Production of poly-β-hydroxybutyrate by thermophilic cyanobacterium, Synechococcus sp. MA19, under phosphate-limited conditions

Synechococcus sp. MA19, grown autotrophically under phosphate-limited conditions at 50 °C, produced poly--hydroxybutyrate (PHB) when intracellular phosphate content was 0.043–0.076mmol per g of cellular components. In the culture for 260h using Ca₃(PO₄)₂ as a phosphate source, strain MA19 accumulated PHB at 55% (w/w) of the dry cells and the amount of PHB produced was 2.4gl^–1 which was almost twice that without Ca₃(PO₄)₂ addition.     

Suitability of commercial beet molasses fractions as substrates fro polyhydroxyalkanoate production byAzotobacter vinelandii UWD

Summary Beet molasses that had been fractionated commercially by ion exclusion resulted in two waste-streams: extract molasses (EM) and concentrated separator by-product (CSB). Only EM at 4–5% w/v contained sufficient sugar to promote polyhydroxybutyrate (PHB) formation byAzotobacter vinelandii UWD, but the yield of PHB/protein was less than that obtained in unfractionated beet molasses. EM and especially CSB added at 0.5–2.0% w/v to media containing a variety of sugar sources promoted an increased yield of PHB/protein. The best use of these beet molasses fractions was, therefore, as a minor addition to media containing sugars to increase PHB yield, but not as a primary substrate for PHB production.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii strain UWD during growth on molasses and other complex carbon sources

Summary Azotobacter vinelandii strain UWD formed >2 mg/ml poly--hydroxybutyrate (pHB) during exponential growth in media containing ammonium acetate and 1% w/v glucose, fructose, sucrose, or maltose, and >1.5 mg/ml with 1% w/v sodium gluconate or glycerol. After acetate exhaustion, pHB formation accompanied carbohydrate utilization and pHB rapidly accounted for 53%–70% of the cell mass. Strain UWD also formed >2 mg/ml pHB when it was grown with 2% w/v corn syrup, cane molasses, beet molasses, or malt extract. Beet molasses had a growth stimulatory effect which promoted higher yields of pHB/ml and a high ratio of pHB/protein. Malt extract also promoted higher yields of pHB/ml. In this case, pHB formation was no longer subject to acetate repression and the cells contained a higher ratio of pHB/protein. This study shows that unrefined carbon sources support pHB formation in strain UWD and that the yields of pHB were comparable to or better than those obtained with refined carbon sources.     

Growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] as affected by nitrogen source

A study was conducted to elucidate the effect of N form, either NH₄ ⁺ or NO₃ ^–, on growth and solute composition of the salt-tolerant kallar grass [Leptochloa fusca (L.) Kunth] grown under 10 mM or 100 mM NaCl in hydroponics. Shoot biomass was not affected by N form, whereas NH₄ ⁺ compared to NO₃ ^– nutrition caused an almost 4-fold reduction in the root biomass at both salinity levels. Under NH₄ ⁺ nutrition, salinity had no effect on the biomass yield, whereas under NO₃ ^– nutrition, increasing salinity from 10 mM to 100 mM caused 23% and 36% reduction in the root and shoot biomass, respectively. The reduced root growth under NH₄ ⁺ nutrition was not attributable to impaired shoot to root C allocation since N form did not affect the overall root sugar concentration and the starch concentration was even higher under NH₄ ⁺ compared to NO₃ ^– nutrition. The low NH₄ ⁺ (2 mM) and generally higher amino-N concentrations in NH₄ ⁺- compared to NO₃ ^–-fed plants indicated that the grass was able to effectively detoxify NH₄ ⁺. Salinity had no effect on Ca²⁺ and Mg²⁺ levels, whereas their concentration in shoots was lower under NH₄ ⁺ compared to NO₃ ^– nutrition (over 66% reduction in Ca²⁺; over 20% reduction in Mg²⁺), but without showing deficiency symptoms. Ammonium compared to NO₃ ^– nutrition did not inhibit K⁺ uptake, and the K⁺-Na⁺ selectivity either remained unaffected or it was higher under NH₄ ⁺ than under NO₃ ^– nutrition. Results suggested that while NH₄ ⁺ versus NO₃ ^– nutrition substantially reduced root growth, and also strongly modified anion concentrations and to a minor extent concentrations of divalent cations in shoots, it did not influence salt tolerance of kallar grass.     

Poly(3-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> in fed-batch culture

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l⁻¹). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH₄Cl and K₂HPO₄ on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH₄Cl and 0.22% (w/v) K₂HPO₄ and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l⁻¹, respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH₄Cl, and K₂HPO₄ were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l⁻¹, PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l⁻¹ h⁻¹.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45°C and in 250 g/l molasses although the optimal growth temperature was 35–37°C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25–35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.     

Effect of nutritional conditions on dye removal from textile effluent by <Emphasis Type="Italic">Aspergillus lentulus</Emphasis>

The nutritional conditions supporting growth and maximum dye removal by Aspergillus lentulus have been investigated. Initially a composite media containing yeast extract, glucose and mineral components was used and the effect of various components on dye removal was studied. For maximum dye removal (≈100%), ≥0.5% (w/v) glucose and ≥0.25% (w/v) yeast extract were essential. While glucose played an important role in pellet formation, which in turn was important for dye removal, yeast extract contributed towards higher biomass production. Mineral components (except NH₄NO₃) did not affect dye removal significantly. Next the alternate sources of carbon (molasses, jaggery, starch and sodium acetate) and nitrogen (peptone, urea, ammonium nitrate, sodium nitrate and ammonium chloride) were tested. Among carbon sources, all the sources produced almost complete dye removal in 48 h (more than 97% in 24 h), except sodium acetate (64% in 48 h). All the tested nitrogen sources resulted in >90% dye removal in 48 h. Yeast extract and peptone gave best results with high dye removal rate (9.8 and 8.1 mg/l/h, respectively). However, among the low cost alternates, urea and NH₄Cl came out to be suitable sources due to the high uptake capacity of the biomass produced coupled with high dye removal rate in case of NH₄Cl. Therefore, a combination of urea and NH₄Cl was tested, which produced complete dye removal with a high dye removal rate (10 mg/l/h). Finally the modified composite media containing urea and NH₄Cl as nitrogen sources and glucose as carbon source was utilized for effluent treatment. Results indicated that performance of modified composite media was at par with composite media for supporting growth of A. lentulus and dye removal from the textile effluent.     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Effect of light intensity and ammonium-N on carotenogenesis ofTrentepohlia odorata andDunaliella bardawil

AxenicTrentepohlia odorata was cultured at three different NH₄Cl levels (3.5 × 10^–2, 3.5 × 10^–3, 3.5 × 10^–4 M) and three different light intensities (48, 76, 122 µmol m^–2 s^–1). Chloride had no effect on growth over this range of concentration. High light intensity and high NH₄Cl concentration enhanced the specific growth rate. The carotenoid content increased under a combination of high light intensity and low N concentration. WhenD. bardawil was exposed to the same combination of growth conditions, there was an increase in its carotenoid content. The light saturation and the light inhibition constants (K _s andK _i, respectively) for growth, and the saturation constant (K _m) for NH₄Cl were determined. TheK _s andK _i values were higher inT. odorata (66.7 and> 122 mol m^–2 s^–1, respectively) than inD. bardawil (5.1 and 14.7 µmol m^–2 s^–1, respectively). TheK _m value determined at 122 µmol m^–2 s^–1, however, was lower inT. odorata (0.048 µM) than inD. bardawil (0.062 µM).Author for correspondence     

Cellular and whole-plant chloride dynamics in barley: insights into chloride–nitrogen interactions and salinity responses

The first analysis of chloride fluxes and compartmentation in a non-excised plant system is presented, examining ten ecologically pertinent conditions. The short-lived radiotracer couple ³⁸Cl/³⁹Cl was used as a Cl^– tracer in intact barley (Hordeum vulgare L. cv. Klondike) seedlings, which were cultured and investigated under four external [Cl^–], from abundant (0.1 mM) to potentially toxic (100 mM). Chloride–nitrogen interactions were investigated by varying N source (NO₃ ^– or NH₄ ⁺) and strength (0.1 or 10 mM), in order to examine, at the subcellular compartmentation level, the antagonism, previously documented at the influx level, between Cl^– and NO₃ ^–, and the potential role of Cl^– as a counterion for NH₄ ⁺ under conditions in which cytosolic [NH₄ ⁺] is excessive. Cytosolic [Cl^–] increased with external [Cl^–] from 6 mM to 360 mM. Cl^– influx, fluxes to vacuole and shoot, and, in particular, efflux to the external medium, also increased along this gradient. Efflux reached 90% of influx at the highest external [Cl^–]. Half-times of cytosolic Cl^– exchange decreased between high-affinity and low-affinity influx conditions. The relationship between cytosolic [Cl^–] and shoot flux indicated the presence of a saturable low-affinity transport system (SLATS) responsible for xylem loading of Cl^–. N source strongly influenced Cl^– flux to the vacuole, and moderately influenced Cl^– influx and shoot flux, whereas efflux and half-time were insensitive to N source. Cytosolic pool sizes were not strongly or consistently influenced by N source, indicating the low potential for Cl^– to act as a counterion to hyperaccumulating NH₄ ⁺. We discuss our results in relation to salinity responses in cereals.Abbreviations [Cl^–]_cyt cytosolic chloride concentration - [Cl^–]_o external chloride concentration     

Influence of nitrogen species (NH4 + and NO3 −) on the dynamics of P in water–sediment–Salvinia herzogii systems

Water – Salvinia herzogii – sediment systems were exposed to different phosphorus and nitrogen combinations in outdoor experiments. The aim was to estimate the amounts of P immobilized in macrophytes and sediments, as well as to elucidate whether or not the presence of N affects the retention of P. The following components were added: o-P, o-P + NH₄ ⁺, o-P + NO₃ ^– + NH₄ ⁺, o-P + NO₃ ^–. The concentration of nutrients was periodically determined throughout the experiment (28 days). The concentrations of P and N in plant tissues and sediments were determined at the beginning and the end of the experiment. Sequential extractions of P-fractions in sediment were performed using the EDTA method (Golterman, 1996). The removal efficiency of P in water was 95–99%. The removal of NH₄ ⁺ (97–98%) was more effective than that of NO₃ ^– (44–86%). The presence of nitrogen species increased the removal velocity of o-P from water, NH₄ ⁺ was the most effective species. Sediments not only had higher P removal rates than macrophytes but, in the control treatment without macrophytes, they reached the values obtained by macrophytes plus sediments in the other treatments. The adsorption of P takes place at the surface layer of the sediment (1 cm). Most of the P incorporated into the sediment during the experiment was sorbed by the fraction Fe(OOH)P. The addition of nutrients to water modified the leaves/lacinias weight ratio.     

Ammonia-oxidizing bacteria on root biofilms and their possible contribution to N use efficiency of different rice cultivars

Ammonia-oxidizing bacteria (AOB) populations were studied on the root surface of different rice cultivars by PCR coupled with denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH). PCR-DGGE of the ammonium monooxygenase gene (amoA) showed a generally greater diversity on root samples compared to rhizosphere and unplanted soil. Sequences affiliated with Nitrosomonas spp. tended to be associated with modern rice hybrid lines. Root-associated AOB observed by FISH were found within a discrete biofilm coating the root surface. Although the total abundance of AOB on root biofilms of different rice cultivars did not differ significantly, there were marked contrasts in their population structure, indicating selection of Nitrosomonas spp. on roots of a hybrid cultivar. Observations by FISH on the total bacterial community also suggested that different rice cultivars support different bacterial populations even under identical environmental conditions. The presence of active AOB in the root environment predicts that a significant proportion of the N taken up by certain rice cultivars is in the form of NO₃ ^–-N produced by the AOB. Measurement of plant growth of hydroponically grown plants showed a stronger response of hybrid cultivars to the co-provision of NH₄ ⁺ and NO₃ ^–. In soil-grown plants, N use efficiency in the hybrid was improved during ammonium fertilization compared to nitrate fertilization. Since ammonium-fertilized plants actually receive a mixture of NH₄ ⁺ and NO₃ ^– with ratios depending on root-associated nitrification activity, these results support the advantage of co-provision of ammonium and nitrate for the hybrid cultivar.     

Comparison of ethanol production by Zymomonas mobilis from sugar beet substrates

Summary The potential of four sugar beet substrates from the sugar industry [syrup (S), crystallizer effluent 1 (CE1), crystallizer effluent 2 (CE2) and molasses (M)] were compared for ethanol production using an osmotolerant mutant strain of the bacterium Zymomonas mobilis. Sucrose of the substrates was enzymatically hydrolysed to avoid levan formation during fermentation. Nutrient supplementation experiments have shown that reproducible growth and ethanol production could be obtained on the four substrates supplemented only with magnesium sulphate (CE2 and M) or additionally with ammonium sulphate (S and CE1). Thus, addition of costly yeast extract could be avoided. All 20% (w/v) substrates showed nearly complete sugar conversion (>94.9%), good growth (0.16 h^–1) and ethanol production (>40 g 1^–1). However, sorbitol formation reduced the ethanol yield (73–79% of the theoretical value) significantly. Batch kinetic parameters and studies of instantaneous parameters showed that enhanced osmolality of substrates (SZ. mobilis with appropriate supplementation. Offprint requests to: J. Baratti     

Effects of light quality,CO2 tensions and NO 3 +− concentrations on the inorganic nitrogen metabolism of Chlamydomonas reinhardii

The blue light dependent utilization of nitrate by green algae under common air and high irradiances, besides its assimilatory nature, is associated with the release of NO₂ ^– and NH₄ ⁺ to the culture medium. If the CO₂ content of the sparging air was increased up to 2%, previously excreted NO₂ ^– and NH₄ ⁺ were rapidly assimilated. When under air and high irradiances the cell density in the culture reached values corresponding to 25 g Ch 1.ml^-1, no further growth was observed and the highest values of NO₃ ^– consumption and NO₂ ^– and NH₄ ⁺ release were attained. Besides low CO₂ tensions, increasing NO₃ ^– concentrations in the medium stimulated the release of NO₃ ^– and NH₄ ⁺. Under CO₂-free air the consumption of NO₃ ^– and the release of NO₂ ^– and NH₄ ⁺ on a total N bases were almost stoichiometric and their rates saturated at much lower irradiances than under air. Under CO₂-free air high rates of NO₂ ^– release were only observed under the blue radiations that were effectively absorbed by photosynthetically active pigments, i.e. 460 nm, but not under 404 and 630 nm radiations. However, the simultaneous illumination of the cells with 404 and 630 nm monochromatic light showed a remarkable synergistic effect on NO₂ ^– release.The results are discussed in terms of the close relationship between C and N metabolism, the photosynthetic reducing power required to convert NO _inf3 ^sup± -N into R – NH₂-N and the blue light activation of nitrate reductase.     

Effects of different nitrogen forms and combination with foliar spraying with 6-benzylaminopurine on growth,transpiration, and water and potassium uptake and flow in tobacco

NH₄ ⁺-N can have inhibitory effects on plant growth. However, the mechanisms of these inhibitory effects are still poorly understood. In this study, effects of different N forms and a combination of ammonium + 6-benzylaminopurine (6-BA, a synthetic cytokinin) on growth, transpiration, uptake and flow of water and potassium in 88-days-old tobacco (Nicotiana tabacum L. K 326) plants were studied over a period of 12 days. Plants were supplied with equal amounts of N in different forms: NO₃ ^–, NH₄NO₃, NH₄ ⁺ or NH₄ ⁺+6-BA (foliar spraying every 2 days after onset of the treatments). For determining flows and partitioning upper, middle and lower strata of three leaves each were analysed. During the 12 days study period, 50% replacement of NO₃ ^–-N by NH₄ ⁺-N (NH₄NO₃) did not change growth, transpiration, uptake and flow of water and K⁺ compared with the NO₃ ^–-N treatment. However, NH₄ ⁺-N as the sole N-source caused: (i) a substantial decrease in dry weight gain to 42% and 46% of the NO₃ ^–-N and NH₄NO₃ treatments, respectively; (ii) a marked reduction in transpiration rate, due to reduced stomatal conductance, illustrated by more negative leaf carbon-isotope discrimination (¹³C) compared with the NO₃ ^– treatment, especially in upper leaves; (iii) a strong reduction both in total water uptake, and in the rate of water uptake by roots, likely due to a decrease in root hydraulic conductivity; (iv) a marked reduction of K⁺ uptake to 10%. Under NH₄ ⁺ nutrition the middle leaves accumulated 143%, and together with upper leaves 206% and the stem 227% of the K⁺ currently taken up, indicating massive mobilisation of K⁺ from lower leaves and even the roots. Phloem retranslocation of K⁺ from the shoot and cycling through the root contributed 67% to the xylem transport of K⁺, and this was 2.2 times more than concurrent uptake. Foliar 6-BA application could not suppress or reverse the inhibitory effects on growth, transpiration, uptake and flow of water and ions (K⁺) caused by NH₄ ⁺-N treatment, although positive effects by 6-BA application were observed, even when 6-BA (10^–8 M) was supplied in nutrient solution daily with watering. Possible roles of cytokinin to regulate growth and development of NH₄ ⁺-fed plants are discussed.     

Biofiltering efficiency in removal of dissolved nutrients by three species of estuarine macroalgae cultivated with sea bass (Dicentrarchus labrax) waste waters 2. Ammonium

Three estuarine macroalgae (Ulva rotundata,Enteromorpha intestinalis, Gracilariagracilis) of economic potential were cultivated in the laboratory toassess their biofiltering capacities for ammonium in waste effluents from a seabass (Dicentrarchus labrax) cultivation tank. The studywasdeveloped to investigate the functioning of N nutrition of the three species.Atlow water flow (< 2 volumes d^–1) the three species strippedefficiently the ammonium dissolved in the waste water from the fish tank, withaminimum biofiltering efficiency estimate of 61% in unstarved cultures ofG. gracilis at a water flow of 2 volumesd^–1. Maximum velocity for ammonium uptake (89.0 molNH₄ ⁺ g^–1 dry wth^–1) was found in U. rotundata,whereas G. gracilis showed the highest affinity for thisnutrient. The net ammonium uptake rate was significantly affected by the waterflow, being greatest at the highest flow assayed (2 volumesd^–1). Variations of tissue N and C:N ratios during aflow-through experiment suggested that N was not limiting macroalgal growth.However, when ammonium was supplied at a flow rate of 0.5 volumesd^–1, specially in a three-stage design, the marked reductionintissue N and the biomass C:N:P ratios suggested a more general nutrientdeficiency. A significant correlation was found between growth rates and the Nbiomass gained in the cultures. The three-stage design under low water flow(0.5volumes d^–1) showed that the highest ammonium uptake rates (upto 80.9 mol NH₄ ⁺ g^–1 dry wtd^–1 in U. rotundata) were found inthe first stage, with decreasing rates in the following ones. As a result, lowincrements or even losses of total N biomass in these stages were found,suggesting that ammonium was excreted from the algae. We conclude that thesespecies present a potential ability to biofilter the ammonium dissolved inwastewater from a D. labrax cultivation tank, suggesting thatscaling up the biofiltration designs, future practises using these macroalgaemay be implemented in the local fish farms, resulting in both environmental andeconomical advantages.     

Evidence of an association between poly(3-hydroxybutyrate) accumulation and phosphotransbutyrylase expression in<Emphasis Type="Italic"> Bacillus megaterium</Emphasis>

Molecular analysis of a genomic region of Bacillus megaterium, a polyhydroxybutyrate (PHB)-producing microorganism, revealed the presence of a gene coding for the enzyme phosphotransbutyrylase (Ptb). Enzyme activity was measured throughout the different growth phases of B. megaterium and was found to correlate with PHB accumulation during the late-exponential growth phase. Ptb expression was repressed by glucose and activated by the branched amino acids isoleucine and valine. Overexpression of Act_Bm, a ⁵⁴ regulator from B. megaterium whose gene is located upstream from ptb, caused an increase in Ptb activity and PHB accumulation in B. megaterium.