Exopolysaccharide production by Pseudomonas NCIB11264 grown in batch culture.

Fermentation studies using batch culture indicated that exopolysaccharide production by Pseudomonas NCIBI1264 in a chemically defined medium increased under conditions of nitrogen limitation and excess carbon substrate at pH values above 6. The polysaccharide was formed from a variety of carbon substrates and its composition was not affected by the nature of the carbohydrate source. Polysacharide formation did not increase in media containing small amounts of phosphate, and, as in secondary metabolite production, it started late in the exponential growth phase continuing maximally after growth had ceased. The efficiency of glucose conversion into exopolysaccharide was low. Colorimetric, viscometric, and total carbon estimation techniques are described for determining exopolysaccharide levels in cell-free culture supernatants.     

分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响

研究了2．5L罐分批培养时pH和温度对重组大肠杆菌生产谷胱甘肽合成酶系的影响,确定了分批培养时生产谷胱甘肽合成酶系的最佳pH和最佳温度。研究结果表明：在发酵液的pH为7．2和温度为37℃时,谷胱甘肽合成酶系产量和细胞干重达到最大,分别为690．6U／L和3．77g／L。采用变温控制对菌体的生长和谷胱甘肽合成酶系的合成并没有明显的优点。     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Effect of pH, temperature and nutrient limitations on growth and leukotoxin production by Mannheimia haemolytica in batch and continuous culture

AIMS: Quantification of the effects of pH, temperature and nutrient limitations on the growth and leukotoxin (LKT) production parameters of Mannheimia haemolytica in batch and chemostat culture. METHODS AND RESULTS: Mannheimia haemolytica strains OVI-1 and PH12296 were grown aerobically in two semi-defined media. In amino acid-limited cultures, the LKT concentration and yield in terms of biomass (Y(LKT/x)) were up to eightfold greater than in carbon-limited cultures. Supplementing amino acid-limited chemostat cultures with cysteine, glutamine, ferric iron and manganese further enhanced the Y(LKT/x) values up to threefold. Supplementation of an amino acid-limited batch culture of M. haemolytica strain OVI-1 with these nutrients resulted in an LKT concentration of 1.77 g l(-1) that was 45-fold greater than that obtained in RPMI 1640 medium. Aerobiosis enhanced LKT production. High acetic acid concentrations were produced under carbon-sufficient conditions. The highest maximum specific growth rates were recorded in the range of pH 6.8 to 7.8 and 37 to 40 degrees C. CONCLUSIONS: An amino acid-limited culture medium greatly improved LKT production in aerobic batch culture, which could be further enhanced by supplementation with cysteine, glutamine, ferric iron and manganese. SIGNIFICANCE AND IMPACT OF THE STUDY: It was demonstrated that LKT production by M. haemolytica could be dramatically increased through manipulation of the culture medium composition, which could benefit the production of LKT-based vaccines against bovine shipping fever pneumonia.     

Sterols and fatty acids of an antarctic sea ice diatom, Stauroneis amphioxys

Unialgal clonal cultures of the diatom Stauroneis amphioxys Gregory, isolated from sea ice of the Indian Ocean sector ofthe Southern Ocean, were grown at 3° and 20°. The relative abundances offatty acids, sterols and phytol for the two cultures are comparable. The two sterols observed [24-methylcholesta-5,22E-dien-3β-ol (79%) and cholesterol (21%)] did not vary with culture temperature. The-major fatty acid composition is typical ofmost diatoms. A pronounced change of ratio with temperature occurred with the pair 16:4 Δ6, 9, 12, 15 and 16:3 Δ6, 9, 12 followed by 18:4 Δ6, 9, 12, 15:18:3 Δ9,12,15 and 20:5 Δ5,8,11,14,17:20:4 Δ8,11,14,17; thus the relative abundances of 16:4, 18:4, 20:5 and 22:6 increase at the lower growth temperature. The total amounts of unsaturated acids do not change with temperature suggesting an effect on the final desaturase step. No cryoprotective role for such changes in lipid composition was inferred.     

RecD plays an essential function during growth at low temperature in the antarctic bacterium Pseudomonas syringae Lz4W

The Antarctic psychrotrophic bacterium Pseudomonas syringae Lz4W has been used as a model system to identify genes that are required for growth at low temperature. Transposon mutagenesis was carried out to isolate mutant(s) of the bacterium that are defective for growth at 4 degrees but normal at 22 degrees . In one such cold-sensitive mutant (CS1), the transposon-disrupted gene was identified to be a homolog of the recD gene of several bacteria. Trans-complementation and freshly targeted gene disruption studies reconfirmed that the inactivation of the recD gene leads to a cold-sensitive phenotype. We cloned, sequenced, and analyzed approximately 11.2 kbp of DNA from recD and its flanking region from the bacterium. recD was the last gene of a putative recCBD operon. The RecD ORF was 694 amino acids long and 40% identical (52% similar) to the Escherichia coli protein, and it could complement the E. coli recD mutation. The recD gene of E. coli, however, could not complement the cold-sensitive phenotype of the CS1 mutant. Interestingly, the CS1 strain showed greater sensitivity toward the DNA-damaging agents, mitomycin C and UV. The inactivation of recD in P. syringae also led to cell death and accumulation of DNA fragments of approximately 25-30 kbp in size at low temperature (4 degrees ). We propose that during growth at a very low temperature the Antarctic P. syringae is subjected to DNA damage, which requires direct participation of a unique RecD function. Additional results suggest that a truncated recD encoding the N-terminal segment of (1-576) amino acids is sufficient to support growth of P. syringae at low temperature.     

Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris

Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate. Received: 30 September 1996 / Received revision: 21 January 1997 / Accepted: 10 February 1997     

Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C12-C18 fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the zeta-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.     

Kinetics of growth and lactic acid production in continuous and batch culture

Summary In a lactic acid fermentation by Streptococcus faecalis, the specific consumption rate of glucose (v) and the specific production rate of lactic acid () were represented by the following simple equations as functions of the specific growth rate (): 1/=(1/) + 1/ = (1/) + By use of data from a batch culture, these two equations were derived from enzyme kinetics of the product inhibition. These equations were successfully applied to the results of batch culture and chemostat culture. In addition, calculation of ATP yield by these equations agreed with the experimental results better than the conventional Leudeking-Piret type equation, which includes two terms associated with growth and not with growth. Correspondence to: H. Ohara     

Influences of incubation temperature and various saccharides on the production of organic acids and gases by gut microbes of rainbow trout Oncorhynchus mykiss in a micro-scale batch culture

We studied the influence of incubation temperature and additional saccharides on the metabolism of hindgut microbes of the rainbow trout Oncorhynchus mykiss in a 50 microl-scale batch culture system. Intestinal contents of rainbow trout reared at 15 degrees C were incubated with glucose, lactosucrose, sodium alginate or colloidal chitin (each 10 g/l) at 15 degrees C or 25 degrees C for 12 h. Levels of organic acids at 0 h and 12 h of incubation were quantified with HPLC. We also monitored gas release from these cultures during incubation. The main product was iso-butyric acid, except for the cultures with colloidal chitin where no net production of organic acids was observed. We detected higher levels of iso-butyric acid in cultures with lactosucrose than in the other cultures. Net production of this acid was less in cultures with colloidal chitin than in blank cultures. The volume of released gas was larger when incubated at 25 degrees C than at 15 degrees C. Cultures with colloidal chitin released more gas than blank cultures when they were incubated at 15 degrees C. Cultures with sodium alginate released less gas than blank cultures irrespective of incubation temperature. These results indicate that the hindgut microbes of this carnivorous fish mainly produce branched-chain fatty acids, very likely by microbial digestion of nitrogenous materials rather than saccharides. However, additional saccharides affected production of branched-chain fatty acids. The influence of incubation temperature in the present study also suggested that the environmental temperature of host fish should affect microbial digestion in the fish gut.     

Effect of exogenous fatty acids on the growth and production of exopolysaccharides of obligately methylotrophic bacterium Methylophilus quaylei

Accelerating growth and increasing exopolysaccharide production in obligate methylotrophic bacterium Methylophilus quaylei were observed in the presence of C₁₂–C₁₈ fatty acids added to the growth media. Sodium oleate was the best growth factor. Based on data on the composition of the free fatty acids fraction in the cells and the values of the ξ-potential and fluorescence anisotropy of whole cells, we suggested that fatty acids were incorporated in the outer membrane of M. quaylei.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.     

Hibernation in an antarctic fish: on ice for winter

Active metabolic suppression in anticipation of winter conditions has been demonstrated in species of mammals, birds, reptiles and amphibians, but not fish. This is because the reduction in metabolic rate in fish is directly proportional to the decrease in water temperature and they appear to be incapable of further suppressing their metabolic rate independently of temperature. However, the Antarctic fish (Notothenia coriiceps) is unusual because it undergoes winter metabolic suppression irrespective of water temperature. We assessed the seasonal ecological strategy by monitoring swimming activity, growth, feeding and heart rate (f(H)) in N. coriiceps as they free-ranged within sub-zero waters. The metabolic rate of wild fish was extrapolated from f(H )recordings, from oxygen consumption calibrations established in the laboratory prior to fish release. Throughout the summer months N. coriiceps spent a considerable proportion of its time foraging, resulting in a growth rate (G(w)) of 0.18 +/- 0.2% day(-1). In contrast, during winter much of the time was spent sedentary within a refuge and fish showed a net loss in G(w) (-0.05 +/- 0.05% day(-1)). Whilst inactive during winter, N. coriiceps displayed a very low f(H), reduced sensory and motor capabilities, and standard metabolic rate was one third lower than in summer. In a similar manner to other hibernating species, dormancy was interrupted with periodic arousals. These arousals, which lasted a few hours, occurred every 4-12 days. During arousal activity, f(H) and metabolism increased to summer levels. This endogenous suppression and activation of metabolic processes, independent of body temperature, demonstrates that N. coriiceps were effectively 'putting themselves on ice' during winter months until food resources improved. This study demonstrates that at least some fish species can enter a dormant state similar to hibernation that is not temperature driven and presumably provides seasonal energetic benefits.     

Effects of incubation temperature on growth and development of embryos ofAlligator mississippiensis

Summary Eggs ofAlligator mississippiensis were incubated at 30 °C and 33 °C throughout incubation up to hatching. Every four days several eggs were opened and the albumen, yolk and extra-embryonic fluids removed and weighed. The embryo was removed and fixed prior to being staged, weighted and measured for various morphometric criteria. Development at 33 °C was accelerated compared with 30 °C in terms of yolk and albumen utilization and embryo growth. Significant losses in yolk mass did not occur until stage 22 at 33 °C but occurred at stage 18 at 30 °C. Different patterns in growth were observed in embryos at the two temperatures at similar morphological stages: between stages 18 and 22 embryos at 33 °C were smaller (in mass and length) compared with embryos at 30 °C despite being morphologically similar. The differences in growth and physiology between embryos at 30 °C (females) and 33 °C (males) were dependent on incubation temperature but not sex. Incubation at 33 °C accelerated both growth and development inAlligator; initially morphogenesis was accelerated by the higher temperature but later, growth rate was accelerated.     

Protoheme,a dispensable growth factor forBacteroides fragilis grown by batch and continuous culture in a basal medium

The growth yields of 10 strains ofBacteroides fragilis isolated from a variety of clinical sites were determined in (a) basal medium, (b) basal medium plus heme, and (c) basal medium plus heme and menadione. The molar growth yield values, expressed as a function of glucose (Y_G) and ATP produced (Y_ATP) for 24 h and 48 h were used for a comparison of different strains. Considerable variation occurred among strains, but in general only the results from 24-h grown cells were reproducible. After this period, the microscopic appearance of cells changed dramatically from well-formed, intact cells to large collections of extracellular vesicles and lysed cells. All strains were stimulated by heme, but marked differences occurred among strains. The addition of heme and menadione to the basal medium increased the Y_G values of some strains, whereas others were unaffected. Heme-cultured cells produced acetate, propionate, and succinate as major metabolic end products and possessed cytochrome b, menaquinone-10, and fumarate reductase activity. Strain NCTC 9343 grown without added heme by continuous culture or batch culture produced cells that were morphologically and biochemically similar. Under both conditions these cells lacked cytochromes, menaquinones, and fumarate reductase activity, but produced high levels of lactate and fumarate together with lower levels of acetate, propionate, and succinate.     

Effects of melanin on the accumulation of exopolysaccharides by Aureobasidium pullulans grown on nitrate

Aureobasidium pullulans produced pullulan and melanin when grown in medium containing low nitrate levels. With high nitrate concentrations, however, this fungus produced a mixture of exopolysaccharides (EPS) without melanin synthesis. At 0.78 g l(-1) N as nitrate, where no melanin synthesis occurred, maximum EPS yields reached 6.92 g l(-1) and then decreased to the final yield of 2.36 g l(-1). Following melanin addition (0.1 g l(-1)), yields reached 7.02 g l(-1) at 48 h and fell to a final yield of 5.21 g l(-1). The EPS produced in high nitrate medium contained both pullulan and (1-->3)-beta-glucan, but only pullulan was produced with melanin-supplementation. With melanin addition a doubling of (1-->3)-beta-glucanase activity was observed in high nitrate medium compared to that without supplementation. On the other hand amylolytic activities disappeared in medium with melanin production or addition. Culture filtrates sustained a higher reducing capacity (RC) when melanin was present. Low RC appeared to reduce (1-->3)-beta-glucanase activity and increase amylolytic activities. Thus, higher RC appears to inhibit production/activity of amylose-degrading enzymes capable of degrading pullulan, and stimulates (1-->3)-beta-glucanase synthesis/activity, leading to a preferential accumulation of pullulan.     

Effects of temperature and incubation period on production of fumonisin B1 by Fusarium moniliforme.

The kinetics of the production of fumonisin B1 (FB1) by Fusarium moniliforme MRC 826 in corn cultures was investigated as a function of fungal growth at various incubation temperatures. The growth rate of F. moniliforme, as measured by ergosterol concentration, was higher at 25 degrees C than at 20 degrees C, reaching a stationary phase after 4 to 6 weeks in both cases. FB1 production commenced after 2 weeks during the active growth phase, continued to increase during the stationary phase, and decreased after 13 weeks. The overall maximal yield of FB1 (17.9 g/kg, dry weight) was obtained in corn cultures incubated at 20 degrees C for 13 weeks, but it was not significantly (P greater than 0.05) higher than the maximum yield (16.5 g/kg, dry weight) obtained at 25 degrees C after 11 weeks. However, a significantly (P less than 0.05) higher mean yield was detected at 25 degrees C (9.5 g/kg, dry weight) than at 20 degrees C (8.7 g/kg, dry weight). Production reached a plateau after 7 weeks of incubation at 25 degrees C or 9 weeks of incubation at 20 degrees C. The maximal production of FB1 at 30 degrees C was very low (0.6 g/kg, dry weight). FB1 was also found to be heat stable, as there was no reduction in the FB1 concentration after boiling culture material of F. moniliforme MRC 826.