On multiple-nutrient-limited growth of microorganisms,with special reference to dual limitation by carbon and nitrogen substrates

Simultaneous limitation of microbial growth by two or more nutrients is discussed for dual carbon/nitrogen-limited growth in continuous culture. The boundaries of the zone where double-limited growth occurs can be clearly defined from both cultivation data and cellular composition and they can be also predicted from growth yield data measured under single-substrate-limited conditions. It is demonstrated that for the two nutrients carbon and nitrogen the zone of double nutrient limitation is dependent on both the C:N ratio of the growth medium and the growth (dilution) rate. The concept on double-(carbon/nitrogen)-limited growth presented here can be extended to other binary and multiple combinations of nutrients.     

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.     

Accumulation of poly[(R)-3-hydroxyalkanoates] in Pseudomonas oleovorans during growth in batch and chemostat culture with different carbon sources

Pseudomonas oleovorans (ATCC 29347) was grown in batch and chemostat cultures with citrate, hexanoate, heptanoate, octanoate, and nonanoate as single carbon substrates. The growth medium for batch cultures was adjusted such that nitrogen (NH(4)(+)) limitation terminated the exponential-growth phase. During batch cultivation with octanoate or nonanoate the biomass continued to increase after depletion of ammonium due to the accumulation of medium-chain-length poly[(R)-3-hydroxyalkanoates] (mcl-PHAs). Additionally, a significant rate of mcl-PHA accumulation was also observed in the exponential-growth phase of batch cultures. It is well known that the accumulation of reserve materials is strongly dependent on the ratio of nutrients (here of carbon, C, and of nitrogen, N) and that in a batch culture the ratio of C:N is continuously changing. Therefore, we have also investigated the effect of defined ratios of C:N under constant cultivation conditions, namely at a fixed dilution rate (D) in a chemostat fed with different medium C:N ratios. These experiments were performed at a constant D of 0.2 h(-1). The concentration of the nitrogen source in the inflowing medium (N()) was kept constant, while its carbon concentration (C()) was increased stepwise, resulting in an increase of the medium carbon to nitrogen ratio (C()/N() ratio). The culture parameters and the cell composition of steady-state cultures were determined as a function of the C()/N() ratio in the feed medium. Mcl-PHA accumulation was detected during growth with the fatty acids, and three distinct regimes of growth limitation were discovered: In addition to carbon limitation at low, and nitrogen limitation at high C()/N() ratios, an intermediate growth regime of simultaneous limitation by carbon and nitrogen was detected where both substrates were used to completion. The width of this dual-nutrient-limited growth regime was dependent on the change in the yield factors for carbon and nitrogen (Y(X/C), Y(X/N)) measured during single-nutrient-limited growth.     

Factors controlling the production of domoic acid by Pseudo-nitzschia (Bacillariophyceae): A model study

A mechanistic model has been developed to explore the factors controlling the production of domoic acid (DA) by the pennate diatom Pseudo-nitzschia. The idealized model allows consideration of the uncoupling between photosynthesis and growth, while DA production has been set as a secondary metabolism sharing common precursors with growth. Under growth limitation, these precursors can accumulate, resulting in an increased DA production. The model was first evaluated based on its ability to simulate the observed DA production by either silicon (Si) or phosphorus (P) limited batch cultures of Pseudo-nitzschia available in the literature. Sensitivity tests were further performed to explore how the ambient nutrients and the light regime (intensity and photoperiod length) are possibly directing the Pseudo-nitzschia toxicity. The general pattern that emerged is that excess light, in combination with Si or P limitation, favours DA production, provided nitrogen (N) is sufficient. Model simulations with varying nutrient stocks supporting Pseudo-nitzschia blooms under non-limiting light suggest two potential ways for nutrients to control DA production. First, N excess in comparison to available Si and P relieves DA production from its limitation by N, an absolute requirement of the DA molecule. Second, increased nutrient stocks amplify the DA production phase of the blooms (in addition to enhancing Pseudo-nitzschia biomass) which leads to an even more toxigenic bloom. Simulations investigating the light regime suggest a light threshold below which an important delay in DA production could be expected in Pseudo-nitzschia cultures. In the natural environment, the monitoring of light conditions during Pseudo-nitzschia blooms might help to anticipate the magnitude of the toxic event. Pseudo-nitzschia toxicity is indeed linked to the excess of primary carbon that accumulates during photosynthesis under growth limitation by nutrients.     

Effect of Growth Rate on the Synthesis of Penicillin by Penicillium chrysogenum in Batch and Chemostat Cultures

The kinetics of penicillin production by Penicillium chrysogenum Wis 54-1255 in a glucose-limited chemostat and in batch cultures are reported. The specific production rate of penicillin, q_pen (units per milligram of dry weight per hour) was independent of specific growth rate over the range 0.014 to 0.086 hr^-1. Growth was stopped by restricting the glucose supply to the “maintenance ration,” that is, the glucose requirement of the organism at zero growth rate with all other nutrients in excess. Under such conditions, the organism dry weight remained constant, but the q_pen fell approximately linearly to zero at a rate inversely related to the previous growth rate. Glucose supplied in excess of the maintenance ration inhibited the decay of q_pen. At a critical growth rate between 0.009 and 0.014 hr^-1, the decay was completely inhibited. Quantitative expressions for the q_pen of growing and nongrowing cultures were derived and used to predict the steady-state concentrations of penicillin accumulating in one- and two-stage continuous processes. A rational explanation of the kinetics of penicillin accumulation in batch cultures is given, relating the rate of penicillin synthesis to growth rate. It is concluded that an important role of corn steep liquor (CSL), a heterogeneous carbon and nitrogen source commonly used in penicillin production media, is the provision of substrates which allow a high concentration of mold to be reached before the growth rate falls below the critical value. CSL had no significant effect on q_pen.     

Influence of gap size and soil properties on microbial biomass in a subtropical humid forest of north-east India

We examined the effects of treefall gap size and soil properties on microbial biomass dynamics in an undisturbed mature-phase humid subtropical broadleaved forest in north-east India. Canopy gaps had low soil moisture and low microbial biomass suggesting that belowground dynamics accompanied changes in light resources after canopy opening. High rainfall in the region causes excessive erosion/leaching of top soil and eventually soil fertility declines in treefall gaps compared to understorey. Soil microbial population was less during periods when temperature and moisture conditions are low, while it peaked during rainy season when the litter decomposition rate is at its peak on the forest floor. Greater demand for nutrients by plants during rainy season (the peak vegetative growth period) limited the availability of nutrients to soil microbes and, therefore, low microbial C, N and P. Weak correlations were also obtained for the relationships between microbial C, N and P and soil physico–chemical properties. Gap size did influence the microbial nutrients and their contribution to soil organic carbon, total Kjeldhal nitrogen and available-P. Contribution of microbial C to soil organic carbon, microbial N to total nitrogen were similar in both treefall gaps and understorey plots, while the contribution of microbial P to soil available-P was lower in gap compared to the understorey. These results indicate that any fluctuation in microbial biomass related nutrient cycling processes in conjunction with the associated microclimate variation may affect the pattern of regeneration of tree seedlings in the gaps and hence be related with their size. This revised version was published online in June 2006 with corrections to the Cover Date.     

A kinetic model for product formation of microbial and mammalian cells

Growth of microbial and mammalian cells can be classified into substrate-limited and substrate-sufficient growth according to the relative availability of the substrate (carbon and energy source) and other nutrients. It has been observed for a number of microbial and mammalian cells that the consumption rate of substrate and energy (ATP) is generally higher under substratesufficient conditions than under substrate limitation. Accordingly, the product formation under substrate excess often exhibits different patterns from those under substrate limitation. The extent of increase or decrease in product formation may depend not only on the nature of limitation and cell growth rate but also on the residual substrate concentration in a relatively wide range. The product formation kinetic models existing in literature cannot describe these effects. In this study, the Luedeking-Piret kinetic is extended to include a term describing the effect of residual substrate concentration. The extended model has a similar structure to the kinetic model for substrate and energy consumption rate recently proposed by Zeng and Deckwer. The applicability of the extended model is demonstrated with three microbial cultures for the production of primary metabolites and three hybridoma cell cultures for the production of ammonia and lactic acid over a wide range of substrate concentration. The model describes the product formation in all these cultures satisfactorily. Using this model, the range of residual substrate concentration, in which the product formation is affected, can be quantitatively assessed. (c) 1995 John Wiley & Sons, Inc.     

Nitrogen Requirement of Iron-Oxidizing Thiobacilli for Acidic Ferric Sulfate Regeneration

Ammonium was shown to be a limiting nutrient for iron oxidation in cultures of Thiobacillus ferrooxidans. In addition, one strain was also able to assimilate nitrate, but not nitrite, for growth and coupled iron oxidation. Some amino acids (0.5 mM) were tested as a source of nitrogen; none clearly stimulated bacterial activity and inhibition was commonly encountered. Complex nitrogenous compounds were inhibitory at high concentrations (0.1 to 0.5%, wt/vol) and, at low concentrations, some clearly stimulated the bacterial iron oxidation in ammonium-limited cultures. Enhancement of iron oxidation by these compounds was also observed in ammonium-unlimited cultures, suggesting their possible role in providing trace nutrients and possibly carbon for the bacteria.     

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.     

Lysis and “cryptic” growth in wastewater and sludge treatment processes

The primary objectives in biological wastewater and sludge treatment processes are to procedure a minimum quantity of solid, stable residues and a maximum quantity of carbon dioxide from the organisms present in the process feeds. As far as minimizing solids production in activated sludge type processes is concerned, endogenous activity in the recycled biomass is usually considered to be the most important mechanism. However, increased understanding of the growth characteristics of mixed microbial cultures suggests that lysis and “cryptic” growth are probably dominant mechanisms. For pathogen destruction in treatment processes, death and subsequent lysis of pathogens are clearly events that must be promoted. Here, the kinetics for death, lysis and “cryptic” growth in aerobic wastewater treatment and aerobic sludge stabilization processes are examined.     

Influence of Carbon Source on Nitrate Removal by Nitrate-Tolerant Klebsiella oxytoca CECT 4460 in Batch and Chemostat Cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h⁻¹, whereas with glycerol it was 0.45 h⁻¹. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (Y_C) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (Y_N) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h⁻¹ the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h⁻¹ the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

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.     

Charge balance and acidity regulation during growth of sugar-cane cell suspensions

Abstract. An analysis of nutrient uptake by batch cultures of sugar-cane cells was performed to gain information about the ionic balance during uptake of charged metabolites. Whereas younger cultures (up to 1 week old) have to compensate excess cation influx with proton efflux, older cultures show balanced cation–anion uptake.
Younger cells produce a small amount of carboxylic acids to furnish protons for charge compensation at the cytoplasmic membrane. Older cells synthesize organic acids more abundantly to generate protons necessary for the proton demand of nitrate and sulphate assimilation. Despite these assimilation reactions only a small percentage of carbon, which is taken up mainly as hexose, needs to be oxidized to carboxylic acids for that purpose. In contrast, younger cultures preferentially use the amino acids of the medium instead of assimilating nitrate. The use of amino acids as a nitrogen source does not require a significant part of metabolism for biochemical pH-stability, whereas an efficient proton circulation on the cytoplasmic membrane seems to be of major importance.
A balance study of the main metabolized elements, carbon, nitrogen and sulphur was performed to get a quantitative impression of the fate of these nutrients during growth of cell cultures.     

Modulation of glycogen and trehalose levels in Micromonospora echinospora (ATCC 15837)

The growth of Micromonospora echinospora was studied in high and low C/N ratio medium using both batch and continuous culture. Asparagine was consumed rapidly in batch cultures where it served as both a nitrogen and carbon source. Glucose consumption was low suggesting that asparagine functions as the major carbon source under these conditions. The effect of nutrient limitation on the accumulation of storage carbohydrate in batch culture revealed an intimate association between nitrogen limitation and the accumulation of carbonaceous reserves. This study revealed that glycogen constituted the major carbohydrate reserve associated with the onset of sporulation. Intracellular trehalose levels were found to be relatively low and may have been affected by the availability of carbon. Continuous culture studies revealed a correlation between glycogen accumulation and increasing growth rate. It was also found that elevated cellular ATP levels correlated with the increase in glycogen, and reduced glycolytic activity. At the higher growth rates cellular ATP levels were elevated and coincided with reduced activity of the key glycolytic enzyme, phosphofructokinase, suggesting that glycogen can act as a convenient energy reservoir when excess carbon flux dictates.     

利用废水培养微藻的研究进展

微藻被认为是一种有潜力的、可被开发为再生能源的重要生物材料。一些微藻种类具有较强的异养和混养能力,能直接利用有机物作为碳源。工农业生产和城市生活中所排放的废水中通常含有大量的有机碳、氮、磷等营养物质。利用废水培养微藻,一方面可以将废水中的碳、氮、磷等营养物质转化为具有更高价值的微藻生物质,另一方面又可实现废水的净化和营养物质的再利用。本综述了不同种类废水的特点,讨论了两类微藻培养模式的优劣,同时还探讨了微藻对营养元素的利用,并总结了微藻培养需突破的瓶颈。     

Stoichiometry and food-chain dynamics

Traditional models of chemostat systems looking at interactions between predator, prey and nutrients have used only a single currency, such as energy or nitrogen. In reality, growth of autotrophs and heterotrophs may be limited by various elements, e.g. carbon, nitrogen, phosphorous or iron. In this study we develop a dynamic energy budget model chemostat which has both carbon and nitrogen as currencies, and examine how the dual availability of these elements affects the growth of phytoplankton, trophic transfer to zooplankton, and the resulting stability of the chemostat ecosystem. Both species have two reserve pools to obtain a larger metabolic flexibility with respect to changing external environments. Mineral nitrogen and carbon form the base of the food chain, and they are supplied at a constant rate. In addition, the biota in the chemostat recycle nutrients by means of respiration and excretion, and organic detritus is recycled at a fixed rate. We use numerical bifurcation analysis to assess the model's dynamic behavior. In the model, phytoplankton is nitrogen limited, and nitrogen enrichment can lead to oscillations and multiple stable states. Moreover, we found that recycling has a destabilizing effect on the food chain due to the increased repletion of mineral nutrients. We found that both carbon and nitrogen enrichment stimulate zooplankton growth. Therefore, we conclude that the concept of single-element limitation may not be applicable in an ecosystem context.     

Poly-3-hydroxybutyrate metabolism in the type II methanotroph Methylocystis parvus OBBP

Differences in carbon assimilation pathways and reducing power requirements among organisms are likely to affect the role of the storage polymer poly-3-hydroxybutyrate (PHB). Previous researchers have demonstrated that PHB functions as a sole growth substrate in aerobic cultures enriched on acetate during periods of carbon deficiency, but it is uncertain how C(1) metabolism affects the role of PHB. In the present study, the type II methanotroph Methylocystis parvus OBBP did not replicate using stored PHB in the absence of methane, even when all other nutrients were provided in excess. When PHB-rich cultures of M. parvus OBBP were deprived of carbon and nitrogen for 48 h, they did not utilize significant amounts of stored PHB, and neither cell concentrations nor concentrations of total suspended solids changed significantly. When methane and nitrogen both were present, PHB and methane were consumed simultaneously. Cells with PHB had significantly higher specific growth rates than cells lacking PHB. The addition of formate (a source of reducing power) to PHB-rich cells delayed PHB consumption, but the addition of glyoxylate (a source of C(2) units) did not. This and results from other researchers suggest that methanotrophic PHB metabolism is linked to the supply of reducing power as opposed to the supply of C(2) units for synthesis.     

Expression of a Rhizopus oryzae lipase in Pichia pastoris under control of the nitrogen source-regulated formaldehyde dehydrogenase promoter

A Rhizopus oryzae lipase gene has been expressed in Pichia pastoris as a reporter using the formaldehyde dehydrogenase 1 promoter (PFLD1) of this organism, which has been reported to be strongly and independently induced by either methanol as sole carbon source or methylamine as sole nitrogen source. Levels of lipase expressed and secreted under the control of the PFLD1 at different induction conditions have been compared to those obtained with the commonly used alcohol oxidase 1 promoter (PAOX1) in small (shake flask) and 1l bioreactor batch cultures. PFLD1-controlled heterologous gene expression was strongly repressed by excess of either glycerol or glucose-but not sorbitol-during growth using methylamine both as sole nitrogen source and inducing substrate. Co-induction of PFLD1 with methanol and methylamine resulted in a synergistic effect on extracellular lipase expression levels. In all tested conditions, the substitution of ammonium for methylamine as carbon source provoked a clear decrease in the specific growth rate and yield of biomass per gram of carbon source. Overall, this study demonstrates that the PFLD1 promoter is at least as efficient as the PAOX1 for extracellular expression of heterologous proteins in P. pastoris bioreactor cultures and provides a first basis for the further design of methanol-free high cell density fed-batch cultivation strategies for controlled overproduction of foreign proteins in P. pastoris.     

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

The application of compound bi-directional flow diffusion chemostats to the study of microbial interactions

Abstract A multi-stage bi-directional chemostat system has been developed in which solutes but not cells are allowed to diffuse between the individual growth chambers which are separated by 0.2 micron pore size polyvinyledene difluoride membranes. The experimental system enables the generation of physico-chemical gradients which, together with the spatial separation of the individual microbial processes, provides a useful laboratory model to study microbial interactions. This paper describes the construction of a multi-stage diffusion chemostat and its application in studying carbon flow in anaerobic estuarine sediments. Populations of Clostridium butyricum, Desulfovibrio desulfuricans and Chromatium vinosum were grown in the compound diffusion chemostat at a dilution rate of 0.03 h⁻¹ at 25°C, and the effects of inorganic nitrogen source and availability on carbon flow and individual cell populations were determined. C. butyricum and D. desulfuricans both used NO⁻₃ as an e⁻ acceptor with an increase in cell numbers. Under these growth conditions, free S²⁻ concentrations were lower, resulting in more stable cell populations than in comparable cultures grown on NH⁺₄ as nitrogen source.