Heterotrophic production of biomass and lutein by Chlorella protothecoides on various nitrogen sources

The effects of nitrate, ammonium, and urea as nitrogen sources on the heterotrophic growth of Chlorella protothecoides were investigated using flask cultures. No appreciable inhibitory effect on the algal growth was observed over a nitrogen concentration range of 0.85-1.7 g l(-)(1). In contrast, differences in specific growth rate and biomass production were found among the cultures with the various nitrogen compounds. The influence of different nitrogen sources at a concentration equivalent to 1.7 g l(-)(1) nitrogen on the heterotrophic production of biomass and lutein by C. protothecoides was investigated using the culture medium containing 40 g l(-)(1) glucose as the sole carbon and energy source in fermentors. The maximum biomass concentrations in the three cultures with nitrate, ammonium, and urea were 18.4, 18.9, and 19.6 g l(-)(1) dry cells, respectively. The maximum lutein yields in these cultures were between 68.42 and 83.81 mg l(-)(1). The highest yields of both biomass and lutein were achieved in the culture with urea. It was therefore concluded that urea was the best nitrogen source for the production of biomass and lutein. Based on the experimental results, a group of kinetic models describing cell growth, lutein production, and glucose and nitrogen consumption were proposed and a satisfactory fit was found between the experimental results and predicted values. Dynamic analysis of models demonstrated that enhancing initial nitrogen concentration in fermentor cultures, which correspondingly enhances cell growth and lutein formation, may shorten the fermentation cycle by 25-46%.     

Enhancement of cephamycin C production using soybean oil as the sole carbon source

Vegetable oils were investigated to evaluate their potential to act as the sole carbon source for production of cephamycin C in shake and jar-fermentor cultures. Soybean oil was the best carbon source for cephamycin C production. Bioautography and HPLC analyses showed that cephamycin C was exclusively produced even when soybean oil was used as the sole cabon source. The optimal pH and initial concentration of soybean oil was 7.5 and 7 g/l, respectively. Both pH and the pH-control agent affected cephamycin C production, and among phosphoric acid, acetic acid and sulfuric acid, phosphoric acid was associated with the best production. Soybean oil was slowly consumed after the soluble nitrogen source was consumed. When the initial soybean oil concentration was 7 g/l, cephamycin C production was maximal, 2.0 g/l, which was twice as high as that from starch. The product yield from soybean oil was 4.7 times higher than that from starch. These results show that vegetable oils, which are cheaper than other carbon sources, could be used as the sole carbon source in the production of antibiotics. Correspondence to: M. Okabe     

Bioreactor culture of oil palm (Elaeis guineensis) and effects of nitrogen source, inoculum size, and conditioned medium on biomass production

We report the successful culture of oil palm (Elaeis guineensis Jacq.) suspension cells in a bioreactor. In vitro propagation of this perennial monocotyledonous tree is an important part of the oil palm industry's approach to clonal propagation of high-yielding accessions. During culture of oil palm cells in a batch bioreactor, nutrients and extracellular metabolites were monitored, and kinetic parameters and nutrient-to-biomass conversion yields were calculated. The biomass increased approximately 3.5-fold per month, consistent with values reported for shake flask cultures. Although the carbon source was completely depleted by the end of the run, nitrogen sources remained in large excess and the sugar-to-biomass conversion yield remained low. Linear growth indicated that the cells were limited. The results obtained from the bioreactor runs indicated that we should be able to improve biomass production by carrying out optimization studies. Therefore, we initiated multi-factorial analyses using response surface experimental designs to investigate the effects of different nitrogen sources, as well as inoculum size and conditioned medium, on biomass production in flask cultures. Whereas glutamine does not have a significant effect on biomass production, ammonia has a positive effect up to an optimum concentration. Both inoculum density and conditioned medium have positive, synergistic effects on biomass production.     

Catabolite repression in Streptomyces venezuelae. Induction of beta-galactosidase, chloramphenicol production, and intracellular cyclic adenosine 3',5'-monophosphate concentrations

Chloramphenicol production was studied in cultures of Streptomyces venezuelae growing in a simple buffered medium with ammonia as the nitrogen source and glucose, lactose, or a glucose-lactose mixture as the sole source of carbon. With each carbon source the antibiotic was formed during growth. In the glucose-lactose medium, the production pattern was biphasic; a marked decrease in the rate of synthesis was associated with depletion of glucose from the medium and a corresponding diauxie pause in growth. Cells of S. venezuelae contained an inducible beta-galactosidase. Induction by lactose was suppressed by glucose. Measurement of the concentration of intracellular adenosine 3',5'-cyclic monophosphate during growth of cultures with glucose or a glucose-lactose mixture as the source of carbon showed no appreciable changes coinciding with depletion of glucose or the onset of chloramphenicol biosynthesis. It is concluded that the cyclic nucleotide does not mediate selective nutrient utilization or control antibiotic biosynthesis in this organism.     

Role of the carbon source in regulating chloramphenicol production by Streptomyces venezuelae: studies in batch and continuous cultures

Both carbon- and nitrogen-limited media that supported a biphasic pattern of growth and chloramphenicol biosynthesis were devised for batch cultures of Streptomyces venezuelae. Where onset of the idiophase was associated with nitrogen depletion, a sharp peak of arylamine synthetase activity coincided with the onset of antibiotic production. The specific activity of the enzyme was highest when the carbon source in the medium was also near depletion at the trophophase-idiophase boundary. In media providing a substantial excess of carbon source through the idiophase, the peak specific activity was reduced by 75%, although the timing of enzyme synthesis was unaltered. Moreover, chemostat cultures in which the growth rate was limited by the glucose concentration in the input medium failed to show a decrease in specific production of chloramphenicol as the steady-state intracellular glucose concentration was increased. The results suggest that a form of "carbon catabolite repression" regulates synthesis of chloramphenicol biosynthetic enzymes during a trophophase-idiophase transition induced by nitrogen starvation. However, this regulatory mechanism does not establish the timing of antibiotic biosynthesis and does not function during nitrogen-sufficient growth in the presence of excess glucose.     

A possible mechanism of metabolic regulation in Gibberella fujikuroi using a mixed carbon source of glucose and corn oil inferred from analysis of the kinetics data obtained in a stirrer tank bioreactor

A nonstructured model was used to study the dynamics of gibberellic acid production in a stirred tank bioreactor. Experimental data were obtained from submerged batch cultures of Gibberella fujikuroi (CDBB H‐984) grown in varying ratios of glucose‐corn oil as the carbon source. The nitrogen depletion effect was included in mathematical model by considering the specific kinetic constants as a linear function of the normalized nitrogen consumption rate. The kinetics of biomass growth and consumption of phosphate and nitrogen were based on the logistic model. The traditional first‐order kinetic model was used to describe the specific consumption of glucose and corn oil. The nitrogen effect was solely included in the phosphate and corn oil consumption and biomass growth. The model fit was satisfactory, revealing the dependence of the kinetics with respect to the nitrogen assimilation rate. Through simulations, it was possible to make diagrams of specific growth rate and specific rate of substrate consumptions, which was a powerful tool for understanding the metabolic interactions that occurred during the various stages of fermentation process. This kinetic analysis provided the proposal of a possible mechanism of regulation on growth, substrate consumptions, and production of gibberellic acid (GA₃) in G. fujikuroi. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1169–1180, 2013     

Causes of conductance change in yeast cultures.

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rubra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds. Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Causes of conductance change in yeast cultures

The conductance change due to growth of Saccharomyces cerevisiae Y112, Zygosaccharomyces bailii M and Rhodotorula rabra NCYC 63 in culture media containing glucose, tartrate pH buffer and ammonium ions as sole nitrogen source was compared with that in a medium containing L-asparagine as sole nitrogen source. Decreases in conductance were observed in glucose-ammonium cultures of all three yeasts while little change occurred in cultures with L-asparagine as sole nitrogen source. This supports the hypothesis that the metabolic activity primarily responsible for conductance change in yeast cultures is the uptake of charged ammonium ions as nitrogen source and the reaction of protons with pH buffer compounds.
Rhodotorula rubra cultures with L-asparagine as sole carbon source caused large increases in conductance with growth. Chemical analyses of culture filtrates showed that this increase in conductance was due to use of L-asparagine as carbon source and the excretion of nitrogen surplus to biosynthetic needs as ammonium. In addition, the production of aspartate, acetate and bicarbonate contributed to the increase in conductance.     

Some observations on protease production in continuous suspension cultures of Bacillus firmus

Invariance of culture conditions in steady state continuous cultures make these a very valuable tool to study the influence of various culture parameters on cell growth and synthesis of primary and secondary metabolites. The result of a parametric study on production of protease in continuous suspension cultures of Bacillus firmus NRS 783 are reported in this article. This strain is a superior producer of an alkaline protease with major application in the detergent industry. The parameters investigated include dilution rate and concentrations of yeast extract, ammonium, and inorganic phosphate in the bioreactor feed, glucose being the principal carbon source in all experiments. The regulatory effects of the key culture parameters on cell growth, synthesis and secretion of protease, and production of acetic acid are investigated. The relations among the specific cell growth rate, specific utilization rates of the principal carbon, nitrogen, and phosphorous sources, and specific production rates of two nonbiomass products, viz., acetic acid and protease, are examined, and the effects of the manipulated culture parameters on these relations, specific protease activity, and yields of cell mass, protease, and acetic acid on the basis of the principal carbon, nitrogen, and phosphorous sources are studied. An increase in dilution rate led to increases in specific utilization rates of the principal carbon, nitrogen, and phosphorous sources and specific production rates of acetic acid and protease and decreases in bulk activities/concentrations of the three products (acetic acid, cell mass, and protease). As a result, the productivities of the three species were maximized at an intermediate dilution rate. Increased supply of yeast extract (a rich source of amino acids, proteins, and vitamins, besides being an additional source of carbon, nitrogen, and phosphorus) promoted cell mass formation but reduced protease production per unit cell mass. Increased supply of nitrogen and phosphorous sources stimulated protease synthesis up to certain threshold levels and repressed the enzyme synthesis beyond the threshold levels. With increased supply of the nitrogen source, the phosphorous source was more efficiently utilized for cell growth and protease synthesis. Stable maintenance of continuous cultures of B. firmus over prolonged period is demonstrated in this study. (c) 1993 John Wiley & Sons, Inc.     

Application of factorial design to the optimization of medium composition in batch cultures of Streptomyces lividans TK21 producing a hybrid antibiotic

Summary The composition of the liquid medium employed to obtain a hybrid antibiotic in batch cultures of a recombinant strain of Streptomyces lividans TK21 has been studied. Starch and glutamic acid are the most appropriate carbon and nitrogen sources to support respectively cell growth and antibiotic production. A central composite experimental design has been employed to derive a statistical model of the effect of phosphate and glutamic acid on growth and antibiotic production, and an initial concentration of 10 mM phosphate and 52.8 mM glutamic acid have been found optimal to maximize the final antibiotic concentration in batch cultures.     

碳源、氮源对异养单针藻Monoraphidium sp. FXY-10油脂积累和脂肪酸组成的影响

研究了碳源与氮源对单针藻Monoraphidium sp. FXY-10异养培养的影响。以BG-11为基础培养基,通过添加不同类型、浓度梯度碳源和氮源,比较分析微藻生物量、油脂积累以及脂肪酸组成。结果表明,以葡萄糖作碳源,硝酸钠为氮源,微藻细胞积累的油脂是理想的生物柴油制备原料。硝酸钠浓度分别为1.00、3.00和5.00 g/L时,对油脂产量影响不显著(P>0.05)。葡萄糖浓度为10.00 g/L,硝酸钠为氮源油脂产量达到实验最高值0.84 g/L,其油脂脂肪酸组成主要由C16:0和C18:1等短链饱和脂肪酸和单不饱和脂肪酸组成,不饱和度值(DU)为61.98,相对偏低。     

Enniatin production by Fusarium sambucinum: primary, secondary, and unitary metabolism.

Fusarium sambucinum liquid surface cultures on semi-defined medium with glucose as carbon source passed through well-defined phases corresponding to trophophase, idiophase (enniatin production), and a degenerative phase. All the glucose was consumed by day 6, at which time the mycelial dry weight had reached only half its maximum. When glucose was replaced by lactose, there was no separation of trophophase and idiophase. Enniatin production, dry weight, and sugar and nitrogen consumption were in approximate balance throughout the growth period (25 days), after which slow degeneration began. The term 'unitary metabolism' is proposed for this type of unphased behaviour. Unitary metabolism may approximate more closely to that occurring under natural conditions than does the metabolic phase separation observed when rapidly utilized carbon sources are used in laboratory cultures.     

Isolation and Characterization of Biosurfactant-and Bioemulsifier-Producing Bacteria from Petroleum Contaminated Sites in Western Canada

Biosurfactant-producing bacteria were isolated from two petroleum contaminated sites in western Canada. Seven potential biosurfactant/bioemulsifier-producing isolates were screened and characterized. All of the seven isolates were able to form emulsions. Emulsion-stabilizing capacity was also measured up to 48 hrs. Strain C-111-2 and C-203-2 would lead to highly reduced surface tension. For strain C-203-2, the optimum conditions that supported bacteria growth and production were investigated. The influences of carbon sources, medium pH values, and temperature were taken into account. The experimental results indicated that the crude oil and glucose were promising carbon sources for biosurfactants production; the isolated strains produced a maximum concentration of biosurfactant in a neutral pH environment and showed a higher surface activity under the temperature level of 35°C than that under 10°C. To further optimize the carbon and nitrogen source for biosurfactant production, response surface methodology (RSM) was applied to explore the favorable concentration of two carbon sources: glucose, crude oil, and one nitrogen source, NaNO₃. The optimal concentration of 8.1g/L, 4% and 3.9 g/L for glucose, crude oil, and NaNO₃, respectively, which can be obtained through RSM analysis.     

Growth and sporulation of Entomophthora virulenta on semidefined media in liquid culture

Entomophthora virulenta was tested for growth and sporulation under various nutritional conditions in shake cultures containing combinations of dextrose and protein hydrolysates. Darkness, a temperature of 25°C, and a pH near 6.5 induced the formation of the greatest number of zygospores when the carbon and nitrogen sources were held constant. The influence of the total concentration of nutrients and of the ratio of the carbon and nitrogen sources depended on the nitrogen source used: The highest amount of spores under these conditions was produced with yeast extract as nitrogen source, a carbon/nitrogen source ratio of 2, and a 15% total nutrient concentration. The influence of the preinoculum and inoculum on sporulation is discussed.     

Effect of nitrogen source on methanol oxidation and genetic diversity of methylotrophic mixed cultures enriched from pulp and paper mill biofilms

Methanol-oxidizing bacteria may play an important role in the development and use of biological treatment systems for the removal of methanol from industrial effluents. Optimization of methanol degradation potential in such systems is contingent on availability of nutrients, such as nitrogen, in the most favorable form and concentration. To that end, this study examined the variation in growth, methanol degradation, and bacterial diversity of two mixed methylotrophic cultures that were provided nitrogen either as ammonium or nitrate and in three different concentrations. Methanol-degrading cultures were enriched from biofilms sampled at a pulp and paper mill and grown in liquid batch culture with methanol as the only carbon source and either ammonium or nitrate as the only added nitrogen source. Results indicate that growth and methanol removal of the mixed cultures increase directly with increased nitrogen, added in either form. However, methanol removal and bacterial diversity, as observed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) methods, were higher when using nitrate as the nitrogen source for enrichment and growth, rather than ammonium. Based on results described here, nitrate may potentially be a better nitrogen source when enriching or working with mixed methylotrophic cultures, and possibly more effective when used as a nutrient addition to biofilters.     

主要营养成分对悬浮培养玫瑰茄细胞生长和花青素合成的影响

本文研究了培养基中碳源和氮源变化对悬浮培养玫瑰茄细胞生长和花青素合成的影响。在８种不同的碳源中,麦芽糖有利于花青素的积累,而蔗糖和葡萄糖适合细胞生长,并有较高的花青素产率。在１％～１０％蔗糖浓度范围内,４％浓度下细胞生长和花青素产率最高,而６％浓度下细胞花青素含量最高,高渗环境较有利于细胞花青素的积累。１３５ｍＭ的氮源总量已足够维持玫瑰茄细胞生长和花青素合成,氮源总量增加对细胞代谢有抑制作用。ＮＨ＋４对细胞有显著抑制作用。总量１３５ｍＭ,ＮＯ－３与ＮＨ＋４比例２５∶２和２３∶４时细胞生长和花青素合成最佳。     

Bacteriocin production with Lactobacillus amylovorus DCE 471 is improved and stabilized by fed-batch fermentation

Amylovorin L471 is a small, heat-stable, and hydrophobic bacteriocin produced by Lactobacillus amylovorus DCE 471. The nutritional requirements for amylovorin L471 production were studied with fed-batch fermentations. A twofold increase in bacteriocin titer was obtained when substrate addition was controlled by the acidification rate of the culture, compared with the titers reached with constant substrate addition or pH-controlled batch cultures carried out under the same conditions. An interesting feature of fed-batch cultures observed under certain culture conditions (constant feed rate) is the apparent stabilization of bacteriocin activity after obtaining maximum production. Finally, a mathematical model was set up to simulate cell growth, glucose and complex nitrogen source consumption, and lactic acid and bacteriocin production kinetics. The model showed that bacterial growth was dependent on both the energy and the complex nitrogen source. Bacteriocin production was growth associated, with a simultaneous bacteriocin adsorption on the producer cells dependent on the lactic acid accumulated and hence the viability of the cells. Both bacteriocin production and adsorption were inhibited by high concentrations of the complex nitrogen source.     

被孢霉生物合成花生四烯酸的初步研究

花生四烯酸不仅是细胞膜的重要成分,在维持细胞膜的结构与功能上发挥重要作用,而且是人体前列腺素合成的前体物质,对人体生理功能具有重要的调节作用。近年来研究发现,花生四烯酸在保护皮肤、降低胆固醇、抑制血小板聚集、提高免疫能力、促进胎儿发育等方面具有独特的...     

AMMONIA PRODUCTION IN UREA-GROWN CULTURES OF CHLORELLA ELLIPSOIDEA12

Production of ammonia by urea-grown Chlorella ellipsoidea was investigated. Ammonia was produced during the stationary growth phase in cultures with urea as sole nitrogen source and glucose as supplementary carbon source. Ammonia was produced only in medium containing excess urea and limiting amounts of glucose. Ammonia production was accompanied by increase in pH. In cultures with nitrate as sole nitrogen source and glucose as supplementary carbon source, growth and pH changes were similar to those in urea-glucose medium, but no ammonia was detected. Cultures grown in urea-acetate medium were similar to those grown in urea medium without additional organic carbon source. No ammonia was produced under these circumstances and growth was significantly lower than that achieved in glucose-supplemented cultures. C. ellipsoidea evidently produces an enzyme or enzyme system which forms ammonia from urea. This organism was reportedly urease-free because previous workers did not detect ammonia formation from urea. Our findings indicate that special circumstances are required to produce detectable amounts of ammonia from urea. These findings are in agreement with a recent report of urea-splitting, cofactor-requiring enzyme in cell-free extracts of Chlorella.     

Storage ofCandida albicans,C. tropicalis and related species in liquid nitrogen

Storage in liquid nitrogen of a collection ofC. albicans, C. tropicalis and related species checked by numerical and classical taxonomy is described. Strains stored for 3 years in liquid nitrogen were thawed and their survival was tested. After adaptation and regeneration, their fermentation and assimilation spectra, production of chlamydospores and pseudomycelia, appearance and radial growth rate of giant colonies were investigated and compared with the properties of cultures stored under paraffin oil. It follows from the results obtained that two different media —with an increased content of a nitrogen source and with an increased carbon source content —should be used for the post-heating adaptation and regeneration of yeast cells. In some strains it is useful to store them at 4 °C for additional time intervals in order to increase survival of the cells. The above strains can be successfully stored in liquid nitrogen.