Physiological and Biochemical Response of the Photosynthetic Apparatus of Two Marine Diatoms to Fe Stress

Flavodoxin is a small electron-transfer protein capable of replacing ferredoxin during periods of Fe deficiency. When evaluating the suitability of flavodoxin as a diagnostic indicator for Fe limitation of phytoplankton growth, we examined its expression in two marine diatoms we cultured using trace-metal-buffered medium. Thalassio-sira weissflogii and Phaeodactylum tricornutum were cultured in ethylenediaminetetraacetic acid-buffered Sargasso Sea water containing from 10 to 1000 nM added Fe. Trace-metal-buffered cultures of each diatom maintained high growth rates across the entire range of Fe additions. Similarly, declines in chlorophyll/cell and in the ratio of photosystem II variable-to-maximum fluorescence were negligible (P. tricornutum) to moderate (T. weissflogii; 54% decline in chlorophyll/cell and 22% decrease in variable-to-maximum fluorescence). Moreover, only minor variations in photosynthetic parameters were observed across the range of additions. In contrast, flavodoxin was expressed to high levels in low-Fe cultures. Despite the inverse relationship between flavodoxin expression and Fe content of the medium, its expression was seemingly independent of any of the indicators of cell physiology that were assayed. It appears that flavodoxin is expressed as an early-stage response to Fe stress and that its accumulation need not be intimately connected to limitations imposed by Fe on the growth rate of these diatoms.     

Genetic indicators of iron limitation in wild populations of Thalassiosira oceanica from the northeast Pacific Ocean

Assessing the iron (Fe) nutritional status of natural diatom populations has proven challenging as physiological and molecular responses can differ in diatoms of the same genus. We evaluated expression of genes encoding flavodoxin (FLDA1) and an Fe-starvation induced protein (ISIP3) as indicators of Fe limitation in the marine diatom Thalassiosira oceanica. The specificity of the response to Fe limitation was tested in cultures grown under Fe- and macronutrient-deficient conditions, as well as throughout the diurnal light cycle. Both genes showed a robust and specific response to Fe limitation in laboratory cultures and were detected in small volume samples collected from the northeast Pacific, demonstrating the sensitivity of this method. Overall, FLDA1 and ISIP3 expression was inversely related to Fe concentrations and offered insight into the Fe nutritional health of T. oceanica in the field. As T. oceanica is a species tolerant to low Fe, indications of Fe limitation in T. oceanica populations may serve as a proxy for severe Fe stress in the overall diatom community. At two shallow coastal locations, FLD1A and ISIP3 expression revealed Fe stress in areas where dissolved Fe concentrations were high, demonstrating that this approach may be powerful for identifying regions where Fe supply may not be biologically available.     

ACCUMULATION OF FERREDOXIN AND FLAVODOXIN IN A MARINE DIATOM IN RESPONSE TO FE

Despite recognition that Fe availability is significant in regulating oceanic production in some regions, the biogeochemistry of this trace element is poorly understood. To complement contemporary methods of analytical chemistry, we have used an immunological approach to monitor the Fe nutrition of marine phytoplankton. In prokaryotes and numerous microalgae, the redox catalyst ferredoxin is functionally replaced by flavodoxin during periods of Fe deficiency. In this study, antibodies were raised against ferredoxin purified from a marine diatom, and their utility as a diagnostic indicator was assessed. A species survey demonstrated broad reactivity with both pennate and centric diatoms and additionally with several nondiatom taxa. In batch cultures of the diatom Phaeodactylum tricornutum Bohlin, in which Fe levels were varied, accumulation of ferredoxin varied with the physiological state of the culture; in unimpaired cells (F_v/F_m≥ 0.65), ferredoxin levels were high, whereas levels dropped markedly in cells experiencing even slight photochemical impairment. Accumulation of flavodoxin varied inversely with that of ferredoxin. An experiment was performed to demonstrate the temporal pattern of accumulation of ferredoxin upon recovery from Fe limitation. Prior to Fe amendment, cells were physiologically impaired (chlorotic, F_v/F_m < 0.3) and contained flavodoxin but no detectable ferredoxin. Following addition of Fe, constraints on photochemistry were relaxed within hours. Coinciding with this, levels of flavodoxin declined, whereas ferredoxin was accumulated to high levels within 8 h.     

DEVELOPMENT OF IMMUNOASSAYS FOR THE IRON‐REGULATED PROTEINS FERREDOXIN AND FLAVODOXIN IN POLAR MICROALGAE1

While the growth of Southern Ocean phytoplankton is often limited by iron availability, there are no comparable experiments on sea‐ice algae. Here we assess the use of ferredoxin and flavodoxin to investigate the iron nutritional status of sea‐ice algae and describe the development of a quantitative immunoassay for both proteins in marine diatoms. High‐affinity monoclonal antibodies toward both proteins were produced from Cylindrotheca closterium (Ehrenb.) J. M. Lewin et Reimann, and these were used to develop Western blots. Western blots run on whole protein extracts detected both proteins with little cross‐reactivity toward other proteins. The two proteins could be successfully quantitated when applied to gels at between 5 and 50 ng in a volume of 25 μL (0.2–2 μg · mL^?1). Flavodoxin and ferrodoxin expression was examined in the Antarctic diatoms Entomoneis kjellmannii (Cleve) Poulin et Cardinal, Navicula directa (W. Sm.) Ralfs, Fragilariopsis curta (Van Heurck) Hust., Pseudo‐nitzschia sp., Porosira glacialis (Grunow) E. G. Jørg., Fragilariopsis cylindrus (Grunow) Willi Krieg., Fragilariopsis sublinearis (Van Heurck) Heiden et Kolbe, C. closterium, Nitzschia lecointei Van Heurck, and the dinoflagellate Polarella glacialis Montresor, Procaccini et Stoecker. Two Arctic isolates were also examined, Nitzschia frigida (Grunow) and Fragilariopsis oceanica (Cleve) Hasle. Significant heterogeneity of protein expression was observed despite all cultures being grown in iron‐replete f/2 medium. Only one species, F. cylindrus, displayed the expected expression of ferredoxin only in iron‐replete medium. Four were observed to produce both proteins under iron‐replete conditions. Ferredoxin was not detected at all in F. curta and Pseudo‐nitzschia sp., but distinct flavodoxin bands were observed in both of these organisms. All species examined were observed to express either flavodoxin or ferredoxin or both of the proteins as determined by Western immunoblotting.     

INDUCTION OF SPECIFIC PROTEINS IN EUKARYOTIC ALGAE GROWN UNDER IRON-, PHOSPHORUS-, OR NITROGEN-DEFICIENT CONDITIONS1

What limits phytoplankton growth in nature? The answer is elusive because of methodological problems associated with bottle incubations and nutrient addition experiments. We are investigating the possibility that antibodies to proteins repressed by a specific nutrient can be used as probes to indicate which nutrient limits photosynthetic carbon fixation in the ocean. The diatom Phaeodactylum tricornutum Bohlin and the chlorophyte Dunaliella tertiolecta Butcher were grown in batch cultures in artificial seawater and f/2 nutrient lacking either phosphorus, iron, or nitrogen. Chlorosis was induced by nutrient limitation in both species with the exception of phosphorus-limited D. tertiolecta. The synthesis and appearance of specific proteins were followed by labeling with ¹⁴C-bicarbonate. Nutrient limitation in general leads to a decrease in the quantum efficiency of photosystem II, suggesting that deficiency of any nutrient affects the photosynthetic apparatus to some degree: however, the effect of nitrogen and iron limitation on quantum efficiency is more severe than that of phosphorus. A crude fractionation of the soluble and membrane proteins demonstrated that the large proteins induced under limitation by phosphorus and iron were associated with the membranes. However, small iron-repressible proteins were located in the soluble fraction. Isolation with anion-exchange chromatography and N-terminal sequencing of iron-repressible, 23-kDa Proteins from D. tertiolecta, P. tricornutum, and Chaetoceros gracilis revealed that these small soluble proteins have strong homology with the N-terminal sequence of flavodoxins from Azotobacter and Clostridium. The identity of the flavodoxin from D. tertiolecta was confirmed by immunodetection using antiflavodoxin raised against Chlorella. Flavodoxin was detected only under iron deprivation and was absent from nitrogen-and phosphorus-limited algae. Flavodoxin is a prime candidate for a molecular probe of iron limitation in the ocean. The requirements to confirm its utility in nature are discussed.     

A Study of Iron Acquisition Mechanisms of Legionella pneumophila Grown in Chemostat Culture

We recently demonstrated that the virulence of a clinical isolate of Legionella pneumophila is significantly attenuated when cultured in an iron-limited environment. In this study the influence of iron limitation on the expression of enzyme activities and iron-transport mechanisms was investigated. Expression of the important pathogenicity factor, the zinc metalloprotease, was reduced fivefold in response to iron limitation. Ferric citrate reductase activity was demonstrated in both iron-limited and replete cell fractions. Activity was located principally in the cytoplasm and periplasm, and was not enhanced by iron restriction. Optimum activity was observed with NADPH as reductant. Siderophores were not elaborated under these culture conditions. Iron-loaded transferrin enhanced the growth of steady-state, iron-limited cultures, demonstrating that transferrin represents a potentially important iron source for L. pneumophila in vivo. Although cell surface transferrin receptors were not detected, in vitro experiments demonstrated digestion of transferrin by the zinc metalloprotease activity of culture supernatants. Received: 3 July 1996 / Accepted: 3 October 1996     

Growth of harmful marine algae in multispecies cultures

Mixtures of harmful and harmless algae were grown in discontinuouslydiluted batch cultures under ammonium, nitrate and phosphatelimitation, and at different irradiances (20–500 µjnolquanta m^–2 s^–1). The species used were Chrysochromulinapolylepis, Emiliania huxleyi type B, Rhodomonas sp., the dinoflagellalesFibrocapsa japonica, Gymnodinium simplex, Gyrodinium aure-olum,Heterocapsa triquetra, Heterosigma carterae, Prorocentrum micansand Alexandrium tamarense, the diatoms Chaetoceros socialis,Cymatosira belgica, Ditylum brightwellii, Laudcria borealis,Odon-telta aunla, Pseudonitzschia pungens, Streptotheca tamesis,and the cyanobacterium Synechococcus. Their growth responsein the mixed algal cultures is discussed in relation to theirabundance in different natural habitats. In comparison withthe other non-diatoms, the mixotrophic C.polylepis grew fastunder all tested nutrient and light limitations.Emiliania huxleyigrew well under nitrogen (N) limitation (with nitrate as N source)and at irradiance levels from 15 up to 500 µmol quantam^–2 s^–1. No growth of calcifying cells could bedetected under N limitation when ammonium was used as N source.Rhodomonas grew reasonably well under ammonium-N limitationand grew fast at the highest irradi-ance. The dinoflagellateswere poor competitors compared to the Prymnesiophyceae. Theenvironmental fitness of the Prymnesiophyceae appears to beclosely related to the reproductive capacity of the vegetativestage, whereas the natural distribution of dinoflagellates seemsmore closely dependent on the generative reproduction-relatedspecific life cycle characteristics of the individual species.The marine diatoms include a mixture of both types of species.Some marine diatom species clearly have the capability to outcompetenon-diatoms under different types of nutrient and light limitationswhen silicate is in excess. Other diatoms seem to be poor competitors.     

SALINITY TOLERANCE OF DIATOMS FROM THALASSIC HYPERSALINE ENVIRONMENTS

Thirty-four benthic diatom strains were isolated from thalassic hypersaline marine environments and their salinity tolerance characterized in growth experiments conducted at salinities ranging from 0.5% to 17.5% (weight of total salts per volume, g·100 mL ^{− 1}). The results were compared with the patterns of diatom species distribution and abundance in hypersaline evaporation ponds and tidal channels of Guerrero Negro, Baja California Sur, Mexico. The isolated strains were representative of the diatom assemblages present in the saltern ponds but were less so of natural assemblages in tidal channels. In general, we found a clear decreasing trend of diatom diversity in the field and in the isolated strains with increasing salinity. With some exceptions, the upper limit of salinity tolerance in cultivated strains corresponded to their distribution in field samples. However, the relative abundance of species in the field was not correlated with growth rates achieved in culture for the same salinities. Most cultured strains exhibited extreme euryhalinity growing well from brackish to hypersaline conditions, but the particulars of salt tolerance were quite diverse among strains. The most halotolerant taxa, two Amphora species, Amphora cf. subacutiuscula Schoeman, Nitzschia fusiformis Grunow, and Entomoneis sp., grew well in salinities ranging from 0.5% to 15%. Three strains of Pleurosigma strigosum W. Smith that were unable to grow in salinities less than 5% total salts represent the only true halophilic diatoms ever reported. The fact that many strains displayed a remarkable halotolerance, with optimal or near-optimal growth rates at salinities as high as three times that of seawater, implies that diatoms from hypersaline environments are evolutionarily highly adapted to such environments.     

IMPACT OF IRON LIMITATION ON THE PHOTOSYNTHETIC APPARATUS OF THE DIATOM CHAETOCEROS MUELLERI (BACILLARIOPHYCEAE)

Iron starvation induced marked increases in flavodoxin abundance and decreases in light-saturated and light-limited photosynthesis rates in the diatom Chaetoceros muelleri. Consistent with the substitution of flavodoxin for ferredoxin as an early response to iron starvation, increases of flavodoxin abundance were observed before declines of cell division rate or chl a specific photosynthesis rates. Changes in the abundance of flavodoxin after the addition of iron to iron-starved cells indicated that flavodoxin was not actively degraded under iron-replete conditions. Greater declines in light-saturated oxygen evolution rates than dark oxygen consumption rates indicated that the mitochondrial electron transfer chain was not affected as greatly by iron starvation as the photosynthetic electron transfer chain. The carbon:nitrogen ratio was unaffected by iron starvation, suggesting that photosynthetic electron transfer was a primary target of iron starvation and that reductions in nitrate assimilation were due to energy limitation (the C:N ratio would be expected to rise under nitrogen-limited but energy-replete conditions). Parallel changes were observed in the maximum light-saturated photosynthesis rate and the light-limited initial slope of the photosynthesis-light curve during iron starvation and recovery. The lowest photosynthesis rates were observed in iron-starved cells and the highest values in iron-replete cells. The light saturation parameter, I_k, was not affected by iron starvation, nor was the chl-to-C ratio markedly reduced. These observations were consistent with iron starvation having a similar or greater effect on photochemical charge separation in PSII than on downstream electron transfer steps. Declines of the ratio of variable to maximum fluorescence in iron-starved cells were consistent with PSII being a primary target of iron starvation. The functional cross-section of PSII was affected only marginally (<20%) by iron starvation, with the largest values observed in iron-starved cells. The rate constant for electron transfer calculated from fast repetition rate fluorescence was found to covary with the light-saturated photosynthesis rate; it was lowest in the most severely starved cells.     

Diatom elemental and morphological changes in response to iron limitation: a brief review with potential paleoceanographic applications

Diatoms are a major group of phytoplankton that account for approximately 40% of the ocean carbon fixation and the vast majority of biogenic silica production through the construction of their cell walls (termed frustules). These frustules accumulate and are partially preserved in the ocean sediments. Diatom growth and nutrient utilization in high‐nitrate, low‐chlorophyll regions of the world’s oceans are mostly regulated by iron availability. Diatoms acclimate to iron limitation by decreasing cell size. The associated increase in surface area‐to‐volume ratio and decrease in diffusive boundary layer thickness may improve nutrient uptake kinetics. In parallel, cellular silicon (Si) contents are elevated in iron‐limited diatoms relative to nitrogen (N) and carbon (C). Variations in degree of silicification and nutritional requirements of iron‐limited diatoms have been hypothesized to account for higher cellular Si and/or lower cellular N and C, respectively. However, in some diatoms, frustule silicification does not significantly change when cells are iron‐limited. Instead, changes in the Si‐containing valve surface area relative to volume within these diatoms is hypothesized to be responsible for the variations in the cellular Si : N and Si : C ratios. In particular, some examined iron‐limited pennate diatoms have reduced widths relative to their lengths (i.e. lower length‐normalized widths, LNW) compared to iron‐replete cells. In the pennate diatom Fragilariopsis kerguelensis, the mean LNWs of valves preserved in sediments throughout the Southern Ocean (a well‐characterized iron‐limited region) is positively correlated with satellite‐derived, climatological net primary productivity in the overlying waters. Because of the specific morphological changes in pennate diatom frustules in response to iron availability, the valve morphometerics (e.g. LNWs) can potentially be used as a diagnostic tool for iron‐limited diatom growth and relative changes in the Si : N (and Si : C) ratios in extant diatom assemblages as well as those preserved in the sediments.     

Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

EFFECTS OF IRON AND LIGHT STRESS ON THE BIOCHEMICAL COMPOSITION OF ANTARCTIC PHAEOCYSTIS SP. (PRYMNESIOPHYCEAE). I. INTRACELLULAR DMSP CONCENTRATIONS

Iron is essential for phytoplankton growth, as it is involved in many metabolic processes. It controls photosynthesis as well as many enzymatic processes. As such, iron affects the cell's energy supply and contributes to the assimilation of carbon and nitrogen. To determine whether iron limitation would result in energy stress or induced nitrogen deficiency, an Antarctic Phaeocystis sp. (Prymnesiophyceae) strain was studied for its biochemical composition, with the main emphasis on intracellular production of dimethylsulfoniopropionate (DMSP). DMSP is suggested to replace nitrogen containing solutes under conditions of nitrogen deficiency. Batch cultures of Antarctic Phaeocystis sp. were grown under iron-rich and iron-poor conditions and simultaneously subjected to high and low light intensities. Iron depletion induced chlorosis and suppressed growth rates as well as the maximum yield of the cultures; these effects were reinforced by low light intensities. Cell volumes were strongly reduced under iron-limited conditions. However, this reduction in cell volume was accompanied by a reduced DMSP content only in cultures experiencing low light intensities. Under high light conditions, no reduction of DMSP was observed; hence, intracellular DMSP concentrations increased. These observations are discussed relative to carbon and nitrogen metabolism and the biosynthetic pathway of DMSP. It is argued that under high light, low iron conditions, the cells were bordering on nitrogen deficiency induced by iron limitation, whereas under low light, low iron conditions, the cells were energy limited resulting in overall suppressed metabolic rates. Between treatments, DMSP to chlorophyll- a ratios varied by a factor of 5, demonstrating the dependence of this parameter on the physiological state of the cell.     

Consequences of the iron-dependent formation of ferredoxin and flavodoxin on photosynthesis and nitrogen fixation on Anabaena strains

Iron-dependent formation of ferredoxin and flavodoxin was determined in Anabaena ATCC 29413 and ATCC 29211 by a FPLC procedure. In the first species ferredoxin is replaced by flavodoxin at low iron levels in the vegetative cells only. In the heterocysts from Anabaena ATCC 29151, however, flavodoxin is constitutively formed regardless of the iron supply.Replacement of ferredoxin by flavodoxin had no effect on photosynthetic electron transport, whereas nitrogen fixation was decreased under low iron conditions. As ferredoxin and flavodoxin exhibited the same K_m values as electron donors to nitrogenase, an iron-limited synthesis of active nitrogenase was assumed as the reason for inhibited nitrogen fixation. Anabaena ATCC 29211 generally lacks the potential to synthesize flavodoxin. Under iron-starvation conditions, ferredoxin synthesis is limited, with a negative effect on photosynthetic oxygen evolution.     

Metabolomic Profiling of 13 Diatom Cultures and Their Adaptation to Nitrate-Limited Growth Conditions

Diatoms are very efficient in their use of available nutrients. Changes in nutrient availability influence the metabolism and the composition of the cell constituents. Since diatoms are valuable candidates to search for oil producing algae, measurements of diatom-produced compounds can be very useful for biotechnology. In order to explore the diversity of lipophilic compounds produced by diatoms, we describe the results from an analysis of 13 diatom strains. With the help of a lipidomics platform, which combines an UPLC separation with a high resolution/high mass accuracy mass spectrometer, we were able to measure and annotate 142 lipid species. Out of these, 32 were present in all 13 cultures. The annotated lipid features belong to six classes of glycerolipids. The data obtained from the measurements were used to create lipidomic profiles. The metabolomic overview of analysed cultures is amended by the measurement of 96 polar compounds. To further increase the lipid diversity and gain insight into metabolomic adaptation to nitrogen limitation, diatoms were cultured in media with high and low concentrations of nitrate. The growth in nitrogen-deplete or nitrogen-replete conditions affects metabolite accumulation but has no major influence on the species-specific metabolomic profile. Thus, the genetic component is stronger in determining metabolic patterns than nitrogen levels. Therefore, lipid profiling is powerful enough to be used as a molecular fingerprint for diatom cultures. Furthermore, an increase of triacylglycerol (TAG) accumulation was observed in low nitrogen samples, although this trend was not consistent across all 13 diatom strains. Overall, our results expand the current understanding of metabolomics diversity in diatoms and confirm their potential value for producing lipids for either bioenergy or as feed stock.     

东海南部陆缘(莆、泉段)全新世沉积硅藻

对东海南部陆缘莆田、泉州地区两口钻井岩芯系统的沉积硅藻研究,共发现33属117种（或变种）硅藻化石,根据剖面硅藻组合特征的变化,结合计算机的对应分析结果划分了硅藻带,建立该区全新世的7个硅藻组合序列,恢复当时古环境演变的7个阶段,填补该区沉积硅藻系统研究的空白,丰富了海陆过渡沉积硅藻的研究。     

Iron,silicate, and light co-limitation of three Southern Ocean diatom species

The effect of combined iron, silicate, and light co-limitation was investigated in the three diatom species Actinocyclus sp. Ehrenberg, Chaetoceros dichaeta Ehrenberg, and Chaetoceros debilis Cleve, isolated from the Southern Ocean (SO). Growth of all species was co-limited by iron and silicate, reflected in a significant increase in the number of cell divisions compared to the control. Lowest relative Si uptake and drastic frustule malformation was found under iron and silicate co-limitation in C. dichaeta, while Si limitation in general caused cell elongation in both Chaetoceros species. Higher light intensities similar to SO surface conditions showed a negative impact on growth of C. dichaeta and Actinocyclus sp. and no effect on C. debilis. This is in contrast to the assumed light limitation of SO diatoms due to deep wind driven mixing. Our results suggest that growth and species composition of Southern Ocean diatoms is influenced by a sensitive interaction of the abiotic factors, iron, silicate, and light.     

GROWTH OF VITAMIN B12-REQUIRING MARINE DIATOMS IN MIXED LABORATORY CULTURES WITH VITAMIN B12-PRODUCING MARINE BACTERIA12

The vitamin B₁₂ requirement of several marine diatoms can be satisfied in B₁₂^?limited laboratory cultures by heterotrophic marine bacteria isolated from the same waters and from sediments. The bacteria can utilize diatom excretory products, or the remains of dead diatom cells, in the production of the vitamin. The growth of 12 B₁₂¹? requiring diatoms (7 genera) in mixed cultures with 14 different bacteria (without added B₁₂) was compared to the growth of those same diatoms in axenic cultures with excess added B₁₂. Diatom growth was generally rapid in the first few days, followed by sustained, slower growth. The diatom yields in mixed cultures ranged from 0.8 to 84% of the yields in axenic cultures with added B₁₂. In a detailed study of one mixed culture, increases in diatom densities were paralleled by increases in cell densities of the bacterium during the first few days of exponential diatom growth. During the period of slow diatom growth, when diatom densities oscillated but steadily increased, the decreases in diatom densities were associated with increased bacterial growth. This suggests that death of a fraction of the B₁₂-limited diatom population releases sufficient organic matter to stimulate growth of the bacteria and their subsequent excretion of B₁₂; this B₁₂ in turn stimulates further growth of the diatoms. Diatom-bacteria interactions leading to the production of B₁₂ may be important in maintaining viable populations of B₁₂-requiring diatoms in nutrient-poor waters during periods between blooms, when conditions are unfavorable for rapid growth.