Principles of the light-limited chemostat: theory and ecological applications

Light is the energy source that drives nearly all ecosystems on planet Earth. Yet, light limitation is still poorly understood. In this paper, we present an overview of the principles of the light-limited chemostat. The theory for light-limited chemostats differs considerably from the standard theory for substrate-limited chemostats. In particular, photons cannot be mixed by vigorous stirring, so that phototrophic organisms experience the light-limited chemostat as a heterogeneous environment. Similar to substrate-limited chemostats, however, light-limited chemostats do reach a steady state. This allows the study of phototrophic microorganisms under well-controlled light conditions, at a constant specific growth rate, for a prolonged time. The theory of the light-limited chemostat is illustrated with several examples from laboratory experiments, and a variety of ecological applications are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Competition between purple and brown phototrophic bacteria in stratified lakes: sulfide, acetate, and light as limiting factors

Abstract The affinities for sulfide and acetate under mixotrophic conditions have been determined for the brown Chlorobium phaeobacteroides and the purple Thiocapsa roseopersicina isolated from a bloom in Lake Kinneret (Israel) at a depth of about 18 m. C. phaeobacteroides exhibited a far higher affinity for sulfide than T. roseopersicina . For acetate, the opposite was observed.
In light-limited continuous cultures, C. phaeobacteroides preferentially used sulfide, whereas in mixotrophic cultures of T. roseopersicina sulfide could be detected without detectable acetate. Competition experiments under increasingly severe light limitation resulted in co-existence of the two strains. Relatively high light intensities resulted in a dominance of T. roseopersicina over C. phaeobacteroides , whereas at lower intensities C. phaeobacteroides became dominant. However, at light intensities below 2 μEin · m⁻²· s⁻¹, T. roseopersicina was completely excluded.
At low light intensities, C. phaeobacteroides is able to grow at a much higher rate than T. roseopersicina . The maintenance rate constant μ_e of C. phaeobacteroides is −0.001 h⁻¹, whereas that of T. roseopersicina is −0.011 h⁻¹. However, high light intensities inhibit the growth rate of C. phaeobacteroides , but not that of T. roseopersicina .
The explanation of the high numbers of C. phaeobacteroides in Lake Kinneret appears to be the combination of low light intensities and low sulfide concentrations. As a result, the incorporation of acetate is enhanced. The low numbers of T. roseopersicina can be explained by the high maintenance energy requirements of this organism, which exceed the available light at the depth of the bloom.     

Optimization of biomass, vitamins, and carotenoid yield on light energy in a flat-panel reactor using the A-stat technique

Acceleration-stat (A-stat) cultivations in which the dilution rate is continuously changed at a constant acceleration rate, leading to different average light intensities inside the photobioreactor, can supply more information and reduce experimental time compared with chemostat cultivations. The A-stat was used to optimize the biomass and product yield of continuous cultures of the microalgae D. tertiolecta in a flat-panel reactor. In this study, four different accelerations were studied, a pseudo steady state was maintained at acceleration rates of 0.00016 and 0.00029 h(-2) and results were similar to those of the chemostat. An increase in the acceleration rate led to an increase in the deviation between results obtained in the A-stat and in the chemostats. We concluded that it is advantageous to use the A-stat instead of chemostats to determine culture characteristics and optimize a specific photobioreactor. The effect of average light intensity inside the photobioreactor on the production of vitamins C and E, lutein, and beta-carotene was studied using the A-stat. The highest concentrations of these products were 3.48 +/- 0.46, 0.33 +/- 0.06, 5.65 +/- 0.24, and 2.36 +/- 0.38 mg g(-1), respectively. These results were obtained at different average light intensities, showing the importance of optimizing each product on light intensity.     

Growth and enzyme synthesis during continuous culture of Trichosporon cutaneum on phenol

The soil yeast Trichosporon cutaneum was grown in continuous culture with phenol as the sole carbon source. The cultures were operated as carbon-limited chemostats or as steady-state continuous cultures without carbon limitation. Selected comparative runs were also conducted on glucose or acetate as carbon source. In addition to growth parameters, the activities of several intracellular enzymes were determined, comprising those directly involved in the degradation of phenol as well as auxiliary enzymes required for the generation of reducing power. All enzymes were assayed in detergent-permea-bilized cells. Phenol was found to serve as an excellent carbon source, comparable to glucose or acetate. The utilization of phenol in T. cutaneum is very efficient as indicated by a low maintenance requirement (0.01 g phenol/g cells.h). The cell yields obtained were on the order of 0.8 g cells/g phenol. Although the phenol-limited chemostats were run with fully phenol-induced cells, a further increase in the activities of isocitrate DH(NADP(+)), maleate DH and the phenol-degrading enzymes occurred after transition to nonlimiting condition. Enzyme activities increased in parallel with increasing phenol levels in the effluent, as well as with increasing toxicity. The significance of this phenomenon is discussed. The significance of this phenomenon is discussed. This elevation in enzyme activities in not related to an increase in specific growth rate.     

Concerted changes in gene expression and cell physiology of the cyanobacterium Synechocystis sp. strain PCC 6803 during transitions between nitrogen and light-limited growth

Physiological adaptation and genome-wide expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to gradual transitions between nitrogen-limited and light-limited growth conditions were measured in continuous cultures. Transitions induced changes in pigment composition, light absorption coefficient, photosynthetic electron transport, and specific growth rate. Physiological changes were accompanied by reproducible changes in the expression of several hundred open reading frames, genes with functions in photosynthesis and respiration, carbon and nitrogen assimilation, protein synthesis, phosphorus metabolism, and overall regulation of cell function and proliferation. Cluster analysis of the nearly 1,600 regulated open reading frames identified eight clusters, each showing a different temporal response during the transitions. Two large clusters mirrored each other. One cluster included genes involved in photosynthesis, which were up-regulated during light-limited growth but down-regulated during nitrogen-limited growth. Conversely, genes in the other cluster were down-regulated during light-limited growth but up-regulated during nitrogen-limited growth; this cluster included several genes involved in nitrogen uptake and assimilation. These results demonstrate complementary regulation of gene expression for two major metabolic activities of cyanobacteria. Comparison with batch-culture experiments revealed interesting differences in gene expression between batch and continuous culture and illustrates that continuous-culture experiments can pick up subtle changes in cell physiology and gene expression.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

Regulation of beta-glucosidase in Bacteroides ruminicola by a different mechanism: growth rate-dependent derepression.

Bacteroides ruminicola B(1)4, a predominant ruminal and cecal bacterium, was grown in batch and continuous cultures, and beta-glucosidase activity was measured by following the hydrolysis of p-nitrophenyl-beta-glucopyranoside. Specific activity was high when the bacterium was grown in batch cultures containing cellobiose, mannose, or lactose (greater than 286 U/g of protein). Activity was reduced approximately 90% when the organism was grown on glucose, sucrose, fructose, maltose, or arabinose. The specific activity of cells fermenting glucose was initially low but increased as glucose was depleted. When glucose was added to cultures growing on cellobiose, beta-glucosidase synthesis ceased immediately. Catabolite repression by glucose was not accompanied by diauxic growth and was not relieved by cyclic AMP. Since glucose-grown cultures eventually exhibited high beta-glucosidase activity, cellobiose was not needed as an inducer. Catabolite repression explained beta-glucosidase activity of batch cultures and high-dilution-rate chemostats where glucose accumulated, but it could not account for activity at slow dilution rates. Maximal beta-glucosidase activity was observed at a dilution rate of approximately 0.35 h-1, and cellobiose-limited chemostats showed a 15-fold decrease in activity as the dilution rate declined. An eightfold decline was observed in glucose-limited chemostats. Since inducer availability was not a confounding factor in glucose-limited chemostats, the growth rate-dependent derepression could not be explained by other mechanisms.     

The effect of growth temperature on the bioenergetics of photosynthetic algal cultures

Two photosynthetic algal cultures, one Chlorella vulgaris, and the other a Chlorogonium sp., were cultured under light limitations in chemostats. The effects of growth temperature on their energy yield and maintenance energy requirement were studied. It was observed that a lowering in temperature resulted in a lower maximum growth yield from the light energy, Y(G). This was attributed to two reasons. First, at low temperatures there was a change in the algal cell composition with more energy being expended to synthesize a higher biomass protein content. Secondly, at low temperatures, a cyanide-resistant respiratory pathway became operative which led to a decrease in the number of ATP being generated. The maintenance energy coefficient was a function of temperature increasing with decreasing temperature. This might reflect energy wastage by the cell at low temperatures. The maximum specific growth rate dropped with decreasing temperature, and can be described by an Arrhenius type rate-temperature model up to the optimal temperature for growth; i.e., activation energy remained constant.     

CONTINUOUS CULTURE OF MARINE DIATOMS UNDER SILICATE LIMITATION. II. EFFECT OF LIGHT INTENSITY ON GROWTH AND NUTRIENT UPTAKE OF SKELETONEMA COSTATUM1,2

Two replicate experiments were conducted to investigate the effect of light intensity on the growth and nutrient uptake of Skeletonema costatum (Grev.) Cleve in silicate-limited continuous culture. Each experiment began with 4 identical chemostat cultures of S. costatum growing at the normal laboratory light (0.14 ly · min^?1, continuous illumination) under strong silicate limitation. Screens were placed over 3 cultures reducing them to light intensities of 0.042, 0.021 and 0.0018 ly · min^?1. Based on growth rules, nutrient uptake rates, cell morphology and chemical composition, the cultures receiving 0.021, and 0.0018 ly · min^?1 appeared to he light-limited, whereas the culture receiving 0.14 ly.     

Comparison of the light-limited growth of the nitrogen-fixing cyanobacteria Anabaena and Aphanizomenon

The effect of simultaneous N₂ fixation and light limitation on the growth of two strains of Anabaena sp. Bory de St. Vincent and Aphanizomenon flos-aquae (L.) Ralfs was investigated using continuous cultures. Under severely light-limited conditions, Aphanizomenon showed a broader absorption spectrum (due to the presence of phycoerythrin), a higher maximum efficiency of photosynthesis, a higher steady-state N₂ fixation activity and a higher growth affinity for light than did Anabaena . On the other hand, under light saturation, Anabaena showed a higher maximum rate of O₂ production and a higher maximum specific growth rate than Aphanizomenon . These monoculture results characterize Anabaena and Aphanizomenon , in relative terms, as a 'sun' and a 'shade' species respectively, and are in accordance with field observations. The difference between the two species in their acclimatory response is discussed in terms of a species-specific alteration of the PSI:PSII stoichiometry. Besides the species-specific modulation of the accessory pigments, such an acclimation would provide a biochemical basis for the observed physiological differences. The monoculture results were used to differentiate the niches of the two species and suggested that Aphanizomenon would competitively displace Anabaena under N₂-fixing, light-limited conditions. However, when both species were grown together, Anabaena became dominant and seemed to be the superior competitor for light. In order to explain this finding, the possible effects of release of allelopathic compounds, or dynamic aspects of light supply, are discussed.     

Predicting Production in Light-Limited Continuous Cultures of Algae

Equations relating productivity, growth rate, cell concentration, and light absorption lead to the prediction that, when incident light is below saturating intensity, maximal productivity will occur at half the maximal growth rate. The freshwater alga Chlorella pyrenoidosa TX71105 and the marine alga Dunaliella tertiolecta were grown in a small continuous culture apparatus with turbidostatic control. With both cultures, the cell concentration showed a linear decrease with dilution rate. Productivity was maximal at about one-half the maximal dilution rate. Average mass per cell increased near the maximal dilution rate, causing some asymmetry in the productivity versus dilution rate curve. The chlorophyll content per unit mass decreased in this region, but the chlorophyll content per cell remained constant. Best production rate in a light-limited algal culture was obtained when the growth rate at very low cell concentration was determined in the apparatus and the dilution rate was set at one-half that value.     

ALGAE AS COMPETITORS FOR GLUCOSE WITH HETEROTROPHIC BACTERIA1

Dissolved organic carbon (DOC) constitutes the bulk of organic carbon in aquatic environments. The importance of DOC utilization by mixotrophic algae is unclear since heterotrophic bacteria are regarded as more efficient users. We tested the hypothesis that algae decrease the DOC concentration in the light to lower levels than in darkness resulting in competitive exclusion of heterotrophic bacteria according to the mechanistic competition theory. We investigated (a) the uptake kinetics of glucose as a model substrate by two cultured algae and mixed bacteria populations, (b) the competition for glucose between algae and bacteria in chemostats, (c) the effect of discontinuous glucose supply in chemostats, and (d) the minimum glucose concentrations achieved in cultures of algae and bacteria. Bacteria showed higher specific‐glucose‐uptake rates than algae. In chemostats, algae became extinct in the dark and coexisted in the light where they decreased bacteria to lower densities. Discontinuous glucose supply promoted the algae compared to continuous substrate addition. Several algae consumed glucose to lower concentrations in the dark than in the light and showed lower or equal residual glucose concentrations than bacteria. Residual concentrations were not related to allometric traits (cell volume) and photosynthetic potential (chl content). Overall, the hypothesis was not supported, and mechanisms of competition for DOC obviously differed from those for particulate prey. However, since some algae showed lower or equal residual glucose concentrations than bacteria, algal dark uptake of DOC may be important in deep layers of many waters.     

Model simulations of continuous rotifer cultures

Derived from the Monod-model and regulating principles a regulation model of the rotifer development in chemostats was developed. The model was validated in continuous cultures of Brachionus angularis both in steady-states, when undisturbed, and in transient-states after perturbations by step changes of dilution rate or input substrate concentration. Simulations of the simple model monotonically approached steady-states, but cultures show overshoots and damped oscillations before reaching this state. After introducing time-lags into the model it depends on the size of the time lag if model rotifer densities reach stable steady-state values (at low time lags) or stable limit cycles with periodic oscillations (at high time lags). At even higher time lags chaotic conditions occur in the model with final extinction of the rotifers.     

Kinetics and stoichiometry of growth of plant cell cultures of Catharanthus roseus and Nicotiana tabacum in batch and continuous fermentors

Plant cell suspension cultures of Catharanthus roseus and Nicotiana tabacum were grown in stirred tank bioreactors operated in batch and continuous mode. The stoichiometry of growth of both species in steady-state glucose limited chemostats was studied at a range of different dilution rates. A linear relation was applied to describe specific glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate. Specific respiration deviated greatly from the linear relation. An unstructured mathematical model, based on the observed stoichiometry in the glucose limited chemostats, was applied to describe the growth in batch culture. From a comparison between the observed growth pattern in batch fermentors and computer simulations it appeared that the stoichiometry of growth of the C. roseus culture was different under steady-state and dynamic conditions. It was concluded that a mathematical model for the growth of suspension culture plant cells in which the biomass is considered to be a single compound with an average chemical composition is of limited value because large changes in the conmposition of the biomass may occur. (c) 1992 John Wiley & Sons, Inc.     

RELATIONSHIPS BETWEEN NITRATE REDUCTASE ACTIVITY AND RATES OF GROWTH AND NITRATE INCORPORATION UNDER STEADY-STATE LIGHT OR NITRATE LIMITATION IN THE MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1

Although activity of the enzyme nitrate reductase (NR) can potentially be used to predict the rate of nitrate incorporation in field assemblages of marine phytoplankton, application of this index has met with little success because the relationship between the two rates is not well established under steady-state conditions. To provide a basis for using NR activity measurements, the relationships among NR activity, growth rate, cell composition, and nitrate incorporation rate were examined in cultures of Thalassiosira pseudonana (Hustedt)Hasle and Heimdal, growing a) under steady-state light limitation, b) during transitions between low and high irradiance (15 or 90 μmol quanta.m^?2.s^?1), and c) under steady-state nitrate limitation. Using a modified assay for NR involving additions of bovine serum albumin to stabilize enzyme activity, NR activity in light-limited cultures was positively and quantitatively related to calculated rates of nitrate incorporation, even in cultures that were apparently starved of selenium. During transitions in irradiance, growth rates acclimated to new conditions within 1 day; through the transition, the relationship between NR activity and nitrate incorporation rate remained quantitative. In nitrate-limited chemostat cultures, NR activity was positively correlated with growth rate and with nitrate incorporation rates, but the relationship was not quantitative. NR activity exceeded nitrate incorporation rates at lower growth rates (<25% of nutrient-replete growth rates), but chemostats operating at such low dilution rates may not represent ecologically relevant conditions for marine diatoms. The strong relationship between NR activity and nitrate incorporation provides support for the idea that NR is rate-limiting for nitrate incorporation or is closely coupled to the rate-limiting step. In an effort to determine a suitable variable for scaling NR activity, relationships between different cell components and growth rate were examined. These relationships differed depending on the limiting factor. For example, under light limitation, cell volume and cell carbon content increased significantly with increased growth rate, while under nitrate limitation cell volume and carbon content decreased as growth rates increased. Despite the differences found between cell composition and growth rate under light and nitrate limitation, the relationships between NR activity scaled to different compositional variables and growth rate did not differ between the limitations. In field situations where cell numbers are not easily determined, scaling NR activity to particulate nitrogen content may be the best alternative. These results establish a strong basis for pursuing NR activity measurements as indices of nitrate incorporation in the field.     

PATTERNS OF GROWTH AND CELL COMPOSITION OF FRESHWATER ALGAE IN LIGHT-LIMITED CONTINUOUS CULTURES1

Selenastrum minutum (Näg.) Collins and three species of Scenedesmus were grown in light-limited continuous cultures. Comparative growth patterns were explained in terms of differences in the rates of light absorption and utilization. For all species, increasing light intensity or lowering temperature caused chl a/C to decrease and RNA/C to increase. Similar shifts in pigment composition have been widely reported. However, the RNA:light interaction has only been reported infrequently, and the RNA:temperature interaction is believed to be the first reported observation of its kind in algae. Distinct differences in RNA/C among the test algae may be size-dependent. Similar environmentally induced shifts in chl a/C and RNA/C among all test algae point to the existence of a common control mechanism which maximizes the specific growth rate through adjustments to cell composition.     

Effects of attachment of bacteria to chemostat walls in a microbial predator-prey relationship

Mixed cultures of the protozoan Tetrahymena pyriformis and the bacterium Escherichia coli were propagated in chemostats fed with glucose and minimal mineral salts medium. The interior surfaces of some chemostats were treated with a silicone compound but those of other chemostats were not. Data obtained showed that bacteria but not protozoans were attached strongly to the walls of the chemostats, that silicone treatment reduced the density of the attached bacteria by two orders of magnitude or more, and that the presence of the attached bacteria had significant effects on the dynamics of the microbial predator-prey system. Attempts were made to simulate the data by various mathematical models. It was found that a model based on combination of the Topiwala-Hamer model for wall attachment and a multiple saturation model for growth of the protozoans on the bacteria gave a reasonable fit of all the data.     

Relationship between phosphates and alkaline phosphatase of Anabaena flos-aquae in continuous culture

Summary Anabaena flos-aquae was grown in chemostats with phosphate-limiting growth and dilution rate of 0.015–0.03 h^-1. The yields of cells were dependent on dilution rate and a two-fold increase obtained by growth in the presence of 15 mM KNO₃. Alkaline phosphatase activity varied 20-fold, lowest activity with excess phosphate light-limited cells and the highest activity with cells grown in the presence of 15 mM KNO₃. There was no correlation between hot water soluble phosphate of cells and alkaline phosphatase activity.     

Design and Use of Multiplexed Chemostat Arrays

Chemostats are continuous culture systems in which cells are grown in a tightly controlled, chemically constant environment where culture density is constrained by limiting specific nutrients.^1,2 Data from chemostats are highly reproducible for the measurement of quantitative phenotypes as they provide a constant growth rate and environment at steady state. For these reasons, chemostats have become useful tools for fine-scale characterization of physiology through analysis of gene expression^3-6 and other characteristics of cultures at steady-state equilibrium.⁷ Long-term experiments in chemostats can highlight specific trajectories that microbial populations adopt during adaptive evolution in a controlled environment. In fact, chemostats have been used for experimental evolution since their invention.⁸ A common result in evolution experiments is for each biological replicate to acquire a unique repertoire of mutations.^9-13 This diversity suggests that there is much left to be discovered by performing evolution experiments with far greater throughput. We present here the design and operation of a relatively simple, low cost array of miniature chemostats—or ministats—and validate their use in determination of physiology and in evolution experiments with yeast. This approach entails growth of tens of chemostats run off a single multiplexed peristaltic pump. The cultures are maintained at a 20 ml working volume, which is practical for a variety of applications. It is our hope that increasing throughput, decreasing expense, and providing detailed building and operation instructions may also motivate research and industrial application of this design as a general platform for functionally characterizing large numbers of strains, species, and growth parameters, as well as genetic or drug libraries.     

Nitrogen and carbon fixation byAnabaena sp. isolated from a rice paddy and grown under P and light limitations

Anabaena sp., isolated from a rice paddy, was investigated for its nitrogen fixation as measured by acetylene reduction activity (ARA) in P-limited continuous and light-limited semi-continuous cultures. Growth rate (μ) under P limitation was a function of cell P content (q _p). Both the photosynthetic capacity (P_max) and photosynthetic efficiency (α) increased with μ when expressed per cell, but not per unit chla. The ARA of steady-state cells under P limitation increased with μ and was linearly related to C-fixation rate. This was apparently a consequence of the control of C-fixation by P limitation. In light-limited cells, steady state ARA, both at the culture light intensity and in the dark, increased asymptotically with μ, but the activity in the dark was only about 51% of that in the light. When the light level of steady-state cells grown at a high in intensity was switched to a low level, ARA decreased exponentially with time. Dark ARA activity also showed a similar decline, but at much lower levels. Thus, ARA depended not only on light history, but also immediate photosynthesis. Steady-state ARA at the ambient intensity or in the dark showed a strong correlation with¹⁴C-fixation rate. ARA of light-limited cells showed the same light-saturation characteristics as their¹⁴C-fixation, with the same initial saturation intensity,I _k. The ratios of P_max to the maximum ARA (ARA_max), and α to the slope of ARA (α_ara) were identical. A comparison of gross to net photosynthesis and N₂ fixation suggested that there was little leakage or excretion of fixed C or N.