Phosphatases; origin,characteristics and function in lakes

Phosphatases catalyze the liberation of orthophosphate from organic phosphorus compounds. The total phosphatase activity in lake water results from a mixture of phosphatases localized on the cell surfaces of algae and bacteria and from dissolved enzymes supplied by autolysis or excretion from algae, bacteria and zooplankton. External lake water phosphatases usually have pH optima in the alkaline region. Acid phosphatases generally seem to be active in the internal cell metabolism. The synthesis of external alkaline phosphatases is often repressed at high phosphate concentrations and derepressed at low phosphate concentrations. Phosphatase activity has therefore been used as a phosphorus deficiency indicator in algae and in natural plankton populations. The possibilities for this interpretation of phosphatase activity in lake water are limited, however, and this is discussed. The in situ hydrolysis capacity, i.e. the rate by which orthophosphate is released from natural substrates, is unknown. However, we advocate that this process is important and that the rate of substrate supply, rather than phosphatase activity, limits the enzymatic phosphate regeneration.     

Preferential feeding behaviour of Daphnia magna

Preferential feeding behaviour by Daphnia magna was shown when Daphnia were fed on a mixture of ¹⁴C-labelled algae (Chlorella vulgaris or Scenedesmus quadricauda) and ³H-labelled bacteria (Escherichia coli). Daphnia consumption of bacteria was equal or higher in the presence of algae. On the other hand, in the presence of bacteria, algal consumption decreased by 40–70% compared to algal consumption in the absence of bacteria. ¹⁴C radioactive uptake was in good agreement with the chlorophyll content and demonstrates the preferential feeding behaviour of Daphnia.     

Bacteria and phosphorus regeneration in lakes. An experimental study

The aerobic decomposition of the green alga Chlamydomonas reinhardii by a mixed population of lake bacteria was studied in batch and chemostat cultures. Bacterial chemostats were supplied with continuously heatkilled algae. The dead algae rapidly released most of their phosphorus as SRP. In the batch experiments bacteria acted as consumers of the released algal phosphorus. This phosphorus uptake was dependent on the C:P ratio of the algae. During the death phase of the bacteria most of the bacterial phosphorus itself was released. The continuous supply of energy in form of dead algae in the chemostat experiments prevented the death phase of the bacteria and thus any net regeneration of phosphorus. The influence of the C:P stoichiometry of algae and bacteria on the regeneration of algal phosphorus is discussed.     

Phosphorus transformations in the epilimnion of humic lakes: biological uptake of phosphate

SUMMARY. 1. The hypothesis that dissolved humic material (DHM) stimulates bacterial involvement in phosphorus transformations and may thus lead to decreased accessibility of phosphorus to algae was investigated by studying three small forest lakes in southern Finland representing a wide range of concentrations of DHM. 2. Other chemical differences between the three lakes were slight, although the most humic lake exhibited higher concentrations of total phosphorus and of molybdate-reactive phosphorus. Bacterial biomass did not differ significantly between the lakes, but algal biomass was significantly lower at higher DHM concentrations. Consequently the ratio of algal biomass to bacterial biomass was significantly lower in the most humic lake. 3. Uptake of phosphorus from added ³³PO₄ was partitioned between algal and bacterial size fractions by differential filtration. No significant variation between lakes was found in the proportion of particulate ³³P recovered from the algal fraction. 4. Turnover times for phosphate were significantly longer in the most humic lake and also showed lower variability. In general turnover times were long in comparison with values reported from many other lakes. Only briefly in mid summmer did turnover times in two of the lakes shorten to values which would indicate that demand for phosphate was outstripping supply. 5. Short-term storage of samples from the most humic lake stimulated biological incorporation of ³³P, but additions of nitrogen and iron had little effect on phosphate uptake. 6. In these small forest lakes it is probable that no single nutrient consistently limits plankton development. Since no evidence was found that DHM shifts the balance of plankton phosphate uptake away from algae towards bacteria, the influence of DHM on phosphorus transformations may rather be through chemical regulation of free phosphate availability.     

Phosphate uptake by blue green algae in vitro and in a lake during an algal bloom: Useful application of a force-flow relationship

A bioenergetic model, developed by M. Thellier, was applied to the ³²P-phosphate uptake data obtained with blue green algae and allowed the determination of a threshold concentration for the absorption of phosphate. Data obtained during an algal bloom of Oscillatoria rubescens showed that algal growth ceased when the phosphate concentration in the lake dropped below this threshold value. The resulting starvation led to an increase in the permeability of the membrane for phosphate.     

海水中藻菌共培养体系对碳氮磷的吸收转化

海洋环境中,细菌和微藻之间的物质交换是生源要素在自然界中迁移转化的重要方式。为进一步了解生源要素的生物地球化学循环,在实验室模拟条件下,研究了共培养体系中营养盐和有机物在细菌和微藻之间的转换。通过纯培养中肋骨条藻(Skeletonema costatum)、东海原甲藻(Prorocentrum donghaiense)、天然海水中的细菌以及藻菌混合培养,分析了营养盐和有机物随藻菌生物量的变化情况,并计算了溶解有机碳(DOC)和溶解有机氮(DON)的浓度比值[(DOC/DON)a]。结果发现,在共培养体系中,细菌对中肋骨条藻的生长有抑制作用,对东海原甲藻影响不明显;中肋骨条藻有利于细菌生长,东海原甲藻抑制细菌生长,这种不同可能与微藻的粒径有关。海洋细菌在2种藻的指数生长均期均会促进微藻吸收氨氮(NH_4-N),但在生长末期NH_4-N以释放为主。硝氮(NO_3-N)的浓度与藻的生长呈负相关,但在衰亡期NO_3-N略有增加,表明NO_3-N再生所需时间较长。细菌对硝氮的吸收量较少,但对其再生有贡献。细菌和中肋骨条藻对磷酸盐(PO_4-P)的吸收存在竞争,但与东海原甲藻的竞争关系不明显。不同培养体系中DOC浓度变化不同,在藻菌共培养体系中增加较快,纯藻培养体系中增加缓慢,在纯菌培养体系中缓慢减少。通过对DOC与DON浓度比值的分析,发现用判断颗粒有机碳(POC)来源的方法可以分析DOC的来源。     

Algal photosynthetic aeration increases the capacity of bacteria to degrade organics in wastewater

Wastewater treatment is an energy-intensive process and a net emitter of greenhouse gas emissions. A large fraction of these emissions is due to intensive aeration of aerobic bacteria to facilitate break-down of organic compounds. Algae can generate dissolved oxygen at levels in excess of saturation, and therefore hold the potential to partially displace or complement mechanical aeration in wastewater treatment processes. The objective of this study was to develop an internally consistent experimental and modeling approach to test the hypothesis that algal photosynthetic aeration can speed the removal of organic constituents by bacteria. This framework was developed using a simplified wastewater treatment process consisting of a model bacteria (Escherichia coli), a model algae (Auxenochlorella protothecoides), and a single carbon source that was consumable by bacteria only. This system was then tested both with and without the presence of algae. A MATLAB model that considered mass transfer and biological kinetics was used to estimate the production and consumption of O₂ and CO₂ by algae and bacteria. The results indicated that the presence of algae led to 18–66% faster removal of COD by bacteria, and that roughly one-third of biochemical oxygen demand was offset by algal photosynthetic aeration.     

MIXOTROPHIC ALGAE CONSTRAIN THE LOSS OF ORGANIC CARBON BY EXUDATION1

Algae of various taxonomic groups are capable of assimilating dissolved organic carbon (DOC) from their environments (mixotrophy). Recently, we reported that, with increasing biomass of mixotrophs, heterotrophic bacteria did not increase. We hypothesized that algal uptake of external DOC may outweigh their release of DOC by exudation (H1). Here, we addressed an alternative hypothesis that algae did not assimilate external DOC but constrained the release of DOC (H2). In chemostat experiments, we cultured the mixotrophic Chlamydomonas acidophila Negoro together with heterotrophic bacteria. As external substrates, we used glucose, which was potentially available for both bacteria and algae, or fructose, which was available only for bacteria. We increased the biomass of algae by the stepwise addition of phosphorus. Bacterial biomass did not increase in experiments using glucose or when fructose was offered, suggesting that mechanisms other than algal mixotrophy (H1) kept concentrations of bacteria low. Measured exudation rates (percent extracellular release, PER) of mixotrophic algae (Cd. acidophila, Chlorella protothecoides W. Krüger) were very low and ranged between 1.0% and 3.5% at low and moderately high phosphorus concentrations. In contrast, an obligately phototrophic alga (Chlamydomonas segnis H. Ettl) showed higher exudation rates, particularly under phosphorus limitation (70%). The results support H2. If mixotrophy is considered as a mechanism to recycle organic exudates from near the cell surface, this would explain why algae retained mixotrophic capabilities although they cannot compete with bacteria for external organic carbon.     

Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study

Removal of nitrogen and phosphorus from wastewater by two green microalgae (Chlorella vulgaris and Scenedesmus rubescens) was investigated using a novel method of algal cell immobilization, the twin-layer system. In the twin-layer system, microalgae are immobilized by self-adhesion on a wet, microporous, ultrathin substrate (the substrate layer). Subtending the substrate layer, a second layer, consisting of a macroporous fibrous tissue (the source layer), provides the growth medium. Twin-layers effectively separate microalgae from the bulk of their growth medium, yet allow diffusion of nutrients. In the twin-layer system, algae remain 100% immobilized, which compares favourably with gel entrapment methods for cell immobilization. Both microalgae removed nitrate efficiently from municipal wastewater. Using secondary, synthetic wastewater, the two algae also removed phosphate, ammonium and nitrate to less than 10% of their initial concentration within 9 days. It is concluded that immobilization of C. vulgaris and S. rubescens on twin-layers is an effective means to reduce nitrogen and phosphorus levels in wastewater.     

Continuous culture of hydrocarbon-rich microalga Botryococcus braunii in secondarily treated sewage

The hydrocarbon-rich green microalga, Botryococcus braunii, was grown on secondarily treated sewage (STS) in a continuous bioreactor system. The algal biomass increased at a sustained rate of 196 mg dry weight/l per week for 1 month. The hydrocarbon content of algae grown on STS (49%) compared well with that of algae grown on an artificial medium. The concentrations of nitrate and phosphate ions in STS decreased from 5.5 to 4.0 mg nitrogen/l and 0.08 to 0.03 mg phosphorus/l, respectively, by algal consumption. STS would thus appear to suffice as an appropriate medium for continuously sustainable growth of B. braunii and the algal consumption of nitrate and phosphate should help in removing these ions from STS.     

NITRATE AND PHOSPHATE UPTAKE INTERACTIONS IN A MARINE PRYMNESIOPHYTE1

The short- and long-term uptake of nitrate and phosphate ions, and their interactions, were studied as functions of the preconditioning of Pavlova lutheri (Droop) Green. Populations were preconditioned in continuous culture at a variety of growth rates and N:P supply ratios. The maximum uptake rates cell^?1 for nitrate and phosphate were of similar magnitudes, in spite of the forty-fold smaller requirement for phosphorus. Short-term phosphate uptake was independent of the nitrate concentration, but the short-term nitrate uptake rate was reduced in the presence of phosphate. The severity of inhibition of nitrate uptake by phosphate was positively correlated with the preconditioning N:P supply ratio and the preconditioning growth rate. In response to large additions of nutrients, P. lutheri was able to increase its phosphorus content sixty-fold, but was only able to take up enough nitrate to double its nitrogen content. The high rate of phosphate uptake relative to its requirement, the inhibition of nitrate uptake by phosphate, and the large capacity for phosphorus storage relative to its requirement, all of which were observed even under N limitation, may imply that even where nitrogen is limiting there can be interspecific competition for available phosphate.     

Growth promoting and inhibiting effects of extracellular substances of soil microalgae and cyanobacteria on Escherichia coll and Micrococcus luteus

Different taxa of chlorophycean, trebouxiophycean and xanthophycean soil microalgae and of cyanobacteria have been tested for the release of substances that inhibit the growth of either Echerichia coli (Migula) Castellani et Chalmersor Micrococcus luteus (Schroeter) Cohn. Experiments suggest two types of antibacterial effects: one type is constitutive; that is, the antibacterial activity is always present in the algal culture medium, as is the case with the Chroococcus turgidus (medium that inhibits the growth of Escherichia coli). The other type is induced; that is, the antibacterial activity occurs only when algae are in contact with bacteria. This is the case when growth of Micrococcus luteus is inhibited in co‐culture with Chroococcus turgidus (Kützing) Nägeli or with Xanthonema debile (Vischer) Silva and when growth of Escherichia coll is inhibited in co‐culture with Tetracystis sp. As well as inhibition, promotion of bacterial growth was observed. This was probably an unspecific effect resulting from soluble organic and inorganic substances, such as carbohydrates, that are generally present in algal cultures.     

Phosphate Uptake by Eukaryotic Algae in Cultures and by a Mixed Phytoplankton Population in a Lake: Analysis by a Force-Flow Relationship*

The phosphate uptake behaviour of monospecific cultures of green algae in the laboratory and of mixed phytoplankton populations in a mesotrophic lake has been analyzed with the aid of a force-flow relationship. This analysis yields two parameters:

• 1 A conductivity coefficient, that characterizes the activity of the phosphate uptake system.
• 2 An external threshold phosphate concentration, below which uptake of phosphate is excluded on energetic grounds.

When the phosphate concentration lies below the threshold value, the algae show an activation of the uptake system, reflected in an increase in the conductivity coefficient. Correspondingly, excess phosphate above the threshold value leads to a diminuation of the conductivity. Using this simple analysis, phosphate discharge into lake water may be readily monitored.     

The Pho Regulons of Bacteria

Bacterial Pho regulons contain genes whose products are involved in the transport and assimilation of inorganic phosphate. The expression of these genes is regulated by a specific two-component signal transduction system. The paper summarizes data on the organization and function of Pho regulons in gram-negative and gram-positive bacteria, with particular emphasis on the Pho regulons of the best studied bacteria Escherichia coli and Bacillus subtilis.     

Growth and phosphate uptake kinetics of the toxic dinoflagellate Alexandrium tamarense from Hiroshima Bay in the Seto Inland Sea, Japan

Shellfish poisoning by the toxic dinoflagellate Alexandrium tamarense (Lebour) Balech occurred for the first time in Hiroshima Bay, Japan, in 1992. Oyster culture in the bay produces as much as 60% of the total production in Japan, and it suffered severe damage. In the present study, we experimentally investigated the growth rate and phosphate uptake kinetics of A. tamarense, Hiroshima Bay strain. A short-term phosphate uptake experiment revealed that the maximum uptake rate was 1.4 pmol P cell^-1 per h and the half-saturation constant was 2.6 umol L^-1. In semicontin-uous culture, the maximum specific growth rate and the minimum phosphorus cell quota were 0.54 day^-1 and 0.56 pmol P cell^-1, respectively. These uptake rates suggest that A. tamarense is a poor phosphorus competitor compared with other species. However, the large phosphorus storage capacity (Q_pmax/q_o= 36), the surge phosphorus uptake ability (V_s/V_i= 4.1) and the low growth rate would be advantageous for surviving brief periods of phosphorus limitation which frequently occur in Hiroshima Bay.     

Nutrient Utilization Strategies of Algae and Bacteria after the Termination of Nutrient Amendment with Different Phosphorus Dosage: A Mesocosm Case

The impacts of nutrient amendment termination on the growth strategies of algae and bacteria were conducted in experimentally designed mesocosm in which two different phosphorus (P) dosages were treated. The algal community composition did not change greatly in Group A (low phosphorus) and Group B (high phosphorus). In Group A, the secretion of bacterial alkaline phosphatase (AP) after nutrient termination stimulated bacterial phosphorus acquisition, which caused the decrease in algal phosphorus levels, in terms of the increase of bacterial abundance and bacterial production, as well as the decrease in chlorophyll a and particulate organic carbon. The algal collapse resulted in dissolved organic carbon secretion, further fuelling bacterial growth. In Group B, excess phosphorus input urged algae to store phosphorus as poly-phosphate. When phosphorus input ceased, in order to maintain their used high phosphorus demand, algae strengthened to gain phosphorus through the hydrolysis of dissolved organic phosphorus in water column and ploy-phosphate inside the cells by AP, evidenced by high algal alkaline phosphatase activity, algal growth continuation, and bacterial growth decline. These facts indicated that phosphorus content should reduce to a lower level than expected, so that algal bloom can be effectively controlled in eutrophic water bodies.     

The loss of culturability by Escherichia coli cells in seawater depends on availability of phosphate ions and phosphate transport systems

Using strains with or without the PhoE porin or different components of the phosphate regulon, we determined that maintenance of the culturability of Escherichia coli in seawater depended significantly on the presence of structures allowing access of phosphate ions to the periplasm, then to the cytoplasm of cells. Cells totally deprived of the two main phosphate transport systems (Pit, Pst) exhibited the highest loss of culturability. Most of this effect resulted from the loss of the high-affinity Pst system, and more specifically that of the periplasmic phosphate-binding protein PhoS. Survival was enhanced in seawater supplemented with phosphate (0.5 mm), whether or not these structures were present. From an ecological point of view, it is assumed that the presence of phosphate ions, even at low concentrations, can influence the behavior of E. coli cells in seawater. Offprint requests to: M.J. Gauthier     

Phosphate exchange in the pit transport system in Escherichia coli.

The Pit system of phosphate transport in Escherichia coli catalyzes a rapid exchange between the external inorganic phosphate and internal phosphate pools, including some ester phosphates which are in rapid equilibrium with the internal Pi pool. Unlike net energized uptake, the Pi exchange proceeds in energy-depleted cells in the presence of uncouplers and is not accompanied by the movement of potassium ions. In the absence of externally added phosphate, the exit of Pi from the cells is insignificant. The apparent Km for external Pi in the exchange reaction is about 7 mM (2 orders of magnitude higher than that of energized uptake), but the maximal velocity is about the same. The exchange is temperature sensitive and is affected by thiol reagents. The combined observations suggest the operation of a facilitator which is part of the Pit system. The exchange is repressed in cells grown on glucose and other phosphotransferase system substrates, but not in cells grown on other carbohydrate sources. The repression can be reversed by the addition of cyclic AMP to the medium.     

Glucose dehydrogenase of a rhizobacterial strain of <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> involved in mineral phosphate solubilization shares properties and sequence homology with other members of enterobacteriaceae

Glucose dehydrogenase (GDH) of Gram-negative bacteria is a membrane bound enzyme catalyzing the oxidation of glucose to gluconic acid and is involved in the solubilization of insoluble mineral phosphate complexes. A 2.4 kb glucose dehydrogenase gene (gcd) of Enterobacter asburiae sharing extensive homology to the gcd of other enterobacteriaceae members was cloned in a PCR-based directional genome walking approach and the expression confirmed in Escherichia coli YU423 on both MacConkey glucose agar and hydroxyapatite (HAP) containing media. Mineral phosphate solubilization by the cloned E. asburiae gcd was confirmed by the release of significant amount of phosphate in HAP containing liquid medium. gcd was over expressed in E. coli AT15 (gcd::cm) and the purified recombinant protein had a high affinity to glucose, and oxidized galactose and maltose with lower affinities. The enzyme was highly sensitive to heat and EDTA, and belonged to Type I, similar to GDH of E. coli.     

Uptake of phosphate and inorganic nitrogen by a sediment-algal system in a subarctic lake

SUMMARY.

• 1 The uptake of phosphate and inorganic nitrogen by sediment and phytoplankton was studied under natural conditions (1977) and during lake fertilization with phosphorus and nitrogen (1978–79) in Lake Gunillajaure, a small, stratified, subarctic lake in northern Sweden. The experiments were performed in situ in plexiglass cylinders, to which additions of nutrients were made, and the uptake followed by consecutive sampling and analysis of the water phase.
• 2 Additions of HgCl₂ to the experimental vessels reduced the phosphate uptake to the sediment to less than 10% and it could therefore be concluded that the sediment uptake was mainly of biological nature.
• 3 Dark assimilation was 30–40% of that in light. Since light clearly stimulated the sediment uptake the epipelic algae were probably responsible.
• 4 The phosphate uptake to the sediment could be described by Michaelis-Menten kinetics and the calculated constants (V_max, ks) were very alike in 1977 and 1978 but appeared to have increased in 1979.
• 5 The sediment uptake of ammonium and nitrate was very slow indrcating that the epipelic algae were not nitrogen starved.
• 6 Even though the epipelic algae had a potential for efficient uptake of phosphorus, the phytoplankton took up 92–96% of the phosphate added to the lake on each fertilization occasion due to the relatively large water volume in the epilimnion in relation to the bottom area available for the epipelic algae.