Experimental demonstration of the roles of bacteria and bacterivorous protozoa in plankton nutrient cycles

We have used a model food chain composed of a natural bacterial assemblage, a pennate diatom and a bacterivorous microflagellate to investigate the factors controlling the relative importance of bacteria and protozoa as sources for regenerated nitrogen in plankton communities. In bacterized diatom cultures in which diatom growth was nitrogen-limited, the carbon:nitrogen (C:N) ratio of the bacterial substrate greatly affected which population was responsible for the uptake of nitrogen. When nitrogen was added as NH ₄ ⁺ and the cultures were supplemented with glucose, the bacteria competed successfully with the algae for NH ₄ ⁺ and prevented the growth of algae by rapidly assimilating all NH ₄ ⁺ in the cultures. Bacterivorous protozoa inoculated into these cultures grazed the bacterial population and remineralized NH ₄ ⁺ , thus relieving the nitrogen limitation of algal growth and allowing an increase in algal biomass. In contrast, bacteria in cultures supplemented with the amino acid glycine (C:N = 2) were major remineralizers of nitrogen, and the influence of protozoan grazing was minimal. We conclude that the relative importance of bacteria and protozoa as nutrient regenerators in the detrital food loop is dependent largely on the overall carbon:nutrient ratio of the bacterial substrate. The role of bacterivorous protozoa as remineralizers of a growth-limiting nutrient is maximal in situations where the carbon:nutrient ratio of the bacterial substrate is high.     

Interactions between the diatom Thallasiosira pseudonanna and an associated pseudomonad in a mariculture system.

The marine diatom Thallasiosira pseudonanna (3H) and several bacteria associated with it were isolated from batch cultures at the University of Delaware mariculture facility. The interaction between the algae and each of the bacteria was investigated. One of the isolates, T827/2B (Pseudomonas sp.), was incapable of surviving in f/2 culture medium unless the algae were present. When the algae and T827/2B were grown together in the f/2 medium, the bacterial growth was stimulated and the algal growth was inhibited. Bacterial filtrate had a similar effect on the algae, indicating that the bacterial effect is an indirect one most likely resulting from the excretion of a harmful compound into the medium. Preliminary characterization of the material excreted by the bacteria indicates that it s proteinaceous in nature. The interactions observed does not fit into any single category of interactions but can be explained as a combination of competition and indirect parasitism.     

Interactions between the diatom Thallasiosira pseudonanna and an associated pseudomonad in a mariculture system

The marine diatom Thallasiosira pseudonanna (3H) and several bacteria associated with it were isolated from batch cultures at the University of Delaware mariculture facility. The interaction between the algae and each of the bacteria was investigated. One of the isolates, T827/2B (Pseudomonas sp.), was incapable of surviving in f/2 culture medium unless the algae were present. When the algae and T827/2B were grown together in the f/2 medium, the bacterial growth was stimulated and the algal growth was inhibited. Bacterial filtrate had a similar effect on the algae, indicating that the bacterial effect is an indirect one most likely resulting from the excretion of a harmful compound into the medium. Preliminary characterization of the material excreted by the bacteria indicates that it s proteinaceous in nature. The interactions observed does not fit into any single category of interactions but can be explained as a combination of competition and indirect parasitism.     

Cage Culture Turbidostat: a Device for Rapid Determination of Algal Growth Rate

The present cage culture turbidostat consists of a growth chamber and a control unit. The microorganisms (photoautotrophic algae) are kept in the growth chamber by porous membranes (pore size 1 to 3 μm) which retain the algae but allow efficient exchange of the growth medium. Flow rate and composition of the medium can therefore be varied independently of algal population density. A reciprocating pumping mode of the medium is introduced to obtain more gentle clearance of membranes than that provided by rotation or stirring in other membrane fermentors. Pulsed light and a light-emitting diode/light-sensitive transistor couple are used to monitor the turbidity of the culture, independent of external light needed for growth. The control unit keeps the turbidity constant by frequent activation of the dilution pump. Theoretical analysis of growth in the turbidostat shows that integrated activation time of the dilution pump is proportional to the growth rate of the organism. Theoretical analysis was also used to determine minimum flow-rate and nutrient concentration of medium to cover the requirement of the algae. Experiments with three different marine diatoms were carried out, and they demonstrated that the growth rate could be determined every hour and that the cultures could be kept at constant turbidity over 10 to 14 days at least.     

Interactions between freshwater bacteria andAnkistrodesmus braunii in batch and continuous culture

Batch and continuous cultures ofAnkistrodesmus braunii were established in an inorganic medium with growth rate limited by P. In batch culture, inoculation of lake water bacterial isolates ofPseudomonas sp. andFlavobacterium sp. showed that thePseudomonas isolate was capable of more rapid growth on algal exudates of lytic products than was theFlavobacterium isolate. When inoculated singly into a continuous culture (D=0.267 day^–1; P level, 2M), theFlavobacterium isolate initially caused a decrease in the population density of the alga, but then steady states for both organisms were obtained. ThePseudomonas isolate under the same conditions caused a rapid washout of the algal culture, and steady-state conditions were never obtained. When thePseudomonas isolate was added to the two-member, steady-state system ofA. braunii andFlavobacterium, the algal population again washed out of the vessel, followed by theFlavobacterium and then thePseudomonas isolate. A transient increase in the P concentration to 200M in the culture vessel caused the low algal population level to increase, followed by increases in the bacterial isolates when the algal population was high enough to supply the required organic carbon source. The system demonstrated that competition for P between the alga and the bacteria can occur, and the results were dependent on the algal and bacterial relative growth rates. The bacterial growth rates were limited initially by organic substrates produced by the alga, and the different bacterial isolates competed for these substrates.     

Development of a novel artificial medium based on utilization of algal photosynthetic metabolites by symbiotic heterotrophs

Aims:  (i) Quantitative and qualitative analyses of photosynthetic metabolites of Chlorella sorokiniana and elucidation of the mechanism of their utilization by algal symbionts. (ii) Development of artificial medium that imitates photoautotroph–heterotroph interaction and investigation of its suitability for isolation of novel microbes from the environment.
Methods and Results:  Various components, including free dissolved carbohydrates, nitrogenous compounds and vitamin, were detected and together contributed 11·1% (as carbon content) of the total photosynthetic metabolites in the medium. Utilization of these photosynthetic metabolites in algal culture broth by algal symbionts was studied. Many symbionts showed specific utilization patterns. A novel artificial extracellular released organic carbon medium, which imitated the nutritional conditions surrounding algae, was developed based on the pattern of utilization of the algal metabolites by the symbiotic heterotrophs. About 42·9% of the isolates were closely related to photoautotrophic-dependent and oligotrophic bacteria.
Conclusions:  With the novel artificial medium, it was possible to selectively isolate some bacterial strains.
Significant and Impact of the Study:  Synthetic bacterial growth medium is an important and basic tool for bacterial isolation from environmental samples. The current study shows that preferential separation of typical bacterial subset can be achieved by using artificial medium that mimics photosynthetic metabolites.     

A BATCH CULTURE METHOD FOR MICROALGAE AND CYANOBACTERIA WITH CO2 SUPPLY THROUGH POLYETHYLENE MEMBRANES1

A new method for CO₂ supply to photoautotrophic organisms was developed, and its applicability for measuring specific growth rates in shaken batch cultures of cyanobacteria and unicellular algae was shown. Small bags containing a concentrated carbonate buffer with a CO₂ partial pressure of 32 mbar were prepared from a thin foil of low density polyethylene (LDPE). These bags were inserted as CO₂ reservoirs (CRs) into polystyrene culture flasks with gas‐permeable screw caps, which were suitable to photometric growth measurement. CO₂ was released directly into the medium with membrane‐controlled kinetics. The CRs were not depleted within 1 week, although the atmosphere in the culture vessel exchanged rapidly with the ambient air. Rates of initial growth and final densities of the cultures of six different unicellular algal species and one cyanobacterium were markedly increased by diffusive CO₂ supply from the CR. In the presence of a CR, growth was exponential during the first 2 d in all cultures studied. The method described allowed a high number of measurements of specific growth rates with relatively simple experimental setup.     

Photobioreactor engineering: Design and performance

This review summarizes the recent advances in high-density algal cultures in the field of algal biotechnology. Photobioreactor engineering for economical and effective utilization of algae and its products has made impressive and promising progress. Bioprocess engineers have expedited the design and the operation of algal cultivation systems. Many of them in use today are open systems due to cost considerations, and closed photobioreactors have recently attracted a considerable attention for the production of valuable biochemicals or for special applications. For high-density cultures, the optimization of environmental factors in the photobioreactors have been explored, including light delivery, CO₂ and O₂ gas transfer, medium supply, mixing and temperature. It is expected that further advanced photobioreactor engineering will enable the commercialization of noble algal products within the next decade.     

CYANOPHYTA-BACTERIA SYSTEMS: EFFECTS OF ADDED CARBON COMPOUNDS OR PHOSPHATE ON ALGAL GROWTH AT LOW NUTRIENT CONCENTRATIONS1

Planktonic blue-green algae are known to be always associated with bacteria. Earlier work has shown that the addition of a bacteria-assimilable carbon source to a normal Zehnder-Gorham culture medium (No. 11) will produce enhanced growth of these algae when atmospheric CO₂ has become the limiting factor. In new work, phosphate-rich culture media were diluted so that they simulated nutrient levels found in the Great Lakes, e.g., Lake Erie. At these low concentrations and when atmospheric CO₂ was not available in a sufficient supply, the addition of sucrose to either a 1/100 or a 1/1000 diluted Z-G, medium (10 mg or 2 mg of sucrose, respectively) also produced enhanced, growth of the tested blue-green algae. The stimulation of algal growth was apparently due to an increased bacterial production of CO₂ and possibly other carbon compounds approaching the composition of the CO₂ molecule. The literature suggests that during vigorous algal growth in lakes, atmospheric CO₂ will be severely limiting. Productive lakes always contain nonliving organic matter. The presence of bacteria-assimilable matter is probably one of the important factors leading to algal bloom.     

Anaerobic digestates are useful nutrient sources for microalgae cultivation: functional coupling of energy and biomass production

We investigated the extent to which nitrogenous and phosphorus nutrients from liquid anaerobic digestates could be recycled for photosynthetic growth of a microalga, Scenedesmus sp. AMDD. Digestates recovered from the anaerobic digestion of cow manure and swine manure and a co-digestion of swine manure and algal biomass were diluted in distilled water and used for algal growth with and without supplemental CO₂ addition. Nutrient assimilation and final biomass yield were retarded in all but the swine manure/algae co-digestate cultures supplemented with high CO₂. Swine manure digestate cultures supplemented with the typical complement of micronutrients normally added with a commonly used growth medium or with Fe/EDTA failed to grow any better than unamended controls. When the culture medium was prepared by blending swine manure digestate with 25 or 50 % algal biomass digestate, diluting it with lake water or by supplementing with magnesium, nutrient assimilation and final algal biomass yields were maximized, indicating that magnesium was critically limiting for algal growth in swine manure digestates. Magnesium amendment thus appears to be essential if nutrients from swine manure digestates are recycled for algal growth. No such requirement is necessary for recycling nutrients from digestates generated wholly or in part from algal biomass.     

Direct and indirect influences of crustacean zooplankton on bacterioplankton of Lake Constance

Herbivorous crustacean zooplankton may influence bacterial populations of lakes directly by grazing on them or indirectly by grazing on algae. In Lake Constance a regularly observed decrease of bacterial density during periods of high abundance of cladocerans (clearwater phase) indicated bacterial grazing losses. However, cladoceran grazing on bacteria appeared to be less efficient than on algae. Moreover, cladocera reduced grazing pressure on bacteria by grazing on bacterivorous flagellates. Additionally, a shift of bacterial composition from an originally higher percentage of filamentous and aggregate growth forms towards a population of homogenously distributed small single celled bacteria was observed regularly at the beginning of the clearwater phase. Transient increases of bacterial abundance and productivity coinciding with the increase of cladocera at the end of the algal spring bloom were interpreted as field indications of indirect bacteria-zooplankton interactions due to crustacean grazing on phytoplankton. The release of organic carbon during grazing of crustacea on algae was considered as explanation for the observed stimulation of bacterial populations. Thereby, additional, otherwise inaccessible algal carbon would be made available to bacteria by zooplankton. Experimental support for this hypothesis was given by showing that bacteria were able to respond to crustacean grazing on algae by enhanced growth and activities. The possible impact of these direct and indirect crustacea-bacteria interactions on the abundance, activity and composition of bacterioplankton as well as on the structure and function of the total planktonic community is discussed.     

Microbial load in mass cultures of green algaeScenedesmus acutus and its processed powder

Microbial contamination in cultures of the alga,Scenedesmus acutus raised in outdoor open tanks and also in the processed powder of the alga was monitored; The total bacterial population increased with time during the growth period of six days. When a combination of molasses and carbondioxide was employed as carbon source for this alga, the bacterial load increased to 10 colony forming units/ml. Yeast, molds and also coliforms were quantitated. Drum-drying the algae drastically reduced the bacterial load and storing the algal powder for a period of over 3 months did not increase the bacterial load. Pathogens likeSalmonella andStaphylococcus were not detectable either in the open cultures or in the drumdried algal powder. Although there are not set standards available in literature on the permissible level of the microbial contamination in algal biomass for use in foods, the microbial load appears to be within the limits of permissible levels stipulated by Indian Standard Institution standards for baby foods.     

Alga-derived substrates select for distinct Betaproteobacterial lineages and contribute to niche separation in Limnohabitans strains

We examined the proportions of major Betaproteobacteria subgroups within bacterial communities in diverse nonaxenic, monospecific cultures of algae or cyanobacteria: four species of cryptophyta (genera Cryptomonas and Rhodomonas), four species of chlorophyta (genera Pediastrum, Staurastrum, and Chlamydomonas), and two species of cyanobacteria (genera Dolichospermum and Aphanizomenon). In the cryptophyta cultures, Betaproteobacteria represented 48 to 71% of total bacteria, the genus Limnohabitans represented 18 to 26%, and the Polynucleobacter B subcluster represented 5 to 16%. In the taxonomically diverse chlorophyta group, the genus Limnohabitans accounted for 7 to 45% of total bacteria. In contrast, cyanobacterial cultures contained significantly lower proportions of the Limnohabitans bacteria (1 to 3% of the total) than the cryptophyta and chlorophyta cultures. Notably, largely absent in all of the cultures was Polynucleobacter necessarius (Polynucleobacter C subcluster). Subsequently, we examined the growth of Limnohabitans strains in the presence of different algae or their extracellular products (EPP). Two strains, affiliated with Limnohabitans planktonicus and Limnohabitans parvus, were separately inoculated into axenic cultures of three algal species growing in an inorganic medium: Cryptomonas sp., Chlamydomonas noctigama, and Pediastrum boryanum. The Limnohabitans strains cocultured with these algae or inoculated into their EPP consistently showed (i) pronounced population growth compared to the control without the algae or EPP and (ii) stronger growth stimulation of L. planktonicus than of L. parvus. Overall, growth responses of the Limnohabitans strains cultured with algae were highly species specific, which suggests a pronounced niche separation between two closely related Limnohabitans species likely mediated by different abilities to utilize the substrates produced by different algal species.     

VOLATILE ORGANIC SULFIDES FROM FRESHWATER ALGAE1

Dimethyl sulfide (DMS) was found to be the principle biogenic sulfur compound in a freshwater environment. Endemic and non-endemic algae were cultured under axenic and nonaxenic conditions in defined media. Sulfur gas analysis of culture fluids indicated that members of the Cyanophyta were probable sources of DMS whereas representatives of the divisions Chlorophyta, Xanthophyta and Bacillariophyta apparently did not produce this compound. Comparison of gaseous contents of young and old nonaxenic cultures of filamentous algae in the divisions Chlorophyta and Xanthophyta showed DMS occurred only in aged cultures and was probably produced by bacteria utilizing substances from senescent algal cells. Data suggest that the composition of the algal community determines whether DMS is algal and/or bacterial in origin.     

Species-Specific Bacterial Communities in the Phycosphere of Microalgae?

Specific associations of bacteria with phytoplankton have recently been reported in the literature. In our study, we analyzed bacterial communities of microalgal cultures related to algal growth phases. Seven freshly isolated key diatom and dinoflagellate species from Helgoland Roads, North Sea, were investigated. The community composition of associated bacteria as well as the cell numbers, the photosynthetic efficiency of the algae, and the depletion of inorganic nutrients in the medium were recorded over a period of 8 weeks in batch cultures. Diversity and succession of bacterial communities was analyzed by ribosomal intergenic spacer analysis. Phylogenetic analysis of bacterial populations was performed by denaturing gradient gel electrophoresis of 16S rRNA genes followed by DNA sequence analysis. Members of Alphaproteobacteria and Gammaproteobacteria and the Flavobacteria–Sphingobacteria group within the Bacteroidetes phylum predominated in the cultures. Differences in free-living and attached bacterial populations were observed between the phylogenetic groups. Shifts in the bacterial communities could not be correlated to changes of nutrient levels or algal growth phases. Regarding our results, it should not be generalized that the compositions of the bacterial communities are strictly species specific for microalgae. The importance of factors like the composition of exudates is apparent.     

Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures

The optimal flocculating conditions for harvesting high-density cultures of Scenedesmus sp. were investigated using inorganic coagulants and the bioflocculant produced by Paenibacillus polymyxa AM49. The flocculated medium as nutrients for subsequent algal cultivation was also tested. Consecutive treatment with 8.5 mM CaCl(2) and 0.2 mM FeCl(3) as coagulants and 1% bioflocculant from the culture broth of P. polymyxa AM49 showed the highest flocculating activity of up to 95% for high density algal cultures. The medium flocculated with the coagulants and bioflocculant showed less than 8% decrease in the growth yield in the subsequent algal cultivation. Furthermore, a 20% or 50% fresh BG11 medium supplement allowed the flocculated medium to maintain a high growth yield in subsequent algal cultivation. These results suggest that the flocculation method presented here is efficient and bio-friendly, and allows the reuse of the flocculated medium, thereby contributing to the economic cultivation and harvest of microalgae.     

Effect of pH on Inorganic Carbon Uptake in Algal Cultures

Biomass production by the green algae Scenedesmus obliquus and Chlorella vulgaris in intensive laboratory continuous cultures was considerably affected by the pH at which the cultures were maintained. Carbon photoassimilation experiments revealed that pH values in the range of 8 to 9 were important for determining the free CO₂ concentrations in the medium. With higher pH values, additional pH effects were observed involving a decrease in the relative high affinity of low CO₂-adapted algae to free CO₂. The carbon uptake rate by high CO₂-adapted algae after transfer to low free CO₂ medium was characterized by a lag period of about 30 min, after which the affinity of the algae to CO₂ increased considerably. Both continuous growth and carbon uptake experiments indicated that artificially maintained high free CO₂ concentrations are recommended for maximal production in intensive outdoor algal cultures.     

Lysis of a red-tide causing alga,Alexandrium tamarense,caused by bacteria from its phycosphere

There are bacteria coexisting in xenic cultures of Alexandrium tamarense, a red-tide causing alga. However little is known concerning the interactions between the alga and the bacteria in its phycosphere. The objective of the current study was to determine the effect of the bacteria in its phycosphere on the growth of the alga. We added one percent (v/v) Zobell 2216E medium to A. tamarense culture to alter bacterial growth and the results showed that algal cells were all lysed within 14 h. After adding the medium, both the abundance and the extracellular enzyme activity of the bacteria increased by 50–100 times from the 4th to the 10th hour which resulted in lysis of the algae. The 16S rRNA gene fragments of the bacteria were amplified from the DNA extracted from A. tamarense cultures and analyzed by denaturing gradient gel electrophoresis and sequencing. The structure of the bacterial community in phycosphere changed significantly during algal lysis. Two bacterial genera, Alteromonas sp. and Thalassobius aestuarii sp. are key factors that caused the lysis, and the β-glucosidase and chitinase produced by the bacteria in the phycosphere could directly cause the lysis. These experiments provide evidence that bacteria in its phycosphere play a key role in the culture of A. tamarense, and may provide insights into the future biocontrol of red-tides.     

The impact of associated bacteria on morphology and physiology of the dinoflagellate Alexandrium tamarense

Despite their potential impact on phytoplankton dynamics and biogeochemical cycles, biological associations between algae and bacteria are still poorly understood. The aim of the present work was to characterize the influence of bacteria on the growth and function of the dinoflagellate Alexandrium tamarense. Axenic microalgal cultures were inoculated with a microbial community and the resulting cultures were monitored over a 15-month period, in order to allow for the establishment of specific algal–bacterial associations. Algal cells maintained in these new mixed cultures first experienced a period of growth inhibition. After several months, algal growth and cell volume increased, and indicators of photosynthetic function also improved. Our results suggest that community assembly processes facilitated the development of mutualistic relationships between A. tamarense cells and bacteria. These interactions had beneficial effects on the alga that may be only partly explained by mixotrophy of A. tamarense cells. The potential role of organic exudates in the establishment of these algal–bacterial associations is discussed. The present results do not support a role for algal–bacterial interactions in dinoflagellate toxin synthesis. However, variations observed in the toxin profile of A. tamarense cells during culture experiments give new clues for the understanding of biosynthetic pathways of saxitoxin, a potent phycotoxin.     

Bacterial response to extracellular dissolved organic carbon released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) in experimental systems

Responses of bacteria to dissolved organic carbon (DOC) released from healthy and senescent Fragilaria crotonensis (Bacillariophyceae) were examined in experimental systems. The alga released DOC actively, although the concentration fluctuated greatly in both the axenic (the alga alone) and the mixed (the alga plus the enriched bacteria) cultures. In the control (the bacteria alone) cultures, both DOC concentration and bacterial density were low and almost constant throughout the experiment: 5.0 mg C 1^–1 and 1.1 × 10⁵ cells ml^–1, respectively. In the mixed cultures, bacterial growth was negligible during the exponential growth phase of the alga, but rapid proliferation of the bacteria occurred after the onset of the stationary growth phase. As the bacterial population grew, the density of senescent algal cells also increased. When the bacteria were fed on the DOC from healthy algae, their growth rate was relatively low (0.44 d^–1), but the maximum cell density was high (6.4 × 10⁵ cells ml^–1). Conversely, when the bacteria fed on the DOC of senescent algae, they grew at a relatively high rate (0.51 d^–1), but the maximum cell density was low (2.8 × 10⁵ cells ml^–1). These results suggest that DOCs released from dominant phytoplankton species in different physiological states affect the biomass and activity of bacteria.