Nitrogen uptake by heterotrophic bacteria and phytoplankton in Arctic surface waters

We estimated rates of heterotrophic bacterial and phytoplanktonuptake of nitrate, ammonium, and urea using ¹⁵N-labelled nitrogenand specific metabolic inhibitors of prokaryote and eukaryotenitrogen metabolism in the surface waters of the North Water(northern Baffin Bay) during autumn that were characterizedby the absence of cyanobacteria (comprising prochlorophytes).The percentage of nitrate + ammonium uptake by heterotrophicbacteria ranged between 44 and 78% of the measured total uptakeand was the highest when the phytoplankton biomass was relativelylow (<2 µg Chlorophyll a L^–1). Phytoplanktonaccounted for a larger fraction (e.g., 58–95%) of ureauptake than heterotrophic bacteria. When our results are combinedwith those from previous studies carried out in diverse temperateand polar areas, it appears that heterotrophic bacteria accountfor 25% (14–40%; median and interquartile range) of thetotal nitrate uptake in surface waters with chlorophyll biomass<2 µg L^–1. Estimates of new production computedfrom phytoplankton carbon uptake and f-ratios may be stronglyoverestimated in regions where nitrate uptake by heterotrophicbacteria is high and the biomass of phytoplankton is low.     

Grazing of bacteria and phytoplankton by heterotrophic nanoflagellates in a Baltic Sea sample

Grazing by heterotrophic nanoflagellates on bacteria and phytoplankton was studied in a laboratory experiment, using a natural pelagic community originating from the Tvärminne sea area off the southern coast of Finland. Water was prescreened to remove larger grazers. Four experimental treatments were used: light and dark, with and without added nutrients. The growth of the large heterotrophic flagellates was stimulated by increased production of < 3 m phytoplankton. Clearance rates for heterotrophic nanoflagellates were estimated and were found to be within the range of previously reported values.     

A method for the experimental determination of light absorption by aquatic heterotrophic bacteria

A method has been set up for the experimental determinationof the volume coefficient for light absorption in vivo by aquaticheterotrophic bacteria. The application described here is theabsorption measurement of the bacterial fraction that passesthrough the commonly used GF/F ifiter and remains unaccountedfor. The experimental samples were prepared by successive waterfiltra tions through GF/F and 0.22 µm Millipore membranes.Light-transmission and light-reflection measurements of thefilter-retained samples were performed using a dual-beam spectrophotometerequipped with an integrating sphere attachment. Sample absorptionwas derived from the data by a procedure that corrects for thecontamination of the results due to the high degree of lightscattering by the bacteria. The bacterial absorption was discriminatedfrom fine detritus absorption by bleaching the bacterial respiratorypigments using a K₂S₂O₈ solution. The absorption amplificationcaused by multiple scattering in the filter was corrected forby an expression that was obtained experimentally. A test ofthe method, including error analysis, was performed on samplescollected in both marine and inland waters. The relative contributionsto light absorption by heterotrophic bacteria and various typesof particulate matter were also measured for a typical situation.Combining the measured volume absorption coefficients with backscatteringcoefficients computed by Mie theory yields a set of input datato multicomponent optical models that is needed to assess thecontribution of these heterotrophic bacteria to the radiativetransfer process.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity.

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Relationship between bacteria, phytoplankton and heterotrophic nanoflagellates along the trophic gradient

Bacterial and heterotrophic nanoflagellates (HNF) abundance, as well as bacterial production and chlorophylla levels, were measured at five sites extending from the coastal zone toward the open Adriatic in the period from March to October 1995. The investigated areas were grouped into trophic categories according to concentrations of chlorophylla. All the biotic-para-meters increased along the trophic gradient, leading to eutrophy, but they did not increase at the same rate. The bacterial biomass: phytoplankton biomass (BB: chla) ratio decreased from about 10 in the very oligotrophic area to 0.8 at the eutrophic site. In contrast, the bacterial abundance: HNF abundance ratio (B: HNF) increased from 1000 bacteria per 1 flagellate in the oligotrophic system to 1700 bacteria flagellate⁴ in the eutrophic area. Decreasing BB: chla and increasing B: HNF ratios along the trophic gradient might reflect the different structures of the microbial food web. Relationships between bacterial abundance and production, and chla and HNF showed that bacterial abundance along the trophic gradient was regulated by the interplay between nutrient supply and grazing pressure. But in the oligotrophic system, bacterial abundance was more closely related to bacterial production and chla than in the eutrophic system, suggesting stronger control of bacterial abundance by substrate supply. On the other hand, the coupling between bacteria and HNF, and uncoupling between bacterial abundance and production in the eutrophic system, showed that the importance of bacteriovory increased in richer systems.     

Oligotrophic properties of heterotrophic bacteria and in situ heterotrophic activity in pelagic seawates

Abstract The distribution of heterotrophic bacteria in polluted coastal and unpolluted pelagic seawaters was studied using a ¹⁴C-MPN method with either five of seven kinds of ¹⁴C-organic compounds as substrates. The total number of heterotrophic bacteria in pelagic waters ranged from 9.2 × 10³ to 5.4. ¢ 10⁴ cell/ml and more than 85% of the heterotrophic bacteria were represented by obligate oligotrophs. In coastal waters, the number of heterotrophs was one order of magnitude higher (av. 3.5 ¢ 10⁵ cells/ml), and eutrophic and facultatively oligotrophic bacteria were predominant. Oligotrophs in pelagic waters had a high specificity for the utilization of amino acids, especially glycine, and acetate-utilizing bacteria were scarce. The in situ maximum uptake rates of glutamate and glycine were much higher than those of glycolate and acetate. Acetate uptake rates were extremely low or not detectable in pelagic waters. The specificity of uptake kinetics is assumed to depend on the existence of obligate oligotrophs as dominant bacteria in pelagic seawater.     

The uptake of inorganic nutrients by heterotrophic bacteria

It is now well known that heterotrophic bacteria account for a large portion of total uptake of both phosphate (60% median) and ammonium (30% median) in freshwaters and marine environments. Less clear are the factors controlling relative uptake by bacteria, and the consequences of this uptake on the plankton community and biogeochemical processes, e.g., new production. Some of the variation in reported inorganic nutrient uptake by bacteria is undoubtedly due to methodological problems, but even so, uptake would be expected to vary because of variation in several parameters, perhaps the most interesting being dissolved organic matter. Uptake of ammonium by bacteria is very low whereas uptake of dissolved free amino acids (DFAA) is high in eutrophic estuaries (the Delaware Bay and Chesapeake Bay). The concentrations and turnover of DFAA are insufficient, however, in oligotrophic oceans where bacteria turn to ammonium and nitrate, although the latter only as a last resort. I argue here that high uptake of dissolved organic carbon, which has been questioned, is necessary to balance the measured uptake of dissolved inorganic nitrogen (DIN) in seawater culture experiments. What is problematic is that this DIN uptake exceeds bacterial biomass production. One possibility is that bacteria excrete dissolved organic nitrogen (DON). A recent study offers some support for this hypothesis. Lysis by viruses would also release DON.While ammonium uptake by heterotrophic bacteria has been hypothesized to affect phytoplankton community structure, other impacts on the phytoplankton and biomass production (both total and new) are less clear and need further work. Also, even though bacteria account for a very large fraction of phosphate uptake, how this helps to structure the plankton community has not been examined. What is clear is that the interactions between bacterial and phytoplankton uptake of inorganic nutrients are more complicated than simple competition.     

Some factors affecting the heterotrophic activity of bacteria in lake.

The heterotrophic activity of aquatic bacteria was measured by incorporation of 3H-thymidine, uptake of 14C-sodium acetate and mineralization of 14C-acetate. The temperature of water, oxygen content and concentration of chlorophyll a were also measured in the studied lakes. The obtained results of the studied processes in the surface layer and epilimnion were distinguished by negative correlation coefficients. A distinctive feature is the relation between chlorophyll a concentration in the water bodies and heterotrophic activity of bacteria. This indicates a negative dependence between these processes and phytoplankton activity in the photic zone. In contrast a positive relationship between phytoplankton activity and heterotrophic activity of bacteria was found in the metalimnion and hypolimnion of the studied lakes.     

Effect of temperature on mineralization by heterotrophic bacteria.

When pure cultures of the bacteria Pseudomonas fluorescens (a psychrotroph), Escherichia coli (a mesophile), and SRL 261 (a thermophile) were shifted away from temperatures to which they were adapted, the percentage of substrate mineralized increased (percent mineralized = [substrate respired to CO2]/substrate respired to CO2 + substrate incorporated into biomass] X 100). The increase in the percent mineralized was larger for larger temperature shifts. Similar responses were observed when natural heterotrophic bacterial populations from sediments of Lake George, N.Y., and a thermophilic algal-bacterial mat community at the Savannah River Plant, Aiken, S.C., were subjected to temperature shifts. These results suggest that an increase in the percent mineralized may be an indication of thermal stress in bacterial populations.     

Primary production and extracellular release by phytoplankton in some lakes of the Masurian Lake District, Poland.

The primary production and extracellular release by phytoplankton were measured at different depths of Miko?ajskie Lake, Ryńskie Lake and Be?dany Lake (Masurian Lake District, Poland). The release of dissolved organic compounds was found to be related to the rates of primary production. The percentage of extracellular release (PER) of organic matter in the photic zone showed an inverse relationship with particulate production and chlorophyll alpha concentration. The highest PER was determined in the epilimnion of the studied lakes, where also increased release was observed. The higher release of algal products of photosynthesis in the photic zone than in the profundal is probably caused by the inhibition of physiological activity of bacteria by antibacterial substances produced by algae. The substances released by algae in the profundal are taken up by aquatic bacteria which explains the lower release and PER measured.     

Distribution and significance of heterotrophic marine bacteria with antibacterial activity

Bacteria with antibacterial activity were isolated from seawater, sediments, phytoplankton, and zooplankton of Suruga, Sagami, and Tokyo Bays and from soft corals and sponges collected from the Taiwan coast. Of the 726 strains isolated, 37 showed antibacterial activity against either Vibrio parahaemolyticus (ATCC 17802) or Staphylococcus aureus (P209). Sediment harbored the lowest number of these forms of bacteria, and those from Tokyo Bay did not show any activity. Attached isolates showed greater activity compared with free-living forms. Relatively high numbers of strains with antibacterial activity were associated with phytoplankton. Among the zooplankton isolates, cladocerans harbored the maximum number of antibacterial strains. Isolates were more inhibitory to gram-positive test cultures. Autoinhibition was observed only among 8% of the isolates. Marine nonproducers were more susceptible. Pseudomonas/Alteromonas species made up 81.0% of isolates, of which 30% were pigmented strains. The absence or reduction in number of bacteria with antibacterial activity in Tokyo Bay is attributed to its eutrophic nature, which may tend to moderate the production of antibacterial compounds.     

Role of heterotrophic bacteria in promoting N2 fixation byAnabaena in aquatic habitats

Anabaena species are commonly colonized by bacteria, especially during N₂-fixing blooms. Generally these associations do not represent bacterial attack on algal hosts. Instead, the algal N₂-fixing capabilities are increased in the presence of the bacteria. Possible mechanisms promoting the mutual growth of algae and attached bacteria were investigated by observing specific sites of bacterial attachment, by noting reduced microzones created by the bacteria, and by locating sites of bacterial uptake of organics representative of algal excretion products.Attached bacteria show preference for typical algal excretion products and their growth is enhanced by such products. In return, enhancement of algal nitrogenase activity occurs when bacteria create O₂-consuming microzones around the nitrogenase-bearing heterocysts.     

Thiosulfate oxidation by obligately heterotrophic bacteria

Thiosulfate was oxidized stoichiometrically to tetrathionate during growth on glucose byKlebsiella aerogenes, Bacillus globigii, B. megaterium, Pseudomonas putida, two strains each ofP. fluorescens andP. aeruginosa, and anAeromonas sp. A gram-negative, rod-shaped soil isolate, Pseudomonad H_w, converted thiosulfate to tetrathionate during growth on acetate. None of the organisms could use thiosulfate as sole energy source. The quantitative recovery of all the thiosulfate supplied to heterotrophic cultures either as tetrathionate alone or as tetrathionate and unused thiosulfate demonstrated that no oxidation to sulfate occurred with any of the strains tested. Two strains ofEscherichia coli did not oxidize thiosulfate. Thiosulfate oxidation in batch culture occurred at different stages of the growth cycle for different organisms:P. putida oxidized thiosulfate during lag and early exponential phase,K. aerogenes oxidized thiosulfate at all stages of growth, andB. megaterium andAeromonas oxidized thiosulfate during late exponential phase. The relative rates of oxidation byP. putida andK. aerogenes were apparently determined by different concentrations of thiosulfate oxidizing enzyme. Thiosulfate oxidation byP. aeruginosa grown in chemostat culture was inducible, since organisms pregrown on thiosulfate-containing media oxidized thiosulfate, but those pregrown on glucose only could not oxidize thiosulfate. Steady state growth yield ofP. aeruginosa in glucose-limited chemostat culture increased about 23% in the presence of 5–22 mM thiosulfate, with complete or partial concomitant oxidation to tetrathionate. The reasons for this stimulation are unclear. The results suggest that heterotrophic oxidation of thiosulfate to tetrathionate is widespread across several genera and may even stimulate bacterial growth in some organisms.     

High-throughput cultivation of heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

On-going studies of phytoplankton-bacterioplankton interactions at the long-term ecological research site Helgoland Roads have indicated that many of the heterotrophic bacterial taxa have not yet been cultivated. A high-throughput approach combining whole cell matrix-assisted laser desorption ionization – time of flight mass spectroscopy with 16S rRNA gene sequencing was applied to the spring bloom of 2016. Aiming at an assessment of cultivability during a spring bloom, cultivation on solid marine media had to be used since dilution to extinction would not have been feasible for a high-throughput approach, as performed in this study. A total of 5023 isolates were obtained from nine weekly samples on eight different solid media between the early-bloom and post-bloom periods. Most of the 4136 strains identified affiliated with Bacteroidetes (13.3%), Gammaproteobacteria (26.9%), Alphaproteobacteria (40.6%) and Actinobacteria (6.7%). Of the 271 operational phylogenetic units (OPUs) identified, 13 are likely to represent novel genera and 143 novel species. A comparison with 16S rRNA gene tag data indicated that most of the isolates were rather rare in surface waters, with the exception of five OPUs affiliating with Rhodobacteraceae, Polaribacter, Psychromonas and Pseudoalteromonas. The effort yielded many novel isolates, yet most of the abundant heterotrophic bacteria still remained elusive. The large strain collection obtained will not only provide insights into the succession of the cultivable fraction of the bacterioplankton, but also enable fine-tuned taxonomic and physiological follow-up studies for improving our knowledge on heterotrophic bacteria in North Sea waters.     

Cultivation of particle-associated heterotrophic bacteria during a spring phytoplankton bloom in the North Sea

Seawater contains free-living and particle-attached bacteria. Only a small fraction is cultivable on plates. As free-living and particle-associated bacteria differ in their physiological traits, their cultivability on plates may coincide with particle association. Using filtration and Imhoff sedimentation cones, particles were collected during a spring phytoplankton bloom off Helgoland (North Sea) in order to obtain particle-associated bacteria as inocula. Direct dilution plating resulted in 526 strains from 3 µm filtration retentates and 597 strains from settled particles. Motile Gammaproteobacteria from the genera Pseudoalteromonas, Shewanella, Psychrobacter, Vibrio and Colwellia, as well as particle-attached Flavobacteriia affiliating with the genera Tenacibaculum and Gramella, were frequently isolated. As a result, a diverse collection comprised of 266 strains was deposited. Two strains were most likely to represent novel genera and 78 strains were probably novel species. Recently, a high-throughput cultivation study from the same site using seawater as an inoculum had retrieved 271 operational phylogenetic units (OPUs) that represented 88% of the 4136 characterized strains at the species level. A comparison of 16S rRNA gene sequences revealed that the collection obtained matched 104 of the 271 seawater OPUs at the species level and an additional 113 at the genus level. This large overlap indicated a significant contribution of particle-associated bacteria to the cultivable microbiome from seawater. The presence of 49 genera not identified in the larger seawater study suggested that sample fractionation was an efficient strategy to cultivate rare members of the planktonic microbiome. The diverse collection of heterotrophic bacteria retrieved in this study will be a rich source for future studies on the biology of particle-associated bacteria.     

Feeding and grazing impact by small marine heterotrophic dinoflagellates on heterotrophic bacteria

ABSTRACT. We investigated the feeding of the small heterotrophic dinoflagellates (HTDs) Oxyrrhis marina , Gyrodinium cf. guttula , Gyrodinium sp., Pfiesteria piscicida , and Protoperidinium bipes on marine heterotrophic bacteria. To investigate whether they are able to feed on bacteria, we observed the protoplasm of target heterotrophic dinoflagellate cells under an epifluorescence microscope and transmission electron microscope. In addition, we measured ingestion rates of the dominant heterotrophic dinoflagellate, Gyrodinium spp., on natural populations of marine bacteria (mostly heterotrophic bacteria) in Masan Bay, Korea in 2006–2007. Furthermore, we measured the ingestion rates of O. marina , G . cf. guttula , and P. piscicida on bacteria as a function of bacterial concentration under laboratory conditions. All HTDs tested were able to feed on a single bacterium. Oxyrrhis marina and Gyrodinium spp. intercepted and then ingested a single bacterial cell in feeding currents that were generated by the flagella of the predators. During the field experiments, the ingestion rates and grazing coefficients of Gyrodinium spp. on natural populations of bacteria were 14–61 bacteria/dinoflagellate/h and 0.003–0.972 day⁻¹, respectively. With increasing prey concentration, the ingestion rates of O. marina , G . cf. guttula , and P. piscicida on bacteria increased rapidly at prey concentrations of ca 0.7–2.2 × 10⁶ cells/ml, but increased only slowly or became saturated at higher prey concentrations. The maximum ingestion rate of O. marina on bacteria was much higher than those of G . cf. guttula and P. piscicida . Bacteria alone supported the growth of O. marina . The results of the present study suggest that some HTDs may sometimes have a considerable grazing impact on populations of marine bacteria, and that bacteria may be important prey.