. In contrast P. tetradactylus has a basal metabolic rate (0.871 ± 0.027 ml O₂ g⁻¹ h⁻¹) close to expected for body mass, and a broad, low thermoneutral zone.

3. 3. The thermal biology of macroscelids is discussed in terms of their distribution, microhabitat and body size.

     

Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis

The fungus Mortierella alpina LPM 301, a producer of arachidonic acid (ARA), was found to possess a unique property of a growth-coupled lipid synthesis. An increase in specific growth rate (μ) from 0.03 to 0.05 h⁻¹ resulted in a two-fold increase in the specific rate of lipid synthesis (milligram lipid (gram per lipid-free biomass) per hour). Under batch cultivation in glucose-containing media with urea or potassium nitrate as nitrogen sources, the ARA content was 46.0 and 60.4% of lipid; 16.4 and 18.8% of dry biomass; and 4.2 and 4.5 g l⁻¹, respectively. Under continuous cultivation of the strain, the productivity of ARA synthesis was 16.2 and 19.2 mg l⁻¹ h⁻¹ at μ=0.05 and 0.03 h⁻¹, respectively.     

Culture fluorescence dynamics in Xanthomonas campestris

Culture (NAD(P)H) fluorescence dynamics have been used to provide information on culture behaviour when Xanthomonas campestris was grown in a bioreactor. Culture fluorescence decreased by 1150 units in response to an increase in extracellular pH from 3.1 to 7.6. A mathematical model incorporating the effect of pH on the bulk NADH depletion reaction simulated the experimental data. The rates of bulk NADH formation and depletion reactions were 1 s⁻¹ and 719 (M h⁻¹)⁻¹ s⁻¹, respectively. Subsequent to the initial NADH decrease, the culture fluorescence increased to within 200 units of its original value, with a concomitant decrease in oxygen uptake rate (OUR) from 7.3 to 3 mM h⁻¹. A mathematical model incorporating the hypothesis that the culture manipulated its OUR to increase its NADH level, simulated the experimental data. In addition, it was inferred from culture fluorescence that the intracellular oxygen availability becomes insufficient at or below 10% extracellular dissolved oxygen value. Studies on H₂O₂ addition to X. campestris, to optimize the liquid-phase oxygen supply, showed no change in metabolic state, as indicated by NADH fluorescence, until 1.4 mmol H₂O₂ (g cell)⁻¹ and a significant decrease above that. Investigations on the reasons for decreases in NADH fluorescence suggested a DNA-damaging Fenton reaction as the probable reason for the observed NADH decrease on addition of H₂O₂.     

Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.     

Photosynthetic response of Myriophyllum salsugineum A.E. orchard to photon irradiance, temperature and external free CO2

The photosynthetic capacity of Myriophyllum salsugineum A.E. Orchard was measured, using plants collected from Lake Wendouree, Ballarat, Victoria and grown subsequently in a glasshouse pond at Griffith, New South Wales. At pH 7.00, under conditions of constant total alkalinity of 1.0 meq dm⁻³ and saturating photon irradiance, the temperature optimum was found to be 30–35°C with rates of 140 μmol mg⁻¹ chlorophyll a h⁻¹ for oxygen production and 149 μmol mg⁻¹ chlorophyll a h⁻¹ for consumption of CO₂. These rates are generally higher than those measured by other workers for the noxious Eurasian water milfoil, Myriophyllum spicatum L., of which Myriophyllum salsugineum is a close relative. The light-compensation point and the photon irradiance required to saturate photosynthetic oxygen production were exponentially dependent on water temperature. Over the temperature range 15–35°C the light-compensation point increased from 2.4 to 16.9 μmol (PAR) m⁻² s⁻¹ for oxygen production while saturation photon irradiance increased from 41.5 to 138 μmol (PAR) m⁻² s⁻¹ for oxygen production and from 42.0 to 174 μmol (PAR) m⁻² s⁻¹ for CO₂ consumption. Respiration rates increased from 27.1 to 112.3 μmol (oxygen consumed) g⁻¹ dry weight h⁻¹ as temperature was increased from 15 to 35°C. The optimum temperature for productivity is 30°C.     

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-β-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 μM mg dw⁻¹ at a maximum APase activity of 2.57 nM PNP mg dw⁻¹ h⁻¹. In contrary, S. platensis depicted maxima of 1.39 nM PNP mg dw⁻¹ h⁻¹ on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.     

Regulation of fermentative capacity and levels of glycolytic enzymes in chemostat cultures of Saccharomyces cerevisiae

Regulation of fermentative capacity was studied in chemostat cultures of two Saccharomyces cerevisiae strains: the laboratory strain CEN.PK113-7D and the industrial bakers’ yeast strain DS28911. The two strains were cultivated at a fixed dilution rate of 0.10 h⁻¹ under various nutrient limitation regimes: aerobic and anaerobic glucose limitation, aerobic and anaerobic nitrogen limitation on glucose, and aerobic ethanol limitation. Also the effect of specific growth rate on fermentative capacity was compared in glucose-limited, aerobic cultures grown at dilution rates between 0.05 h⁻¹ and 0.40 h⁻¹. Biomass yields and metabolite formation patterns were identical for the two strains under all cultivation conditions tested. However, the way in which environmental conditions affected fermentative capacity (assayed off-line as ethanol production rate under anaerobic conditions) differed for the two strains. A different regulation of fermentative capacity in the two strains was also evident from the levels of the glycolytic enzymes, as determined by in vitro enzyme assays. With the exception of phosphofructokinase and pyruvate decarboxylase in the industrial strain, no clear-cut correlation between the activities of glycolytic enzymes and the fermentative capacity was found. These results emphasise the need for controlled cultivation conditions in studies on metabolic regulation in S. cerevisiae and demonstrate that conclusions from physiological studies cannot necessarily be extrapolated from one S. cerevisiae strain to the other.