Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Phenotypic changes in the chemistry of Aspergillus nidulans: Influence of culture conditions on mycelial composition

A quantitative study was made of macromolecular (nucleic acids, protein), carbohydrate and mineral (magnesium, potassium and phosphorus) components of Aspergillus nidulans in glucose limited chemostat cultures, under varying conditions of dilution rate, temperature, pH and NaCl concentration.The overall mineral content showed greatest variation in response to changes in culture salinity, which also affected the mycelial carbohydrate content. Concomitant and opposite changes in the conent of cations and carbohydrates under conditions of increasing salinity may be interpreted in terms of mycelial osmoregulation. Slight variations in DNA content but gross fluctuations in the level of RNA were noted under the different cultural conditions examined. Co-ordinate changes in RNA and Mg²⁺ contents were evident only under certain conditions: dilution rate from 0.05–0.07 h^-1 or temperature from 22–30° C. The constant molar stoichiometry between RNA and Mg²⁺ characteristic of unicellular microorganisms was not a feature of fungal growth. The protein content was most affected by shifts of temperature and reached minimal values at 25 and 50° C.The growth environment had a marked influence on the protein synthesising activity of RNA, which increased eightfold as the dilution rate was increased from 0.02–0.175 h^-1, doubled within the temperature range 20–30° C and fell by 50% between 40 and 50° C. These observations are discussed in the context of the constant ribosomal efficiency in protein synthesis hypothesis.     

Competition for Ammonium between Nitrifying and Heterotrophic Bacteria in Dual Energy-Limited Chemostats

The absence of nitrification in soils rich in organic matter has often been reported. Therefore, competition for limiting amounts of ammonium between the chemolithotrophic ammonium-oxidizing species Nitrosomonas europaea and the heterotrophic species Arthrobacter globiformis was studied in the presence of Nitrobacter winogradskyi in continuous cultures at dilution rates of 0.004 and 0.01 h⁻¹. Ammonium limitation of A. globiformis was achieved by increasing the glucose concentration in the reservoir stepwise from 0 to 5 mM while maintaining the ammonium concentration at 2 mM. The numbers of N. europaea and N. winogradskyi cells decreased as the numbers of heterotrophic bacteria rose with increasing glucose concentrations for both dilution rates. Critical carbon-to-nitrogen ratios of 11.6 and 9.6 were determined for the dilution rates of 0.004 and 0.01 h⁻¹, respectively. Below these critical values, coexistence of the competing species was found in steady-state situations. Although the numbers were strongly reduced, the nitrifying bacteria were not fully outcompeted by the heterotrophic bacteria above the critical carbon-to-nitrogen ratios. Nitrifying bacteria could probably maintain themselves in the system above the critical carbon-to-nitrogen ratios because they are attached to the glass wall of the culture vessels. The numbers of N. europaea decreased more than did those of N. winogradskyi. This was assumed to be due to heterotrophic growth of the latter species on organic substrates excreted by the heterotrophic bacteria.     

Mixotrophic and heterotrophic growth of Beggiatoa alba in continuous culture

Beggiatoa alba strain B18LD was grown in continuous culture under heterotrophic conditions on acetate or acetate and asparagine and under mixotrophic conditions on acetate plus either 1 mM sodium sulfide or 1 mM sodium thiosulfate. Considerable differences were observed between the yields and the cell compositions of heterotrophic and mixotrophic cultures at all dilution rates tested. The dry weight yield per gram acetate utilized was approximately three times higher in the acetate-sulfide mixotrophic culture than in the acetate heterotrophic culture, whereas the poly--hydroxybutyric acid and carbohydrate contents were much higher in the heterotrophic cultures. The high yields (0.52–0.75, corrected for the weight of the sulfur) obtained with the mixotrophic cultures imply that the acetate was utilized mainly for biosynthesis. Thus, the oxidation of sulfide supplied energy. The addition of catalase to the chemostat cultures increased yields slightly, but it was insufficient to explain the differences between the heterotrophic and the mixotrophic cultures.     

Depression of hydrogenase during limitation of electron donors and derepression of ribulosebisphosphate carboxylase during carbon limitation of Alcaligenes eutrophus

Alcaligenes eutrophus did not form the key enzymes of autotrophic metabolism, the soluble and particulate hydrogenases and ribulosebisphosphate carboxylase (RuBPC), during heterotrophic growth on succinate in batch cultures. During succinate-limited growth in a chemostat, high activities of both hydrogenases were observed. With decreasing dilution rate (D) the steady-state hydrogenase activity (H) followed first-order kinetics, expressed as follows: H = Hmax .e-alpha.D. An identical correlation was observed when autotrophic growth in a chemostat was limited by molecular hydrogen. During autotrophic growth under oxygen or carbon dioxide limitation, the activity if the soluble hydrogenase was low. These data suggested that hydrogenase formation depended on the availability of reducing equivalents to the cells. RuBPC activities were not correlated with the hydrogenase activities. During succinate-limited growth, RuBPC appeared at intermediate activities. During autotrophic growth in a carbon dioxide-limited chemostat, RuBPC was highly derepressed. RuBPC activity was not detected in cells that suffered from energy limitation with a surplus of carbon, as in a heterotrophic oxygen-limited chemostat, nor was it detected in cells limited in carbon and energy, as in the case of complete exhaustion of a heterotrophic substrate. From these data I concluded that RuBPC formation in A. eutrophus depends on two conditions, namely, carbon starvation and an excess of reducing equivalents.     

Formation of polysaccharide depolymerase and glycoside hydrolase enzymes byBacteroides ruminicola subsp.ruminicola grown in batch and continuous culture

The activity of ten polysaccharide depolymerase and glycoside hydrolase enzymes was monitored inBacteroides ruminicola subsp.ruminicola throughout the growth cycle and over a range of dilution rates in carbon-limited continuous (chemostat) culture. The enzymes were principally cell associated, and the specific activities increased throughout the growth cycle to reach maximum levels in the late exponential and stationary growth phases. In chemostat-grown cells the activities were growth rate dependent and were highest at the lowest dilution rates examined (0.025/h), but remained constant over a wide range of growth rates (D=0.05–0.15/h). The specific activities were lower in cells with a generation time of 3 h (D=0.225/h). The major metabolites formed from xylose, in batch and continuous culture, were lactic, acetic, and succinic acids, with traces (1%–2% of total acid production) of branched and straight-chain C₃–C₅ volatile fatty acids. The proportions of the metabolites produced varied with the stage and rate of growth.     

Characteristics of the chemostat culture of murine leukemia L 1210 cells

Mouse leukemia L 1210 cells were cultivated under glucose limitation in a chemostat. More than 20 steady-states were established over 9 different dilution rates ranging from 0.20 day⁻¹ (cell doubling time 83 h) to 2.0 days⁻¹ (cell doubling time 8.3 h). The steady-states were characterized by: a constant cell number, constant cell volume, constant concentrations of DNA, RNA, and L-lactate (in the culture supernatant), a constant percentage of cells labelled by autoradiography, and constant rate of incorporation of [³H]TdR, [³H]uridine, and ¹⁴C-labelled amino acids into cellular acid-precipitable material. Individual steady-states were maintained for periods up to 600 h continuous operation of the chemostat. A maximum output of 66.4 × 10⁶ cells/h was obtained at a dilution rate of 1.3 day⁻¹. The glucose substrate constant was determined as 0.0063 mg/ml. The relationships between dilution rate and the steady-state cell concentration, glucose concentration, and output of L 1210 cells from the chemostat, were in general agreement with the theoretical curves. It was found that the principles of continuous culture derived from the study of microorganisms are to a large extent applicable to the cultivation of animal cells.     

Competition for ethanol between sulfate-reducing and fermenting bacteria

Competition for ethanol between the sulfate-reducing bacteria Desulfobulbus propionicus, Desulfotomaculum orientis, Desulfovibrio vulgaris Marburg, Desulfovibrio gigas, Desulfovibrio desulfuricans Essex and the fermenting bacteria Pelobacter propionicus and Acetobacterium carbinolicum were studied in batch culture. A number of these bacteria was also chosen for competition experiments under ethanol limitation in chemostat culture. The maximum growth rates determined by washout experiments were higher for the fermenting bacteria (_max=0.096 resp. 0.335h^–1) than for the sulfate-reducing bacteria (_max0.03h^–1). In contrast, the saturation concentrations for half maximum growth rates (K_s values) for ethanol were lower for the sulfate-reducing bacteria (K_s5 M) than for the fermenting bacteria (K_s50 M). In batch culture competition experiments the fermenting bacteria turned out to be the better competitors, degrading 51–80% of the ethanol added. In competition experiments with ethanollimited chemostat cultures the sulfate-reducing bacteria Desulfobulbus propionicus and Desulfovibrio vulgaris outcompeted Pelobacter propionicus at dilution rates below their maximum specific growth rates. At a high dilution rate, a fast growing population of Desulfobulbus propionicus originated and was enriched in the chemostat during the competition experiment.     

The regulatory effect of fermentable sugar levels on the production of leukotoxin by Actinobacillus actinomycetemcomitans

The relationship between sugar availability and RTX (repeats in toxin) cytotoxin (leukotoxin) production in the periodontopathic bacterium, Actinobacillus actinomycetemcomitans, was investigated using a chemostat. A. actinomycetemcomitans 301-b produced significant amounts of leukotoxin in anaerobic fructose-limited chemostat cultures at a dilution rate of 0.15 h⁻¹ and at pH 7.0. When the growth limitation was relieved by pulsing the cultures with 50 or 150 mM fructose (final concentrations), leukotoxin production immediately stopped and the amount of cellular leukotoxin decreased until the culture was returned to fructose-limited conditions. Leukotoxin synthesis was also repressed in the chemostat cultures by pulsing with glucose but not with the non-fermentable sugar analog, α-methyl-d-glucoside. Leukotoxin production was also repressed by fructose in chemostat cultures of ATCC 33384, which is generally recognized as a non-leukotoxin-producing or minimally leukotoxic strain.     

A stoichiometric organic matter decomposition model in a chemostat culture

Biodegradation, the disintegration of organic matter by microorganism, is essential for the cycling of environmental organic matter. Understanding and predicting the dynamics of this biodegradation have increasingly gained attention from the industries and government regulators. Since changes in environmental organic matter are strenuous to measure, mathematical models are essential in understanding and predicting the dynamics of organic matters. Empirical evidence suggests that grazers’ preying activity on microorganism helps to facilitate biodegradation. In this paper, we formulate and investigate a stoichiometry-based organic matter decomposition model in a chemostat culture that incorporates the dynamics of grazers. We determine the criteria for the uniform persistence and extinction of the species and chemicals. Our results show that (1) if at the unique internal steady state, the per capita growth rate of bacteria is greater than the sum of the bacteria’s death and dilution rates, then the bacteria will persist uniformly; (2) if in addition to this, (a) the grazers’ per capita growth rate is greater than the sum of the dilution rate and grazers’ death rate, and (b) the death rate of bacteria is less than some threshold, then the grazers will persist uniformly. These conditions can be achieved simultaneously if there are sufficient resources in the feed bottle. As opposed to the microcosm decomposition models’ results, in a chemostat culture, chemicals always persist. Besides the transcritical bifurcation observed in microcosm models, our chemostat model exhibits Hopf bifurcation and Rosenzweig’s paradox of enrichment phenomenon. Our sensitivity analysis suggests that the most effective way to facilitate degradation is to decrease the dilution rate.

     

Accumulation of Poly[(R)-3-Hydroxyalkanoates] in Pseudomonas oleovorans during Growth with Octanoate in Continuous Culture at Different Dilution Rates

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C₀/N₀ ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH₄⁺ in excess at low C₀/N₀ ratios, (ii) purely nitrogen-limited growth conditions at high C₀/N₀ ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C₀/N₀ ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C₀/N₀ ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C₀/N₀ ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C₀/N₀ ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.     

Respiration rate, growth rate and the accumulation of streptomycin in Escherichia coli

Using chemostat cultures of Escherichia coli it was possible to vary respiration rates while maintaining a constant growth rate. This allowed the effect of variations in respiration rates on the accumulation of streptomycin to be studied in cultures at constant growth rates. At a particular dilution rate cultures exhibited higher respiration rates when phosphate limited growth than when carbon limited growth. A ubiquinone-deficient strain had a lower rate of respiration at a particular dilution rate than a related ubiquinone-sufficient strain. In spite of these differences in respiratory activity, the accumulation of streptomycin was identical in carbon- and in phosphate-limited chemostat cultures of ubiquinone-deficient and ubiquinone-sufficient strains. Moreover, accumulation of streptomycin in an anaerobic chemostat culture occurred at the same rate as that in an aerobic chemostat. There was however a lag of 1.5 h before accumulation commenced in the anaerobic culture, a feature that was not apparent in the aerobic culture. These results indicate that the lower rates of respiration in slow-growing bacteria are not responsible for the decreased accumulation of streptomycin in slow-growing compared to fast-growing cultures. Moreover, it seems unlikely that quinones are involved directly (e.g. as carriers) in streptomycin accumulation, since removal of 90% of cellular ubiquinone, or replacement of ubiquinone with a structural analogue, did not affect accumulation as long as mutant and parent cultures grew at the same rate.     

Bottom-up and top-down controls of the microbial food web in the Southern Ocean: experiments with manipulated microcosms

Summary We have studied bottom-up and top-down control of the Southern Ocean microbial food web by microcosm experiments. Water from the Weddell Sea and Weddell Scotia Confluence were used for the experiments. Microcosms were manipulated by nutrients and light, and by size-selective screening. Incubation at the higher light level doubled phytoplankton growth rates from 0.12 to 0.24 day^–1 in the Weddell experiment and from 0.15 to 0.30 day^–1 in the Confluence experiment. Nutrient enrichment had no significant effect on growth rates in either of the experiments, indicating that phytoplankton growth was not nutrient-limited. In the microcosms where dinoflagellate growth rate was different, high dinoflagellate numbers were reflected as depressed nanoflagellate growth as well as depressed growth of phytoplankton, suggesting that dinoflagellates controlled both heterotrophic nanoflagellates and autotrophic nanoplankton. Only during short periods, when dinoflagellate numbers were low, could exponential growth of nanoflagellates be demonstrated. Bacterioplankton growth rates were, on average, 0.26 day^–1 in the Weddell experiment and 0.22 day^–1 in the Confluence experiment. Bacteria were controlled by heterotrophic nanoflagellates. Potential growth rates up to 0.75 day^–1 were measured from batch cultures without predators. With the microcosm experiments, we could demonstrate a strong top-down control by dinoflagellates on phytoplankton and on heterotrophic nanoflagellates, and a control by heterotrophic nanoflagellates on bacteria. We could also demonstrate weak nutrient limitation on autotrophs and substrate limitation on heterotrophic bacteria. In the two study areas, biomass production and carbon flow were mediated mainly by organisms that passed through a 20 m net and had growth rates in the order of 0.20 to 0.30 day^–1.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Accumulation of Poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth with octanoate in continuous culture at different dilution rates

Pseudomonas oleovorans ATCC 29347 was grown in chemostat culture at different dilution rates with mineral media varying in their ratios of octanoate to ammonia (C(0)/N(0) ratio). At all dilution rates tested, three distinct growth regimes were observed: (i) carbon limitation with NH(4)(+) in excess at low C(0)/N(0) ratios, (ii) purely nitrogen-limited growth conditions at high C(0)/N(0) ratios with residual octanoate in the culture supernatant, and (iii) an intermediate zone of dual-nutrient-limited growth conditions where both the concentration of octanoate and that of ammonia were very low. The dual-nutrient-limited growth zone shifted to higher C(0)/N(0) ratios with decreasing dilution rates, and the extension of the dual-nutrient-limited growth zone was inversely proportional to the growth rate. The cells accumulated the storage compound medium-chain-length poly[(R)-3-hydroxyalkanoate] (mcl-PHA) during dual (C and N)-nutrient-limited and N-limited growth conditions. Within the dual-nutrient-limited growth zone, the cellular mcl-PHA contents increased when the C(0)/N(0) ratio in the feed was increased, whereas the cellular mcl-PHA level was independent from the feed C(0)/N(0) ratio during N-limited growth. The monomeric composition of the accumulated mcl-PHA was independent of both the dilution rate and the feed C(0)/N(0) ratio and consisted of 12 mol% 3-hydroxyhexanoic acid and 88 mol% 3-hydroxyoctanoic acid. Accumulation of mcl-PHA led to an increase in the cellular C/N ratio and to changes in elemental growth yields for nitrogen and carbon.     

Enhanced anaerobic degradation of benzene by enrichment of mixed microbial culture and optimization of the culture medium

A heterogeneous mixed culture, originally collected from two different sources, namely cow-drug and sludge from the mineral medium containing 1% glucose and then adapted on benzene as the carbon and energy source. Under anaerobic conditions benzene was degraded via benzoic acid as a major intermediate in the benzene degradation pathway. The degradation rate of benzene was improved stepwise by the number of enrichments and optimization of the culture medium. The effects of microaerobic conditions and/or physicochemical treatment with H₂O₂ prior to anaerobic degradation were studied with respect to variations in benzene degradation rate, growth of biomass and gas produced is less than the theoretical value expected and the percentage of methane in the product gas was very small (3%–3.5%). The reason for this is not well understood but it is presumed that the major group of benzene-degrading bacteria present in the culture medium are sulphate reducers and the mixed consortium is unable to degrade certain complex aromatic intermediates in the benzene degradation pathway under the experimental conditions. For an actual explanation of the situation arising in this study, further investigations must be carrie out. However, the mixed culture is capable of oxidizing benzene more rapidly to intermediate compounds and also partly into gas under the culture conditions, compared to the published data for the anaerobic degradation of benzene.     

Production of gramicidin S synthetases by Bacillus brevis in continuous culture.

The effects of different nutrient limitations on the production of the two enzymes of gramicidin S biosynthesis were studied during continuous culture of Bacillus brevis. Gramicidin S synthetases I and II were produced in the chemostat under carbon, nitrogen, phosphorus or sulphur limitation. The growth rate, rather than the nature of the limitation, was the major controlling factor in regulating the level of the gramicidin S synthetases. Synthetase production was low at high dilution rates (0.45 to 0.50 h-1) but increased as the dilution rate was lowered. The highest specific activities occurred at dilution rates that were different for each type of limitation: 0.40 h-1 for nitrogen, 0.32 h-1 for carbon, 0.24 h-1 for sulphur and 0.20 h-1 for phosphorus. Phosphorus limitation gave the highest specific activities. At low dilution rates (0.10 to 0.15 h-1), enzyme activities were again low. Sporulation occurred under carbon limitation, but at a lower dilution rate than that which supported optimal gramicidin S synthetase formation. The specific productivity of the synthetases in the chemostat was higher than the highest productivity obtained in batch growth.     

Large-scale production of bacterial biomass from methanol for use as milk-replacer

Summary A mixed methanol-utilizing bacterial culture was utilized to produce bacterial biomass as milk replacer. This culture comprised three pure strains: KISRI-5 (NCIB 12135), KISRI-512 (NCIB 12137) and KISRI-5112 (NCIB 12138). Optimal concentrations of methanol (15 g 1^–1) and medium elements as well as optimal growth conditions, e.g., pH (6.8), temperature (38°C), dissolved oxygen and dilution rate, were established. The maximum biomass yield coefficient obtained under optimized conditions was 0.48 g g^–1.· Large-scale production was successfully carried out in a 1500 1 fermenter under chemostat conditions. A good product was obtained having high true protein content (59–62%) and low polysaccharides (5%) without microbial contamination.     

Glucose uptake rates of single E. coli cells grown in glucose-limited chemostat cultures

To evaluate the extent to which single-cell glucose uptake rates determine the overall specific growth rate of a culture, dilute chemostat cultures of Escherichia coli BL21 were grown in defined medium under glucose limitation. The glucose uptake dynamics of the cell population was examined at the single-cell level using the fluorescent glucose analog, 2-NBDG. Between dilution rates of 0.12 h(-1) and 0.40 h(-1), mean cellular protein content and steady-state, extracellular glucose concentrations increased with increasing dilution rate. However, the distribution of 2-NBDG uptake rates in the population remained constant over the range of dilution rates studied. This indicates that the growth of cells in continuous culture is not limited by the maximum rate of uptake of glucose but by the availability of glucose for transport. The work represents an example of how quantitative flow cytometry can be applied to gain detailed insight into microbial growth physiology.     

Carbon mass balance methodology to characterize the growth of pigmented marine bacteria under conditions of light cycling

A carbon mass balance methodology employing minimal measurements was applied to heterotrophic and photoheterotrophic marine bacteria grown under constant dilution and exposed to 12-h intervals of light or darkness. Carbon mass balance calculations using measurements taken every 3 h closed to within 93–103% using dissolved organic carbon, biomass carbon and CO₂ production data only, indicating that background interference from dissolved inorganic carbon variations in the amended seawater medium was not significant. Neither strain was observed to sustain a net CO₂ fixation using paramagnetic measurement of oxygen uptake rates (OUR), indicating a need for more sensitive on-line measurement techniques for OUR. Photoheterotrophic growth demonstrated lower carbon-mole biomass yields (0.41±0.026 vs. 0.64±0.013 mol mol^–1) despite higher specific glucose uptake rates (0.025 vs. 0.02 mol mol^–1 h^–1), suggesting that bioreactor-based study of marine bacteria can present growth modes that are different from those encountered in the marine environment.     

Reassessment of the bioenergetic yield of Arthrospira platensis using continuous culture

Reassessement of bioenergetic growth yield of Arthrospira platensis was performed by using continuous culture under both autotrophic and mixotrophic conditions. Continuous culture was carried out at dilution rates of 0.017, 0.023 and 0.030 h^–1. Under these dilution rates bioenergetic yields ranged between 4.45–6.03 × 10^–3 g biomass kJ^–1 and between 5.42–7.46 × 10^–3 g biomass kJ^–1, under autotrophic and mixotrophic conditions respectively. A maximum bioenergetic yield of 8.1 × 10^–3 g biomass kJ^–1 using an autotrophic culture can be calculated. Pigment accumulation (chlorophyll a and carotenoids) may be related to light irradiance, reaching a maximum pigment concentration under light saturation irradiance. Phycocyanin concentration increased during light limitation.