Effect of pH on Inorganic Carbon Uptake in Algal Cultures

Biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably affected by the pH at which the cultures were maintained. Carbon photoassimilation experiments revealed that pH values in the range of 8 to 9 were important for determining the free CO₂ concentrations in the medium. With higher pH values, additional pH effects were observed involving a decrease in the relative high affinity of low CO₂-adapted algae to free CO₂. The carbon uptake rate by high CO₂-adapted algae after transfer to low free CO₂ medium was characterized by a lag period of about 30 min, after which the affinity of the algae to CO₂ increased considerably. Both continuous growth and carbon uptake experiments indicated that artificially maintained high free CO₂ concentrations are recommended for maximal production in intensive outdoor algal cultures.     

An algD-bioluminescent reporter plasmid to monitor alginate production in biofilms

A broad-host range algD-lux bioluminescent reporter plasmid was developed to examine the role of exopolymer production in biofilm function. The algD-lux reporter plasmid will allow rapid on-line in situ detection of environmental factors that induce alginate biosynthesis. The algD promoter was stimulated by factors previously reported to induce alginate production, including ethanol and NaCl, and differences were observed with different nitrogen sources. With growth on minimal media with either glucose or succinate as a carbon source, succinate had a greater inductive effect on the algD promoter. An increase in light output of 1.3-fold and 1.7-fold was seen with cultures amended with 50 and 150 mM NaCI, respectively, compared to cultures with succinate alone. NaCl induction of the algD promoter was confirmed by algD RNA slot blots. Light output increased 2.0-fold and 1.7-fold with 0.25% and 0.5% ethanol, respectively, compared with controls grown with succinate only. While the rate of algD promoter response was initially similar when either NH₄ or NO₃ was used as a nitrogen source, NH₄-grown cultures maintained a higher light output during late log phase compared to NO₃-grown cultures. Correspondence to: G.S. Sayler.     

Acidic Conditions Are Not Obligatory for Onset of Butanol Formation by Clostridium beijerinckii (Synonym, C. butylicum)

Factors that may initiate the metabolic transition for butanol production were investigated in batch cultures of Clostridium beijerinckii (synonym, Clostridium butylicum) VPI 13436. Cultures maintained at pH 6.8 produced nearly as much butanol as those incubated without pH control, indicating that neither a change in the culture pH nor acid conditions per se are always required to initiate solvent formation. Acetate and butyrate levels at the onset of butanol production were dependent on the pH at which the cultures were maintained. Cultures maintained at pH 6.8 could be accelerated into solvent production by artificially lowering the pH to 5.0 or by the addition of acetate plus butyrate without a pH change (but neither acid alone was effective). Solvent production was associated with slower rates of growth and general metabolism, and it did not show a requirement for mature spore formation. We speculate that a slowdown in metabolism, which may be brought about by several conditions, is mechanistically related to the onset of butanol production. Extracts of solvent-producing cells contained acetoacetate decarboxylase activity as well as higher NADP⁺-linked butanol dehydrogenase and lower hydrogenase activities than extracts of acid-producing cells. Solvent production did not appear to involve an enhanced ability to catalyze H₂ oxidation.     

Effect of anoxic conditions on wood-decay fungi treated with argon or nitrogen

The effects of low-oxygen conditions, achieved with either argon or nitrogen gas, on the viability of wood-decay fungi Coniophora puteana and Antrodia vaillantii, grown on artificial growth medium, were tested. Initial tests for viability were run after 1, 2, 3 and 4 weeks of exposure to low oxygen conditions, at oxygen levels in the vessels maintained below 10 ppm. Treatment was later extended to 10 and 16 weeks. Tests included measurements of CO₂ production and determination of the ability of fungal tissue to regenerate. The effect of anoxic conditions on the mycelia of treated fungal species was expressed as an increased time needed for regeneration or as a complete absence of growth of inocula taken from the exposed cultures. The cultures that were retarded by the low-oxygen environment consequently produced less CO₂ per hour. For C. puteana cultures, the effects of anoxic treatment became apparent in the second week of the treatment. The number of affected cultures rose steadily with the prolongation of anoxic treatment. By the 16th week of the experiment, 80% of the inocula of C. puteana did not regenerate. A. vaillantii inocula regeneration was not affected until after the fourth week of treatment. The influence of anoxic treatment on the cultures of this species was more pronounced in the test on the 10th and especially on the 16th week, when 67% of inocula did not regenerate. Argon or nitrogen gas caused the same degree of loss of viability in both the fungal species tested. In general, A. vaillantii mycelial cultures proved to be less sensitive to anoxic conditions, caused by either argon and nitrogen gas.     

Glutamine Involvement in Nitrogen Control of Gibberellic Acid Production in Gibberella fujikuroi

When the fungus Gibberella fujikuroi ATCC 12616 was grown in fermentor cultures, both intracellular kaurene biosynthetic activities and extracellular GA₃ accumulation reached high levels when exogenous nitrogen was depleted in the culture. Similar patterns were exhibited by several nonrelated enzymatic activities, such as formamidase and urease, suggesting that all are subject to nitrogen regulation. The behavior of the enzymes involved in nitrogen assimilation (glutamine synthetase, glutamate dehydrogenase, and glutamate synthase) during fungal growth in different nitrogen sources suggests that glutamine is the final product of nitrogen assimilation in G. fujikuroi. When ammonium or glutamine was added to hormone-producing cultures, extracellular GA₃ did not accumulate. However, when the conversion of ammonium into glutamine was inhibited by L-methionine-DL-sulfoximine, only glutamine maintained this effect. These results suggest that glutamine may well be the metabolite effector in nitrogen repression of GA₃ synthesis, as well as in other nonrelated enzymatic activities in G. fujikuroi.     

Physiological aspects of surface-immobilized Catharanthus roseus cells

Summary Growth and alkaloid production of surface-immobilized C. roseus cells were studied in a 2-1 bioreactor. Media designed to maximize cell growth or alkaloid production were employed. Nitrate and carbohydrate consumption rates as well as growth rates and biomass yields of immobilized cultures were equal or somewhat lower than for cell suspension cultures. Respiration rate (O₂ consumption and CO₂ production rates) of immobilized C. roseus cell cultures was obtained by on-line analysis of inlet and outlet gas composition using a mass spectrometer. Respiration rate increased during the growth phase and decreased once the nitrogen or the carbon source was depleted from the medium. The respiration rate of immobilized C. roseus cells resembled rates reported in the literature for suspension cultures. Offprint requests to: Denis Rho     

Photosynthesis and nitrogen utilization in exponentially growing nitrogen-limited cultures of Lemna gibba

The photosynthetic performance and nitrogen utilization of Lemna gibba L. G3 adapted to limited nitrogen supply was studied. The plants were adapted to two levels of nitrogen limitation where the nitrogen addition rates were calculated to sustain relative growth rates (RGR) of 0.15 day^?1 and 0.25 day^?1, respectively. The photosynthetic performance of these cultures was compared to nitrogen-sufficient cultures with an average RGR of 0.32 day^?1. Plants transferred from nitrogen-sufficient conditions attained RGR values corresponding to the nitrogen addition rates after 6 to 10 days. Light-saturated net photosynthesis declined during adaptation according to the drop in growth rate, and a concomitant decrease in the respiration rate was recorded. The efficiency of net photosynthesis on a dry weight basis increased with increased nitrogen supply, whereas it was the same in all cultures when expressed on a chlorophyll basis. The light compensation point was unaffected by the nitrogen regime. Limited nitrogen supply resulted in an increased proportion of dry matter in the roots, which led to decreased leaf area ratios. The net assimilation rates also decreased, but not to the same extent as the leaf area ratios. Growth-limiting amounts of nitrogen were added to the cultures once daily, and the net influx of N was higher than the requirement for N, also in adapted cultures with a steady growth rate. This resulted in transient, periodic fluctuations in the NO₃^?, NH₄⁺ and amino acid pools. Also the rates of NO₃^? reduction and NH₄⁺ assimilation fluctuated as did the amino acid assimilation which paralleled NH₄⁺ assimilation. The role of flux rates over the plasmalemma and tonoplast for control of nitrogen assimilation rates are discussed.     

OBSERVATIONS ON CELL DEVELOPMENT IN CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) AT LOW AND HIGH CARBON DIOXIDE TENSION1

Some cell cycle events were compared in Chlamydomonas segnis Ettl during its development in synchronous cultures (12:12 LD) supplied with air and air enriched with 5% CO₂. In cultures bubbled with air, growth resulted in production of 2 relatively small zoospores. In cultures provided with 5% CO₂, 4 large zoospores were formed but not released in darkness unless the cultures were bubbled with CO₂-free air and/or exposed to light. Respiration in zoospores was inhibited by high CO₂ tension. In cultures maintained under continuous illumination for one cell cycle, provision of 5% CO₂ led to enhanced growth, a relatively long S-phase and a 4 h delay of the second cell division. In such cultures, the DNA content of parental cells (12 h L) was insufficient to support two cell divisions. The RNA/DNA ratio of the resulting zoospores was 10 compared to 4 in air cultures. These results provided evidence that the delay of the second cell division was a consequence of the delay in DNA production.     

Enhancement of poly-3-hydroxybutyrate (PHB) productivity by the two-stage supplementation of carbon sources and continuous feeding of NH4Cl

Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB accumulation. Via the feeding of 0.03 mmol/h of NH₄Cl solution prior to the exhaustion of the initial amount of NH₄Cl, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture evidenced a higher level of NADPH during the NH₄Cl-exausted PHB accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. NH₄Cl feeding following the depletion of initial NH₄Cl resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

Production of citric acid with immobilized Aspergillus niger

Summary The spores of Aspergillus niger were entrapped in calcium-alginate beads and precultivated in growth media with various amounts of nitrogen. During the following citric acid production in shaking cultures an optimum of acid formation and yield was observed after the precultivation with 100–200 mg/l NH₄NO₃. The productivity of the immobilized Aspergillus was found to be 1.5 times higher than in the case of free pellets. The outgrowth of free mycelia into the medium could be provided by increasing the ratio particle-volume: medium volume, using a 1-l air-lift fermenter, by which means the productivity was increased twice as much as obtained in shaking culture.     

Growth and toxin production of tropical <Emphasis Type="Italic">Alexandrium minutum</Emphasis> Halim (Dinophyceae) under various nitrogen to phosphorus ratios

Effects of nitrogen to phosphorous (N/P) ratios of two nitrogen sources (nitrate and ammonium) on growth and toxin production of a tropical estuarine dinoflagellate, Alexandrium minutum Halim, were examined using a strain isolated from a bloom at Tumpat Estuary, Malaysia in September 2001. Experiments were carried out in batch cultures, using either nitrate (N-NO₃) or ammonium (N-NH₄) as the nitrogen source at a constant amount, and with initial N/P ratios ranging from 5 to 500. Cell density, residual N and P in the medium, cellular toxin quota (Q _t), and toxin composition were analyzed throughout the growths. Our results showed that cell densities and growth rates of A. minutum were severely suppressed under high N/P ratios (>100) in both N-NO₃ and N-NH₄ treatments. Cells tended to be larger at lower growth rate and P-limited cultures. Toxin profile was relatively constant throughout the experiments, with GTX4/GTX1 as the dominant toxin congeners. Cellular toxin quota (Q _t) increased with elevated N/P ratios in both N-NO₃ and N-NH₄ treatments. Toxin production rate, R _tox, however was enhanced in N-NH₄-grown cultures when P was limited, but showed no difference between N-NO₃- and N-NH₄-grown cultures when P was replete. Our results clearly showed that N/P ratios as well as the nitrogen compounds not only affected the growth of A. minutum, but also the cellular toxin quota and its toxin production rate.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Effect of specific amino acids on growth and aflatoxin production by Aspergillus parasiticus and Aspergillus flavus in defined media.

Four amino acids were used as sole nitrogen sources or as supplements to ammonium sulfate, and casein and ammonium sulfate were used as sole nitrogen sources to examine their effects on aflatoxin production by Aspergillus parasiticus NRRL 2999 and Aspergillus flavus 3357 grown on synthetic liquid media. In general, when proline, asparagine, casein, and ammonium sulfate were used as sole nitrogen sources, they supported more growth and toxin production than tryptophan or methionine. However, proline stimulated more toxin production per gram of mycelium in stationary cultures than the other nitrogen sources, including the amino acid asparagine, which is generally recognized as supporting good aflatoxin production. The exact responses to individual nitrogen sources were influenced by the species of fungus and whether cultures were stationary or shaken. In shake cultures, but not in stationary cultures, increased growth was generally associated with increased toxin production.     

Nitrogen-Gas Bubbling during the Cultivation of Clostridium tetani Produces a Higher Yield of Tetanus Toxin for the Preparation of Its Toxoid

We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N₂) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid. N₂ was bubbled through nine 10-liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling. We found that treatment of the C. tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.     

Selection of Protease-Positive and Protease-Negative Variants of Streptococcus cremoris

Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E₈, HP, and Wg₂ at pH values higher than 6.0 in milk. For S. cremoris E₈ this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not found in all media tested. The poor growth of the protease-negative variant at low pH was not due to lower internal pH values. By growing S. cremoris E₈ and Wg₂ in acidified milk (pH 5.9) the proteolytic activity of the cultures could be stabilized. In continuous cultures under amino acid limitation the wild type S. cremoris E₈ and HP strains had a selective advantage over the protease-negative variants at low dilution rates (D < 0.2) at all pH values of the medium. This was apparently due to a lower affinity-constant (K_s) of the protease-positive variants for amino acids. Finally, a high fraction of protease-positive variants could be maintained in continuous cultures by using a growth medium with low concentrations of casein as a nitrogen source. At high dilution rates nearly all cells were protease positive.     

Optimization of lipid production in the oleaginous yeastApiotrichum curvatum in wheypermeate

Summary Lipid production of the oleaginous yeastApiotrichum curvatum was studied in wheypermeate to determine optimum operation conditions in this medium. Studies on the influence of the carbon to nitrogen ratio (C/N-ratio) of the growth medium on lipid production in continuous cultures demonstrated that cellular lipid content in wheypermeate remained constant at 22% of the cell dry weight up to a C/N-ratio of about 25. The maximal dilution rate at which all lactose is consumed in wheypermeate with excess nitrogen was found to be 0.073 h^-1. At C/N-ratios higher than 25–30 lipid content gradually increased to nearly 50% at C/N=70 and the maximal obtainable dilution rate decreased to 0.02 h^-1 at C/N=70. From these studies it could be derived that maximal lipid production rates can be obtained at C/N-ratios of 30–35 in wheypermeate. Since the C/N-ratio of wheypermeate normally has a value between 70 and 101, some additional nitrogen is required to optimize the lipid production rate. Lipid production rates ofA. curvatum in wheypermeate were compared in four different culture modes: batch, fed-batch, continuous and partial recycling cultures. Highest lipid production rates were achieved in culture modes with high cell densities. A lipid production rate of nearly 1 g/l/h was reached in a partial recycling culture. It was calculated that by using this cultivation technique lipid production rates of even 2.9 g/l/h may be reached when the supply of oxygen can be optimized.Nomenclature C/N-ratio carbon to nitrogen ratio of the growth medium (g/g) - C/N_crit C/N-ratio at which there is just enough nitrogen to allow all carbon source to be converted to biomass - D dilution rate=volume of incoming medium per unit time/volume of medium in the culture vessel (h^-1) - D_max maximum dilution rate (h^-1) - DW cell dry weight - L lipid yield (g storage lipid/g carbon source) - specific growth rate (h^-1) - _max maximum specific growth rate (h^-1) - Q_L lipid production rate (g/l/h) - Y_i molecular fraction of carbon substrate that is converted to storage carbohydrate (C-mol/C-mol) - Y_ls maximal amount of storage lipid that can be produced per mol carbon source (C-mol/C-mol)     

Effect of plant growth regulators and medium composition on cell growth and saponin production during cell-suspension culture of mountain ginseng (Panax ginseng C. A. mayer)

We have established cell-suspension cultures of mountain ginseng (Panax ginseng G A. Mayer), and have attempted to increase the yield of saponin by manipulating our processing method and culturing factors (e.g., media strengths; the presence of plant growth regulators or sucrose; ratios of NO⁺ ₃/ NH^- ₄). Maximum biomass yield was obtained in media containing 2,4-D. However, saponin productivity was much higher in a medium comprising either IBA or NAA; 7.0 mg/L IBA was optimal for promoting both cell growth (10.0 g/L dry weight) and saponin production (7.29 mg/g DW total ginsenoside). Although the addition of cytokinins (BA and kinetin) did not affect cell growth, the level of saponin (particularly in the Rb group) was enhanced when the media were supplemented with either 0.5 mg/L BA or 0.5 mg/L kinetin. Half- and full-strength MS media were equally suitable for inducing both biomass as well as saponin production. We also investigated the effect of various concentrations of sucrose and nitrogen, and found that 30 g/L sucrose enhanced biomass yield as well as saponin content However, further increases (i.e., up to 70 g/L) led to a decrease in saponin accumulation and biomass production. Maximum growth and saponin productivity were reported from treatments with an initial nitrogen concentration of 30 mM. In general, the amount of saponin increased when the test media had high NO⁺ ₃/ NH^- ₄ ratios; in fact, saponin production was greatest when nitrate was the sole nitrogen source.     

In vitro thiophene production by transformed root cultures of Tagetes laxa (Cabrera)

The effect of different concentrations of carbohydrates, nitrogen, sulphate, plant growth regulators and elicitors on growth and thiophene accumulation by transformed root cultures of Tagetes laxa (Cabrera) was studied. The combinations of sucrose (30 g/l), nitrogen (60 mM), sulphate (150 mM) and the ratio N_ox:N_red 2:1 are the most appropriate combination to support growth and thiophene accumulation, which was increased by 90% when the cultures were elicited with homogenate of Sclerotinia sclerottiorum. The plant growth regulators used produced dedifferentiation with a decrease in thiophene biosynthesis.