Utilization of Organic Nitrogen Sources by Two Phytoplankton Species and a Bacterial Isolate in Pure and Mixed Cultures

Algal production of dissolved organic carbon and the regeneration of nutrients from dissolved organic carbon by bacteria are important aspects of nutrient cycling in the sea, especially when inorganic nitrogen is limiting. Dissolved free amino acids are a major carbon source for bacteria and can be used by phytoplankton as a nitrogen source. We examined the interactions between the phytoplankton species Emiliania huxleyi and Thalassiosira pseudonana and a bacterial isolate from the North Sea. The organisms were cultured with eight different amino acids and a protein as the only nitrogen sources, in pure and mixed cultures. Of the two algae, only E. huxleyi was able to grow on amino acids. The bacterium MD1 used all substrates supplied, except serine. During growth of MD1 in pure culture, ammonium accumulated in the medium. Contrary to the expectation, the percentage of ammonium regenerated from the amino acids taken up showed no correlation with the substrate C/N ratio. In mixed culture, the algae grew well in those cultures in which the bacteria grew well. The bacterial yields (cell number) were also higher in mixed culture than in pure culture. In the cultures of MD1 and T. pseudonana, the increase in bacterial yield (number of cells) over that of the pure culture was comparable to the bacterial yield in mixed culture on a mineral medium. This result suggests that T. pseudonana excreted a more-or-less-constant amount of carbon. The bacterial yields in mixed cultures with E. huxleyi showed a smaller and less consistent difference than those of the pure cultures of MD1. It is possible that the ability of E. huxleyi to use amino acids influenced the bacterial yield. The results suggest that interactions between algae and bacteria influence the regeneration of nitrogen from organic carbon and that this influence differs from one species to another.     

Consequences of protist-stimulated bacterial production for estimating protist growth efficiencies

The trophic link between bacteria and bacterivorous protists is a complex interaction that involves feedback of inorganic nutrients and growth substrates that are immeadiately available for prey growth. These interactions were examined in the laboratory and in incubations of concentrated natural assemblages of bacterioplankton. Growth dynamics of estuarine and marine bacterivorous protists were determined in laboratory culture using Vibrio natriegens as prey and were compared to growth of protists on bacterioplankton assemblages concentrated by tangential flow filtration from four northwest Florida Estuaries. Biomass transfers from bacteria to protists were monitored by tracing elemental carbon and nitrogen in particulate fractions of protist added and grazer free controls. Gross growth efficiencies of the protists on naturally occurring bacteria were within the range determined in lab estimates of growth efficiency on cultured bacteria (50%). However, bacterial response to protist excretion products was different in the lab and field incubations, and bacterial growth contributed to the biomass available to protists in the field incubations. As determined by radioisotope-labeled substrate incorporation, a time lag in bacterial reponse to protist excretion products was observed for laboratory batch cultures, allowing accurate estimation of growth efficiency. In incubations with concentrated natural bacterial assemblages, bacterial growth response coincided with protist growth and excretion. The additional bacterial production on protist excretion products reached a maximum of 2–3-fold higher than protist-free controls. In addition, ammonium concentrations increased with protist grazing and growth in lab cultures, but ammonium excreted by protists in concentrates did not accumulate. The C:N values for the bacterial concentrates suggests that these bacteria were nitrogen limited. It is speculated that dissolved organic carbon, concentrated by tangential flow filtration (> 100,000 MW membrane) with the bacterioplankton, was utilized by bacteria when nitrogen was supplied as ammonium and amino acids from protist excretion. Thus, estimates of protist growth efficiency on naturally occurring bacterioplankton, corrected for protist-stimulated bacterial production, were in the range of 13–21%.     

蚂蚁筑巢对西双版纳热带森林土壤易氧化有机碳时空动态的影响

蚂蚁筑巢定居能够形成与巢穴周围显著不同的微生境和土壤养分环境,从而对土壤易氧化有机碳(EOC)产生重要影响.本研究以中国科学院西双版纳勐仑热带植物园白背桐群落为研究对象,比较蚂蚁巢地与非巢地土壤EOC时空分布特征,并分析蚂蚁筑巢引起土壤理化性质的改变对土壤EOC时空动态的影响.结果表明: 研究区蚁巢和非蚁巢地土壤EOC随月份均呈明显的单峰型变化规律,表现为6月>9月>3月>12月;土壤EOC沿土层呈逐渐降低的变化趋势,在0～5 cm土层,蚁巢土壤EOC显著大于非巢地,在5～10和10～15 cm土层的差异不显著.蚂蚁筑巢显著提高了土壤温度、土壤有机碳、土壤易氧化有机碳、土壤微生物生物量碳、全氮、硝态氮和水解氮含量,显著降低了土壤含水率和容重,但对铵态氮、pH值的影响不显著.土壤有机碳、土壤微生物生物量碳是调控蚁巢和非巢地土壤EOC时空变化的主要因子,土壤温度、含水率、全氮和硝态氮等土壤指标对土壤EOC的影响次之.蚂蚁筑巢主要通过改变微生境(土壤温度和水分)及土壤养分(主要是土壤有机碳和微生物生物量碳)的状况,进而调控热带森林土壤易氧化有机碳的时空动态.     

Influence of metazooplankton on interactions of bacteria and phytoplankton in an oligotrophic lake

Commensalism based on organic carbon supplied by phytoplanktonand competition for mineral nutrients are important interactionsbetween bacteria and phytoplankton in oligotrophic clear-watersystems. Both interactions are influenced by zooplankton activity.To examine the relation ship between algae and bacteria in LakeLa Caldera, we studied: the correlations among phyto plankton,bacteria and phosphorus (P) dynamics; the ratio of organic carbonsupplied by algae to organic carbon demand by bacteria; andthe importance of P remineralized by metazooplankton for bothcommunities. Phytoplankton and bacteria had a similar seasonaldynamics, and there was a sig nificant and positive relationshipbetween bacterial abundance and algal biomass (P<0.01). However,the release of organic carbon from phytoplankton was usuallyhigher than the bacterioplankton carbon requirement. P availablevia zooplankton remineralization satisfied between 74 and 316%of the minimum P demands of algae and bacteria. To elucidatewhether zooplankton operate similarly on algae and bacterialgrowth or indirectly influence bacterial growth through phytoplanktonmetab olism, we performed zooplankton manipulation experiments.High zooplankton biomass in these experiments stimulated bothprimary and bacterial production, but release of organic carbonfrom phytoplankton declined. These results suggest a directstimulus of bacterial growth, so algae and bac teria can balancegrazing losses by compensatory growth. Further, the algal decreaseof the organic carbon supply for bacteria could, over time,lead to a change in the algae-bacteria interaction from competitionto commensalism. This reduction in organic carbon excretioncould affect the balance of the competitive interaction.     

Root exudation and rhizoplane bacterial abundance of barley (Hordeum vulgare L.) in relation to nitrogen fertilization and root growth

The abundance of bacteria in the rhizoplane of barley varieties was investigated at different soil nitrogen levels. Increased amendments of nitrogen resulted in higher bacterial numbers in the rhizoplane of barley seedlings of different varieties. A negative correlation was found between nitrogen level in the soil and the growth rate of the seedling roots. The effect of nitrogen on the bacterial abundances could be indirect through changed root growth and thereby changed exudation. The exudation of soluble organic carbon componds from barley seedling roots were measured in hydroponic culture. The effect of natural variation in root growth rate and of different concentrations of nitrogen in the nutrient solution was investigated. The amount of exudates consituted 2–66% of the dry weight increase in root biomass, depending on the root growth. Slower growing roots released considerably more organic carbon per unit root weight than faster growing roots. The variation in root exudation appeared to be mainly explained by differences in root growth, rather than of the nitrogen concentration in the nutrient solution. A significantly higher exudation rate was found during day time compared to night.     

Modelling the influence of food C:N ratio, and respiration on growth and nitrogen excretion in marine zooplankton and bacteria

Zooplankton and bacteria use food to synthesize new biomassand meet respiratory demands. A steady-state bioenergetic modelis presented which explores the effects of the quality of assimilatedfood and respiration on growth and nitrogen excretion in marineheterotrophs. Processes of grazing and assimilation are notconsidered. Assimilated food is divided into two groups: nitrogenous(proteins) and non-nitrogenous (carbohydrates, lipids), andassumptions used to determine how each group is utilized forgrowth and respiration. Zooplankton growth is predicted to becarbon-limited, in contrast to existing experimental evidencewhich suggests nitrogen limitation. Excretion of nitrogen increaseswith the nitrogen content of food, and quantities of food consumed.A dissolved organic matter carbon to nitrogen ratio of 10.2:1was predicted below which bacteria remineralize nitrogen, andabove which ammonium is taken up from the environment and usedas a growth substrate. This compares well with experimentalstudies. The model provides valuable insight into the use ofsimpler nutrient-balancing models: critical assumptions aboutorganisms' carbon net growth efficiency must be made in orderto apply them successfully. In general, the study highlightedthe importance of having a proper understanding of respirationin marine organisms and how it is related to their biomass andgrowth.     

Carbon budgets in batch and continuous cultures: How can we understand natural physiology of marine phytoplankton?

Monitoring of respiratory and organic losses from carbon assimilationfor the diatom Thalassiosira pseudonana shows very slight (about5%) total carbon losses in continuous culture. Although correlationbetween batch and continuous culture cellular chemistry wasless than ideal, batch culture exponential phase cells showedcarbon metabolism similar to that for the continuous culture.In both cases, growth rates were in excess of 1.5 divisionsd^–1 and total carbon losses in the batch exponential phasewere similar (<10%) to those in the continuous culture. Estimatesof growth rates from C-14 uptake and particulate carbon matchedthose from changes in cell numbers in the batch culture andthat from the dilution rate in the continuous culture. In batchculture, immediately after starting the culture and in stationaryphase, carbon losses from respiration, organic excretion, andcellular degradation were large (>50%). To understand phytoplanktonphysiology in nature, it is necessary to find what effects growthrate and population density have on carbon losses and to ascertainwhether or not steady state conditions really pertain to theocean.     

Effect of nitrogen source on methanol oxidation and genetic diversity of methylotrophic mixed cultures enriched from pulp and paper mill biofilms

Methanol-oxidizing bacteria may play an important role in the development and use of biological treatment systems for the removal of methanol from industrial effluents. Optimization of methanol degradation potential in such systems is contingent on availability of nutrients, such as nitrogen, in the most favorable form and concentration. To that end, this study examined the variation in growth, methanol degradation, and bacterial diversity of two mixed methylotrophic cultures that were provided nitrogen either as ammonium or nitrate and in three different concentrations. Methanol-degrading cultures were enriched from biofilms sampled at a pulp and paper mill and grown in liquid batch culture with methanol as the only carbon source and either ammonium or nitrate as the only added nitrogen source. Results indicate that growth and methanol removal of the mixed cultures increase directly with increased nitrogen, added in either form. However, methanol removal and bacterial diversity, as observed by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR–DGGE) methods, were higher when using nitrate as the nitrogen source for enrichment and growth, rather than ammonium. Based on results described here, nitrate may potentially be a better nitrogen source when enriching or working with mixed methylotrophic cultures, and possibly more effective when used as a nutrient addition to biofilters.     

Growth dynamics of Azospirillum lipoferum at steady and transitory states in the presence of NH

AIMS: The objective of this paper was to study the adaptation dynamics and biochemical response of Azospirillum lipoferum grown in a continuous culture at various environmental shifts. METHODS AND RESULTS: The kinetics of A. lipoferum Sp 59b grown at steady states in a microaerobic chemostatic environment deviated from a typical Monod kinetics in both low and high dilution rates (D) due to several metabolic shifts that occurred in the microbial cell. When NH4Cl was exhausted (at low D), the microbial cell partitioned carbon flow in order to sustain growth, nitrogen fixation and assimilation processes (occurred via the glutamate synthase reaction). Increasing D the specific activities of the enzymes involved in the tricarboxylic acid cycle and the respiration rate were decreased. At transitory states, under optimal for nitrogen fixation dissolved oxygen (DO) concentrations, ammonium nitrogen negatively affected, besides nitrogen fixing activity, the bacterial growth. At sub-optimal for nitrogen fixation DO concentration (i.e. 1.56 microM) and 0.1 g l(-1) NH4Cl in the fed medium, the activities of citrate synthase and succinate dehydrogenase were significantly reduced. CONCLUSIONS: Important shifts in both carbon and nitrogen metabolism occur in A. lipoferum grown in the presence of the ammonium nitrogen, while the boundaries of ammonium nitrogen concentration in which A. lipoferum can be adapted depend on the DO concentration in the growth environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on growth dynamics and physiology of A. lipoferum, grown in experimental model systems, can contribute to an efficient application of these bacteria as plant-growth-promoting-agents.     

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.     

混合培养对固氮菌和纤维素分解菌生长及固氮的影响

对筛选的自生固氮菌和纤维素分解菌进行混合培养 ,研究了菌数与菌液含氮量的变化情况 ,并与其单独培养进行了比较。实验证明 :在混合培养条件下 ,两种菌能相互利用、相互促进 ,混合培养液的菌数增加 ,固氮菌的固氮能力提高。这两种菌可混合培养制成混合菌剂。。     

Experimental Approach to the Role of Protozoa in Aquatic Ecosystems

In enrichment batch experiments, samples from three water bodieswere alternatively supplemented by various amounts of organicmaterial and incubated at 20 C. Colpidium campylum reached itshighest total cell volumes in cultures with the highest initialtotal cell volumes in cultures with the highest initial concentrationsof organics; Cyclidium glaucoma preferred lower concentrations;and Glaucoma chattoni occupied the intermediate position. Noneof the species preferred any special type of organic material.In two-stage continuous-flow units, a mixed culture of bacteriawas kept in stage I and the clones of ciliates were maintainedin stage II. The interrelations between the total cell volumesof ciliates at various concentrations of bactopeptone were inaccordance with the results from the enrichment experiments.Since the growth of bacteria continued in the presence of ciliates,a four-stage apparatus was constructed in which a bacterialculture was raised and diluted in three stages before enteringthe culture ofColpidium. The bacterial growth in the presenceof Colpidium was not eliminated even by this arrangement, asdemonstrated by dosing antibiotics along with bacteria. An effectof ciliate metabolites on bacterial growth rate is suggested,completing a metabolic cycle in the bacteria-protozoa system.     

A comparison of carbon/energy and complex nitrogen sources for bacterial sulphate-reduction: potential applications to bioprecipitation of toxic metals as sulphides

Detailed nutrient requirements were determined to maximise efficacy of a sulphate-reducing bacterial mixed culture for biotechnological removal of sulphate, acidity and toxic metals from waste waters. In batch culture, lactate produced the greatest biomass, while ethanol was more effective in stimulating sulphide production and acetate was less effective. The presence of additional bicarbonate and H₂ only marginally stimulated sulphide production. The sulphide output per unit of biomass was greatest using ethanol as substrate. In continuous culture, ethanol and lactate were used directly as efficient substrates for sulphate reduction while acetate yielded only slow growth. Glucose was utilised following fermentation to organic acids and therefore had a deleterious effect on pH. Ethanol was selected as the most efficient substrate due to cost and efficient yield of sulphide. On ethanol, the presence of additional carbon sources had no effect on growth or sulphate reduction in batch culture but the presence of complex nitrogen sources (yeast extract or cornsteep) stimulated both. Cornsteep showed the strongest effect and was also preferred on cost grounds. In continuous culture, cornsteep significantly improved the yield of sulphate reduced per unit of ethanol consumed. These results suggest that the most efficient nutrient regime for bioremediation using sulphate-reducing bacteria required both ethanol as carbon source and cornsteep as a complex nitrogen source.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.     

Bacterial Growth in Mixed Cultures on Dissolved Organic Carbon from Humic and Clear Waters

Interactions between bacterial assemblages and dissolved organic carbon (DOC) from different sources were investigated. Mixed batch cultures were set up with water from a humic and a clear-water lake by a 1:20 dilution of the bacterial assemblage (1.0 μm of prefiltered lake water) with natural medium (sterile filtered lake water) in all four possible combinations of the two waters and their bacterial assemblages. Bacterial numbers and biomass, DOC, thymidine incorporation, ATP, and uptake of glucose and phenol were followed in these cultures. Growth curves and exponential growth rates were similar in all cultures, regardless of inoculum or medium. However, bacterial biomass produced was double in cultures based on water from the humic lake. The fraction of DOC consumed by heterotrophic bacteria during growth was in the same range, 15 to 22% of the total DOC pool, in all cultures. Bacterial growth efficiency, calculated from bacterial biomass produced and DOC consumed, was in the order of 20%. Glucose uptake reached a peak during exponential growth in all cultures. Phenol uptake was insignificant in the cultures based on the clear-water medium, but occurred in humic medium cultures after exponential growth. The similarity in the carbon budgets of all cultures indicated that the source of the bacterial assemblage did not have a significant effect on the overall carbon flux. However, fluxes of specific organic compounds differed, as reflected by glucose and phenol uptake, depending on the nature of the DOC and the bacterial assemblage.     

Comparison of different algal carbon estimates by use of the Droop-model for nutrient limited growth

Three different estimates of algal carbon were derived fromfield experiments in plastic enclosures and tested as measureof algal biomass in the growth model of Droop. Fresh weightderived carbon and total paniculate organic carbon correctedfor detritus were both found to behave as good estimates ofalgal biomass in the model, while total paniculate organic carbondid not. The study also provided results which suggested thatthe relation between growth rate and endogenous phosphorus differedfor two species involved. Growth was maintained at lower Q_p-ratiosin Staurastrum dominated communities compared with communitiesconsisting mainly of Anabaena flos-aquae.     

A simulation model for the effect of predation on bacteria in continuous culture

A simulation model was developed for the carbon (C), nitrogen (N), and phosphorus (P) content of bacteria and their medium in a chemostat. Cell components distinguished included the structural component, synthetic machinery, building blocks and intermediates, C reserves, ammonium (NH₄), orthophosphate (PO₄), and polyphosphate. Growth, incorporation of substrates, and production of waste products were related to physiological status, as indicated by the amounts of various cell components. The model was fitted to data from chemostats on the chemical composition of bacteria growing in C-, N-, and P-limiting media and was used to explore the consequences of predation on bacterial populations. In C-limiting media predation (without the return of nutrients to the medium by the predator) increased NH₄ uptake in spite of a decrease in bacterial biomass. In N-limiting media predation decreased both biomass and the rate of N uptake. These results were accounted for by the effect of growth rate on bacterial N demand. In C-limiting media the return of NH₄ and PO₄ by the predator did not change the effect of predation on bacteria. But in N-limiting media the return of nutrients decreased the effect of predation on biomass, and stimulated respiration and NH₄ uptake by the bacteria. The effect of growth rate on the chemical composition of bacteria was proposed as a possible explanation of the stimulatory effect of predators on bacteria.     

Can bacteria outcompete phytoplankton for phosphorus? a chemostat test

Although the bacterioplankton of lakes are usually considered primarily in terms of mineralization processes, recent studies suggest that they may also strongly compete for phosphorus with the phytoplankton. In the present study, we have tested in chemostat culture, and found support for the hypotheses that (1) a freshwater bacterium (Pseudomonas paucimobilis), whose carbon source is excretion from a phosphorus-limited alga (Synedra ulna var.danica), can outcompete that alga for phosphorus (P) under widely varied P supply rates; (2) exogenously-supplied organic carbon positively influences bacterial biomass and negatively influences algal biomass; (3) the ratio of bacterial to algal phosphorus uptake in short-term³²P orthophosphate uptake experiments is an accurate predictor of their relative long-term phosphorus assimilation (i.e., growth) in mixed culture.     

Estimation of ammonium regeneration efficiencies associated with bacterivory in pelagic food webs via a 15N tracer method

Phagotrophic protists are major components of pelagic food webs,both as consumers of bacterial and phytoplankton cells, andas regenerators of inorganic nutrients. In this study, we estimatedthe efficiency of ammonium regeneration by protists feedingon bacteria within natural plank-tonic assemblages, using a¹⁵N tracer method, in which the excretion of ¹⁵N-labeled ammoniumdue to grazing on ¹⁵N pre-labeled bacteria was followed overtime. We tested this approach in experiments based on the additionof heat-killed ¹⁵N-labeled bacteria to laboratory cultures andto samples of coastal seawater. During two experiments, variationin abundance of bacterivores and bacterioplankton resulted innon-constant grazing rates. Deterministic computer models thatused abundance of bacteria and protists as variables were developedto estimate best-fit values of grazing mortality (g, h^–1)and of ammonium regeneration efficiency (R_E, fraction of theinitial ¹⁵N label in added bacteria which is released as ammonium).Estimated ammonium R_E were 0.30–0.35 for one trophic linksystems with both a monospecific culture and a mixed speciesassemblage of bacterivorous flagellates. R_E was higher for multi-trophicstep food webs: 0.60 for 5 µm pre-screened coastal seawaterand 0.90 for whole coastal seawater.     

Model of copepod growth influenced by the food carbon:nitrogen ratio and concentration, under the hypothesis of strict homeostasis

This study describes a model which addresses the processes of ingestion, assimilation, respiration, excretion and growth of copepods as a function of the concentration of food and its elemental composition in terms of carbon and nitrogen (N). Two experimental data sets are used to estimate several parameters of the model concerned with the influence of food quality. The results of the model suggest that the concentration of food and its quality (i.e. the C:N ratio) largely determine copepod growth. Both the experimental data sets and the model output show that low carbon relative to the nitrogen content of food does not limit the production of copepods. Comparing the results of the model to those of a previous model on bacteria suggests large differences between bacterial and copepod physiological responses to a variable quality of the substrate or food. The results of these models suggest that the regeneration of ammonium performed by copepods always favors regenerated primary production, whereas that performed by bacteria, depending on the quality of assimilated substrates, can favor or limit regenerated production.