A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

Microcalorimetric investigations of the metabolism of yeasts VII. Flow-calorimetry of aerobic batch cultures

Summary The heat evolution of aerobic batch cultures of growing yeast (Saccharomyces cerevisiae) in glucose media was investigated by a combination of a flow-microcalorimeter with a fermentor vessel. The course of heat production, cell production and the rate of oxygen consumption were qualitatively the same for all glucose concentrations between 10 mM and 100 mM. Under optimal aerobic conditions a triphasic growth was observed due to the fermentation of glucose to ethanol, respiration of ethanol to CO₂ and acetate, and respiration of acetate to C0₂. Energy and carbon were found to be in balance for all glucose concentrations.     

High cell density production of <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> under optimized conditions

Deinococcus radiodurans is a bacterium being investigated for mechanisms of extreme radiation resistance and for bioremediation of environmental radioactive waste sites. In both fundamental and applied research settings, methods for large-scale production of D. radiodurans are needed. In this study, a systematic investigation was carried out to optimize D. radiodurans production at the 20-L fermentor scale. In defined medium, the phosphate buffer typically used was found to be inhibitory to D. radiodurans growth, and caused cell aggregation. Substitution of HEPES and MOPS buffers for phosphate buffer improved D. radiodurans growth characteristics. Several antifoaming agents were investigated to support large-scale production with submerged aeration, and the defoamer KFO 673 was chosen based on its ability to prevent foaming without affecting D. radiodurans growth. The conventional undefined rich medium tryptone/glucose/yeast extract (TGY) maximally supported D. radiodurans growth to an OD₆₀₀ of 10. Using a ‘design of experiments’ approach, we found glucose, Mg and Mn to be critical in supporting high-density growth of D. radiodurans. The optimal pH and temperature for D. radiodurans growth in large-scale preparations were 7.0 and 37°C, respectively. Growth was carried out in a 20-L fermentor using the newly developed media under the optimal conditions. With addition of 10 g/L glucose, 0.5 g/L MgSO₄ · 7H₂O, 5 μM MnCl₂ into TGY media, an OD₆₀₀ of 40 was achieved.     

Pyranose 2-oxidase (P2O): Production from Trametes versicolor in Stirred Tank Reactor andits Partial Characterization

Abstract

Optimization of pyranose-2-oxidase (P2O) production conditions from Trametes versicolor was carried out in shaking cultures containing glucose, malt, and yeast extracts; the optimum concentration values were found to be 1.5% glucose, 1.0% yeast extract, and 1.0% malt extract, pH 5.0, temperature, 26°C, and agitation rate 150 rpm. For the first time, P2O production was also carried out in a stirred tank reactor (STR) with 2.2 L working volume in the optimized medium composition, and biomass, P2O activity, protein, nitrogen and glucose concentrations were also monitored besides pH and dissolved oxygen (DO). In the STR, P2O activity peaked on day 9. Partial enzyme characterization occurred and optimum pH and temperature were detected as 7.0 and 37°C, respectively. K _m value was found to be 1.009 mM.     

Oxygen Consumption by Pediococcus halophilus

The behavior toward oxygen of several strains of Pediococcus halophilus was studied. Although these organisms are generally regarded as facultative anaerobes, this investigation showed that resting cells of P. halophilus consumed oxygen at the expense of p-giucose or L-lactate as substrate.

The oxygen consuming activities among strains of soy pediococci varied from 7.06 to 11.63 (nmol/min/mg dry cells) with glucose and 5.52 to 6.59 with L-lactate, respectively. Oxidative metabolism of glucose increased acetate production with a corresponding decrease in lactate formation. Lactate oxidation with O₂. led to the formation of acetate. The oxygen consuming activity was not inhibited by any of the respiratory inhibitors tested such as KCN or NaN₃

NADH oxidase activity was found iri cell-free extracts of P. halophilus No, 51, which is capable of lowering the redox potential of the growth medium. A direct correlation between the abilities to consume oxygen and to reduce the redox potential has not been found so far, but this enzyme is considered to be involved in the aerobic metabolism.     

Studies on Production of Biotin by Microorganisms

The utilization of hydrocarbons by microorganisms was studied in many fields, but the production of biotin vitamers by hydrocarbon-utilizing bacteria has never been reported.

We have screened many hydrocarbon-utilizing bacteria which produce biotin vitamers in the culture broth. The effects of cultural conditions on biotin vitamers production by strain 5–2, tentatively assigned to the genus Pseudomonas, were studied.

More than 98% of biotin vitamers produced from hydrocarbons by strain 5–2 was chromatographically determined as desthiobiotin. As nitrogen source, natural nutrients were more effective than inorganic nitrogen sources. The production of biotin vitamers was increased under the condition of good aeration. Exogenous pimelic or azelaic acid enhanced biotin vitamers production by strain 5–2.

The production of biotin vitamers from n-alkanes, n-alkenes or glucose by an isolated bacterium, strain 5-2, tentatively assigned to the genus Pseudomonas, was investigated. Among these carbon sources, n-undecane was the most excellent for biotin vitamers production.

The biosynthetic pathway of biotin vitamers, especially desthiobiotin, from n-undecane was also studied. It was found by thin-layer and gas-liquid chromatographical methods that pimelic and azelaic acids were the main acid components in n-undecane culture.

This result, together with previously reported enhancement of biotin vitamers production by these acids, suggests that pimelic and azelaic acids may be the intermediates of biotin vitamers biosynthesis from n-undecane.     

Optimization of exopolysaccharide production from Armillaria mellea in submerged cultures

Aims: To study the optimization of submerged culture conditions for exopolysaccharide (EPS) production by Armillaria mellea in shake‐flask cultures and also to evaluate the performance of an optimized culture medium in a 5‐l stirred tank fermenter. Methods and Results: Shake flask cultures for EPS optimal nutritional production contained having the following composition (in g l^?1): glucose 40, yeast extract 3, KH₂PO₄ 4 and MgSO₄ 2 at an optimal temperature of 22°C and an initial of pH 4·0. The optimal culture medium was then cultivated in a 5‐l stirred tank fermenter at 1 vvm (volume of aeration per volume of bioreactor per min) aeration rate, 150 rev min^?1 agitation speed, controlled pH 4·0 and 22°C. In the optimal culture medium, the maximum EPS production in a 5‐l stirred tank fermenter was 588 mg l^?1, c. twice as great as that in the basal medium. The maximum productivity for EPS (Q_p) and product yield (Y_P/S) were 42·02 mg l^?1 d^?1 and 26·89 mg g^?1, respectively. Conclusions: The optimal culture conditions we proposed in this study enhanced the EPS production of A. mellea from submerged cultures. Significance and Impact of the Study: The optimal culturing conditions we have found will be a suitable starting point for a scale‐up of the fermentation process, helping to develop the production of related medicines and health foods from A. mellea.     

Effects of culture conditions on the co-fermentation of a glucose and xylose mixture to ethanol by a mutant of Saccharomyces diastaticus associated with Pichia stipitis

Substrates that contain hexose as well as pentose sugars can form an interesting substrate for the production of ethanol. Pichia stipitis and a respiratory-deficient mutant of Saccharomyces diastaticus were used to convert such a substrate into ethanol under continuous culture conditions. With a sugar mixture (glucose 70%/xylose 30%) at 50 g/l, the xylose was entirely consumed when the dilution rate (D) did not exceed 0.006 h^–1 whereas the glucose was entirely consumed whatever the D. The study of influence of initial substrate concentration (S₀) was performed at D = 0.015 h^–1. Under these conditions the substrate was entirely consumed when its initial concentration did not exceed 20 g/l. With S₀ = 80 g/l the residual xylose concentration reached 20.5 g/l. At low D or at low S₀, P. stipitis was the dominant species in the fermentor. Increasing the D or S₀ resulted in the wash-out of P. stipitis mainly because of its low ethanol tolerance. Correspondence to: J. P. Delgenes     

Enhanced stability of a rumen-derived xylanase using SpyTag/SpyCatcher cyclization

A phosphate solubilizing bacterium ZB was isolated from the rhizosphere soil of Araucaria, which falls into the species Pantoea agglomerans. Optimization for phosphate solubilization by strain ZB was performed. At optimum culture conditions, the isolate showed great ability of solubilizing different insoluble inorganic phosphate sources viz. Ca₃(PO₄)₂ (TCP), Hydroxyapatite (HP), CaHPO₄, AlPO₄, FePO₄ along with rock phosphates (RPs). Inoculation with planktonic cells was found to enhance dissolved phosphorous as compared to that achieved by symplasma inoculation. Besides inoculation with different status of cells, pre-incubation could also exert a great effect on phosphate solubilization ability of P. agglomerans. When isolate ZB was cultured with glucose as carbon sources, phosphorous was more efficiently dissolved from HP and RP without pre-incubation in comparison to that obtained with pre-cultivation. Pre-cultivation, however, was more suitable for P solubilization than no pre-cultivation when bacteria were grown with xylose. A positive correlation was detected between the production of organic acids and phosphate solubilization. P. agglomerans ZB possessed many plant growth promotion traits such as N₂ fixation and production of indole 3-acetic acid, phytase, alkaline phosphatase. Pot experiment showed inoculation with single isolate ZB or biofertilizer prepared from semi-solid fermentation of isolate ZB with spent mushroom substrate (SMS) compost could enhance plant growth with respect to number of leaves, plant leave area, stem diameter, root length, root dry mass, shoot dry mass and biomass when compared to the abiotic control, revealing strain ZB could be a promising environmental-friendly biofertilizer to apply for agricultural field.

     

Pentose Metabolism in Candida utilis

In attempts to obtain GMP producing strains, Brevibacterium ammoniagenes was treated with UV, N.T.G. or D.E.S. as a mutagen. Adenine-guanine requiring mutants were obtained from an adenine-requiring mutant of Brev. ammoniagenes, KY 3482–9 and two of them, presumably adenine-xanthine requiring mutants, were then reverted to mutants which required only adenine for their growth.

Although these revertants were not able to accumulate a copious amount of GMP, most of them and of adenine-guanine requiring mutants produced larger amounts of IMP than the parent adenine-requiring strain.

Effects of Mn²⁺ and purine bases in the medium on IMP production by these mutants were examined and IMP productivities of these mutants were compared with the parent strain under optimal conditions.

These mutagenic treatments were thus proved to be effective for the increase of de novo IMP production by Brev. ammoniagenes mutants.

Brevibacterium ammoniagenes ATCC 6872 accumulates 5′-GDP and -GTP, or 5′-ADP and -ATP together with GMP or AMP in nucleotide fermentation by salvage synthesis.

With cell free extract of this strain, transphosphorylating reactions of AMP or GMP were investigated.

ATP-AMP transphosphorylating enzyme(s) was partially purified to 21.7 fold with acid treatment, salting-out and column chromatography.

In ATP-AMP and ATP-GMP transphosphorylating reactins, optimal conditions were decided such as for concentrations of enzyme, of MgCl₂ and of phosphate donor, pH and cell age as the enzyme sources.

Specificities of phosphate donors and acceptors were examined with both the partially purified enzymes or the sonicate. AMP and GMP were phosphorylated by ATP rapidly, but IMP and XMP were not, therefore supporting our previous finding that Brev. ammoniagenes could not accumulated IDP, ITP, XDP and XTP in IMP and XMP fermentation, respectively.

Although ATP was the best donor for both AMP and GMP phosphorylations, other nucleoside triphosphates and PRPP were used as phosphate donors.

Furthermore, phosphorylation of ADP to ATP was investigated and possible mechanisms of nucleoside di- or triphosphates synthesis in the nucleotide fermentation were discussed.

From these results, it is suggested as a possible mechanism for nucleoside di- and triphosphate accumulation by Brev. Ammoniagenes, that a nucleoside monophosphate formed is phosphorylated to a nucleoside di-phosphate with ATP or other phosphate donors and then the nucleoside diphosphate is converted to a triphosphate with these phosphate donors.

Both AMP and GMP were transphosphorylated rapidly to the corresponding nucleoside-diphosphates and triphosphates by ATP and by other high energy phosphate compounds with cell free extracts of Brevibacterium ammoniagenes.

Some enzyme inhibitors, such as metals and PCMB were shown to inhibit the phosphorylations of AMP and GMP. Higher levels of ATP, ADP, GTP and GDP also inhibited the activity of the partially purified ATP-AMP transphosphorylating enzyme(s).

In guanine nucleotides fermentation by salvage synthesis with this strain, addition of these inhibitors to the medium increased the amounts of GMP and total guanine nucleotides accumulated.

On the contrary, supplement of xylene or of other organic solvents to the medium stimulated the accumulation of both GTP and total guanine compouuds in this fermentation. From enzymatic studies, these solvents are presumed to have the ability to change cell permeability.

Such findings give an effective method for controlling the amounts of nucleotides accumulated in these fermentations.     

Improvement of Bacillus thuringiensis Bacteriocin Production Through Culture Conditions Optimization

Abstract

BUPM4 is a Bacillus thuringiensis subsp. kurstaki strain, isolated from Tunisian soil, producing an original bacteriocin named Bacthuricin F4. The optimization of the latter production conditions was carried out under several physicochemical conditions. It was found that the highest bacteriocin activity was reached at low aeration while bacteriocin synthesis yields were strongly reduced at higher ones. A balance between growth and bacteriocin synthesis, both highly dependent on aeration, was taken into account for the overproduction of bacteriocin. Both glucose and glycerol were shown to be necessary for Bacthuricin F4 maximal synthesis. In addition, the optimal carbon/nitrogen ratio for bacteriocin production is 9. In such optimal conditions, more than 4-fold greater bacteriocin production was obtained than when using TSB medium.     

Nutritional and cultural conditions for production of poly-3-hydroxybutyric acid byAzotobacter chroococcum

Free-living nitrogen-fixing bacteria have been identified as a potential source of poly-3-hydroxybutyric acid (PHB). Systematic study of this ability of N₂-fixing organisms has lead to the isolation of an efficient strain, identified asAzotobacter chroococcum. Nutritional requirements and cultural conditions for optimal production of PHB by this strain under laboratory conditions were determined. In N-free liquid medium containing 2% glucose, the strain accumulated PHB up to 68% of its cell dry mass. Glucose and mannitol were found to be the best carbon sources, while organic nitrogen compounds were preferred as nitrogen source. Maximum yield (3.3 g/L) was obtained with 0.2% bactopeptone supplementation. Inorganic phosphate at a concentration suboptimal for growth had some growth-promoting effect. Under oxygen limiting conditions, biomass production was enhanced but a different response was obtained for PHB production.     

Malformin A1 as a Mammalian Toxicant from Aspergillus niger

Culture conditions for guanosine production were studied with Bacillus subtilis MG–1 that exclusively accumulated guanosine. Of components investigated, KH₂PO₄, KC1, Fe⁺⁺, Mn⁺⁺, NH₄NO₃ and sodium glutamate have played important roles for guanosine production. The optimal concentrations in the culture medium were 2.0 g, 0.9 g, 7.5 mg, 7.5 mg, 20. 4 g and 6.0 g per liter, respectively.

In particular, the extremely minor concentration of Mn⁺⁺ 0.01 ppm completely repressed guanosine production although the cells grew sufficiently. Amino acids mixture was necessary for cell growth, but not essential for guanosine production.

Under these conditions, MG–1 accumulated 15 g of guanosine per liter in a weight yield of 18.8% of consumed sugar. However, a large amount of acetoin was also found as a byproduct.     

Culture condition ofPseudomonas aeruginosa F722 for biosurfactant production

Pseudomonas aeruginosa F722 produces a biosurfactant (BS) during its degradation of carbon and hydrocarbon compounds. The culture conditions for upgrading the biosurfactant productivity were investigated. The concentration of the biosurfactant produced byP. aeruginosa F722 was 0.78 g/L in C-medium; however, this increased to 1.66 g/L in BS medium, which was experimentally adjusted to optimal conditions. NaNO₂ was found to be most effective for microbial growth, with an O.D_600nm of 1.18 for 0.1% NaNO₂. Microbial growths, according to the O.D_600nm were 2.53, 2.68, 2.89, and 2.87 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Clear zone diameters (cm), indicating biosurfactant activity, were 9.0, 8.8, 5.7, and 8.5 for glucose, glycerol,n-C₁₀, andn-C₂₂, respectively. Microbial growth was not consistent with the biosurfactant activity. The best biosurfactant activity was found with a C/N ratio of 20. Under optimal culture condition, the average surface tension decreased from 70 to 30 mN/m after 5 days. With aeration of 1.0 vvm, the biosurfactant produced increased to 1.94 g/L (up to 20%) compared to that of 1.66 g/L with no aeration. With aeration, the velocities of glucose degradation during both the log and stationary growth phases increased from 0.25 and 0.18 h⁻¹ to 0.33 and 0.29 h⁻¹, respectively, and the time for the culture to arrive at the maximum clear zone diameter became shorter, from 80 down to 60 h with no aeration.     

Optimization of Fermentation Medium and Conditions for Mycelial Growth and Water-soluble Exo-polysaccharides Production by Isaria farinosa B05

Abstract

The optimal fermentation medium and conditions for mycelial growth and water-soluble exo-polysaccharides production by Isaria farinosa B05 were investigated. The medium components and fermentation conditions were optimized according to the one at a time method, while the concentration of medium components was determined by the orthogonal matrix method. The results showed that the optimal fermentation medium was as follows: sucrose 3.5% (w/v), peptone 0.5%, yeast extract 0.2%, K₂HPO₄ 0.1%, and MgSO₄ 0.05%. The suitable fermentation conditions were as follows: initial pH 7.0, temperature 25°C, medium volume 75 mL/250 mL, inoculum volume 5% (v/v), time 5d. In such optimal nutrition and environmental conditions, the maximal mycelial yield was 2.124 g/100 mL after 4 day's fermentation, while maximal water-soluble exo-polysaccharides production reached 2.144 g/L after 5 day's fermentation.     

Yeast mutants without phosphofructokinase activity can still perform glycolysis and alcoholic fermentation

Summary Mutants of Saccharomyces cerevisiae without detectable phosphofructokinase activity were isolated. They were partly recessive and belonged to two genes called PFK1 and PFK2. Mutants with a defect in only one of the two genes could not grow when they were transferred from a medium with a nonfermentable carbon source to a medium with glucose and antimycin A, an inhibitor of respiration. However, the same mutants could grow when antimycin A was added to such mutants after they had been adapted to the utilization of glucose. Double mutants with defects in both genes could not grow at all on glucose as the sole carbon source. Mutants with a single defect in gene PFK1 or PFK2 could form ethanol on a glucose medium. However, in contrast to wild-type cells, there was a lag period of about 2 h before ethanol could be formed after transfer from a medium with only nonfermentable carbon sources to a glucose medium. Wild-type cells under the same conditions started to produce ethanol immediately. Mutants with defects in both PFK genes could not form ethanol at all. Mutants without phosphoglucose isomerase or triosephosphate isomerase did not form ethanol either. Double mutants without phosphofructokinase and phosphoglucose isomerase accumulated large amounts of glucose-6-phosphate on a glucose medium. This suggested that the direct oxidation of glucose-6-phosphate could not provide a bypass around the phosphofructokinase reaction. On the other hand, the triosephosphate isomerase reaction was required for ethanol production. Experiments with uniformly labeled glucose and glucose labeled in positions 3 and 4 were used to determine the contribution of the different carbon atoms of glucose to the fermentative production of CO₂. With only fermentation operating, only carbon atoms 3 and 4 should contribute to CO₂ production. However, wild-type cells produced significant amounts of radioactivity from other carbon atoms and pfk mutants generated CO₂ almost equally well from all six carbon atoms of glucose. This suggested that phosphofructokinase is a dispensable enzyme in yeast glycolysis catalyzing only part of the glycolytic flux.     

Enhanced production of heteropolysaccharide-7 by <Emphasis Type="Italic">Beijerinckia indica</Emphasis> HS-2001 in repeated batch culture with optimized substitution of culture medium

In this study, a process of removing a half volume of culture broth and replacing it with an equal volume of substituted solution was developed to enhance the production of heteropolysaccharide-7 (PS-7) by Beijerinckia indica HS-2001. The optimal substitution time and volume of the substituted solution were found to be 48 h after cultivation and 50% of the initial volume of the culture broth. The optimal composition of the substituted solution was determined to be 20.0 g/L glucose, 10.0 g/L soybean pomace, 0.1 g/L MgSO₄·7H₂O, 0.9 g/L NH₄NO₃, and 5.0 g/L potassium phosphate, which was the same composition as the medium developed in a previous study for the production of PS-7 by B. indica HS-2001. The total amount and productivity of PS-7 by B. indica HS-2001 with a substitution under optimized conditions in a 7 L bioreactor for 96 h were 49.28 g and 0.51 g/h, respectively, which were 1.76 and 1.31-foldgreater values than those without a substitution for 72 h.     

Optimization of culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Cordyceps militaris in liquid culture

Both crude exo-biopolymers and mycelial biomass, produced by liquid culture of Cordyceps species, are believed to possess several potential health benefits. As a result of its known biological activities, Cordyceps militaris has been extensively characterized in regards to potential medicinal applications. However, optimized liquid culture conditions for enhanced polysaccharide productivity have yet to be developed, which is a necessary step for industrial applications. Therefore, in this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide (EPS) by C. militaris. The effects of medium composition, environmental factors, and C/N ratio were investigated. Among these variables 80 g, glucose; 10 g, yeast extract; 0.5 g, MgSO₄·7H₂O; and 0.5 g, KH₂PO₄ in 1 L distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 24°C, uncontrolled pH, 200 rpm, and 1.5 vvm, respectively. Under these optimal conditions, mycelial growth in shake flask cultures and 5 L jar bioreactors was 29.43 and 40.60 g/L, respectively, and polysaccharide production in shake flask cultures and 5 L jar bioreactors was 2.53 and 6.74 g/L, respectively.     

Studies on Sugar-isomerizing Enzyme

A glucose isomerase which reversibly catalyzes the reaction between d-glucose and d-fructose was demonstrated in the cell-free extracts of a strain of Streptomyces sp. isolated from soil. The enzyme was produced when the strain was grown in the medium containing xylan or xylan-containing material such as wheat bran. A medium which consists of 3% of wheat bran, 2% of corn steep liquor and 0.024% of CoCl₂·6H₂O is recommendabie for the production of the glucose isomerase enzyme with the strain. With the enzyme, some conditions for the conversion of d-glucose to d-fructose were also studied. The method is very useful for the production of invert sugar from d-glucose and is now on the way to be applied to the practical use.     

Light control on phytoplankton production in a shallow and turbid estuarine system

Tagus estuary is one of the largest estuaries of Western Europe. With the aim of unravelling the drivers of primary production in this shallow and turbid nutrient replete estuary, we tested the hypothesis that light availability is a major factor controlling phytoplankton production. Environmental parameters, phytoplankton biomass, community composition, and photosynthetic parameters were monitored at two sites in the estuary during a complete annual cycle. Despite the fact that nutrient concentrations were always above growth-limiting values, Chl a concentrations were relatively low throughout the study period. High water column turbidity, due to riverine inputs, promoted a rapid attenuation of light and created a compressed profile with optimal photosynthetic conditions. Therefore, the phytoplankton community, dominated by small cells, such as diatoms and cryptophycean flagellates, displayed highly photosynthetic efficiency and low light-saturated photosynthetic rates as a photo-acclimation response to low light conditions year-round. Primary production rate was unimodal, peaking in the summer months. In such estuarine system, gross primary production could thus be predicted by an existing robust empirical model based on pigment standing crop (Chl a), surface irradiance (E ₀) and optical depth (Z _eup). Compared to other shallow estuaries, the Tagus can be classified as a low- to moderately productive estuary, being the turbidity-induced low light conditions the principal factor limiting phytoplankton growth.