DIATOM MINERALIZATION OK SILICIC ACID. I Si(OH)4 TRANSPORT CHARACTERISTICS IN NAVICULA PELLICULOSA

Silicic acid transport was studied in the photosynthetic diatom Navicula pelliculosa (Bréb.) Hilse using [⁶⁸Ge] germanic acid (⁶⁸Ge(OH)₄) as a tracer of silicic acid (Si(OH)₄). The initial uptake rate of Si(OH)₄ was dependent on cell number, pH, temperature, light and was promoted by certain monovalent cations in the medium. Na⁺ was more effective than K⁺, whereas Li⁺ and NH⁺₄ were ineffective at promoting uptake. Uncouplers and inhibitors of oxidative phosphorylation and of photophosphorylation reduced uptake by 40–99% of control values. Uptake was also especially sensitive to the sulfhydryl blocking agents at 10^?5 M and to the ionophorous compound valinomycin (10^?7 M) which inhibited uptake by 82%. The Si(OH)₄ transport system displayed Michaelis-Menten-type saturation kinetics with kinetic parameters of K_S= 4.4 p. mol Si(OH)₄· 1^?1, V_max= 334 pmol Si(OH)₄· 10⁶ cells^?1· min^?1. Calculations of the acid soluble silicic acid pool size based on 60 s uptake at 20 μM Si(OH)₄ suggested that intracellular levels of Si could reach 20 mM and as much as 5 mM could exist as free silicic acid, representing maintenance of a 250-fold concentration gradient compared with the medium. Efflux from preloaded cells was dependent on temperature and the Si(OH)₄ concentration of the external medium. In the presence of 100 μMM “cold” Si(OH)₄, approximately 30% of the Si(OH)₄ in preloaded cells was exchanged in 20 min. The initial uptake rate of Si(OH)₄ in logarithmic phase cells was constant, but the uptake rate increased in a linear fashion for 6 h in stationary phase cells. These results suggest that the first step in silica mineralization by diatoms is the active transmembrane transport of Si(OH)₄ by an energy dependent, saturable, membrane-carrier mechanism which requires the monovalent cations Na⁺ and K⁺ and is sensitive to sulfhydryl blocking agents. Silicic acid transport activity also appears to be regulated during different growth stages of the diatom.     

A STUDY OF Si DEPOSITION SYNCHRONY IN RHIZOSOLENIA (BACILLARIOPHYCEAE) MATS USING A NOVEL 32Si AUTORADIOGRAPHIC METHOD

A ³²Si autoradiographic technique using a liquid photographic emulsion was developed for the study of diatom silica deposition in culture or in natural water samples. The method was used in the Central North Pacific to study silica deposition by diatoms of the genus Rhizosolenia. The species examined form centimeter-sized aggregates commonly referred to as mats. The Rhizosolenia mats examined were composed of a matrix of R. fallax Sundström chains, embedded with chains of larger cells, either R. debyana H. Peragallo or R. acuminata H. Peragallo. The autoradiographs revealed distinct rings of labeled intercalary bands and/or labeled valves. A greater proportion of the frustule of the larger species was labeled during the incubations with ³²Si, implying higher rates of silicification by R. debyana and R. accuminata compared to R. fallax. A quantitative consideration of these differences in species-specific Si production combined with abundance and surface area estimates for each species indicates that cells of the larger species carry out the majority of silica production in Rhizosolenia mats. The large cell size (pervalvar axis 240 to 3000 μm) and elongate frustule morphology of Rhizosolenia cells enabled us to localize the deposition of silica along the pervalvar axis. Positions of labeled bands along this axis indicate progress through the Si deposition cycle, and the results suggest that cell division is phased, with either a bimodal or unimodal age distribution of cells within the cell cycle for all species in a mat. Species-specific doubling times from 25 to 60 h were implied by the mean fractions of frustule that were labeled. ³²Si autoradiography revealed unique species-specific differences in diel patterns of cell division and silica deposition and has potential for studies of Si deposition by other diatom species and assemblages.     

METHYLAMINE UPTAKE IN THE GREEN ALGA CHLORELLA PYRENOIDOSA1

Methylamine uptake in nitrogen-starved Chlorella pyrenoidosa Beij. follows Michaelis-Menten kinetics: maximum uptake is about 1.6 nmol μl^?1· cells · min^?1, half-saturation occurs at 4 μM methylamine, and the slope in the range where uptake is proportional to concentration is 0.4 nmol μl^?1· min^?1·μM^?1. In cells grown in the presence of a non-limiting nitrogen concentration, methylamine uptake is directly proportional to concentration up to at least 0.5 mM, and the slope is 1/500 that for starved cells. Similar uptake kinetics have been reported for Penicillium chrysogenum and attributed to an inducible “ammonium permease.” Apparently, a similar permease occurs in algae.     

SIMULTANEOUS ASSIMILATION OF AMMONIUM AND NITRATE BY GELIDIUM NUDIFRONS (GELIDIALES: RHODOPHYTA)1

Simultaneous assimilation of NH₄ and NO₃ by Gelidium nudifrons Gardner was observed in culture experiments of 4 possible combinations of NH₄ and NO₃. The combinations tested were those in which the concentration of both N sources were in the range of 3.0–4.0 μg-atN · l^?1; both in the range of 0.5–1.0 μg-atN · l^?1; one in the 3.0–4.0 μg-atN · l^?1 range and the other in the 0.5–1.0 μg-atN · l^?1 range; and, visa versa. The data suggest that the pools of both NH₄ and NO₃ are simultaneously available for algal assimilation.     

Diatom mineralization of silicic acid

Silicic acid binding, possibly at a membrane site is suggested as the first step in Si(OH)₄ metabolism in the marine diatom Nitzschia angularis. We estimate 1738±133 Si(OH)₄ binding sites·m^-2 cell surface and a turnover of 4–12 Si(OH)₄ molecules·s^-1 at maximum transport velocity. All studies were carried out using ⁶⁸Ge(OH)₄ as a tracer for Si(OH)₄.After Si(OH)₄ uptake, rapid (<1 min) transformation or intracellular binding was demonstrated; yet the extractable pool size after 1 min was indicative of transport against a concentration gradient. Initial uptake kinetics were linear for 150 s and saturation kinetics were demonstrated with kinetic parameters of 560 pmol Si(OH)₄·10⁶ cells^-1·min^-1 (V _max) 4.2 mol Si(OH)₄·L^-1(K_s). A metabolic energy requirement for transport was suggested by inhibition of uptake by agents that uncouple or inhibit phosphorylation; transport also was sensitive to agents that block sulfhydryl groups. Such characteristics are consistent with Si(OH)₄ transport being an active carrier mediated process.Rates of Si(OH)₄ transport were regulated during various growth stages and during the synchronized cell cycle. In Si(OH)₄ starved cells, blocked at the initiation of silica frustule formation, cycloheximide treatment caused a rapid decline of transport rate. Blocked cells, placed in the dark, maintained high transport rates for 6 h after which there was a loss of activity during the following 18 h. Light dependent recovery of transport ability in 12 h predarkened cells was dependent on de novo protein synthesis.Abbreviations ASW artificial sea water - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DCMU 3(3,4-dichlorophenyl-1,1-dimethyl urea) - 2,4-DNP 2,4-dinitrophenol - ESWT enriched sea water with tryptone - NEM n-ethylmaleimide - pCMB p-chloromercuribenzoic acid     

Control of chitin nanofiber production by the lipid‐producing diatom Cyclotella Sp. through fed‐batch addition of dissolved silicon and nitrate in a bubble‐column photobioreactor

Diatoms are single‐celled algae that make cell walls of nanopatterned biogenic silica called frustules through metabolic uptake of dissolved silicon and its templated condensation into biosilica. The centric marine diatom Cyclotella sp. also produces intracellular lipids and the valued coproduct chitin, an N‐acetyl glucosamine biopolymer that is extruded from selected frustule pores as pure nanofibers. The goal of this study was to develop a nutrient feeding strategy to control the production of chitin nanofibers from Cyclotella with the coproduction of biofuel lipids. A two‐stage phototrophic cultivation process was developed where Stage I set the cell suspension to a silicon‐starved state under batch operation, and Stage II continuously added silicon and nitrate to the silicon‐starved cells to enable one more cell doubling to 4 × 10⁶ cells mL^?1. The silicon delivery rate was set to enable a silicon‐limited cell division rate under cumulative delivery of 0.8 mM Si and 1.2 mM nitrate (1.5:1 mol N/mol Si) over a 4‐ to 14‐day addition period. In Stage II, both cell number and chitin production were linear with time. Cell number and the specific chitin production rate increased linearly with increasing silicon delivery rate to achieve cumulative product yields of 13 ± 1 mg chitin/10⁹ cells and 33 ± 3 mg lipid/10⁹ cells. Therefore, chitin production is controlled through cell division, which is externally controlled through silicon delivery. Lipid production was not linearly correlated to silicon delivery and occurred primarily during Stage I, just after the complete co‐consumption of both dissolved silicon and nitrate. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:407–415, 2017     

EFFECT OF SILICATE LIMITATION ON VALVE MORPHOLOGY IN THALASSIOSIRA AND COSCINODISCUS (BACILLARIOPHYCEAE)1

Coscinodiscus radiatus Ehrenb. and Thalassiosira eccentrica (Ehrenb.) Cleve were grown in a silicate-limited chemostat at silicate concentrations below 1 μg-atoms · l^?1. The resulting abnormal valves of C. radiatus lacked a thickened ring around the foramina; their pore membranes were thinner and their loculi shallower than those in normal cells. Abnormal valves of T. eccentrica had a fasciculate areolae pattern; they lacked a silica covering over the foramina and some tangential areolae walls. Neither abnormal valve could be termed a new species.     

EFFECTS OF Si:P CONCENTRATION RATIOS AND NUTRIENT LIMITATION ON THE CELLULAR COMPOSITION AND MORPHOLOGY OF ASTERIONELLA FORMOSA (BACILLARIOPHYCEAE)1

The freshwater diatom Asterionella formosa Haas. was grown in semicontinuous culture at 20°C under continuous cool-white fluorescent light of ca. 20 μEin · m^?2· s ·^?1 in a medium containing Si: P in various concentration ratios. The cell quotas of P and Si changed in relation to the available concentrations of P and Si at constant μ= 0.11 and 0.16 d^?1. Under Si-limitation, the P cell quota increased by over an order of magnitude as the influent [Si:P] decreased. The Si cell quota increased with increase in [Si] in the influent medium, and it increased as [P] increased at a specific [Si]. Under P-limitation, the P cell quotas were fairly constant and low; the Si cell quotas were relatively high and decreased slightly as influent [P] and [Si] increased. Asterionella stored up to 28 times more P and 2 times more Si than needed. The number of Asterionella cells per colony varied as a function of the influent [Si:P] and nutrient limitation being usually less than or equal to 6 when P-limited, and greater than 10 when Si-limited.     

EFFECTS OF ENVIRONMENTAL VARIATION ON SINKING RATES OF MARINE PHYTOPLANKTON1

The effects of environmental variables, particularly irradiance, on the sinking rates of phytoplankton were investigated using cultures of Chaetoceros gracilis Schütt and C. flexuosum Mangin in laboratory experiments; these data were compared with results from assemblages in the open ocean and marginal ice zone of the Greenland Sea. In culture experiments both the irradiance under which the diatom was grown and culture growth rate were positively correlated with sinking rates. Sinking rates (ψ) in the Greenland Sea were smallest when determined from chlorophyll (mean ψ_chl= 0.14 m · d^?1) and biogenic silica (ψ_si= 0.14 m · d^?1) and greatest when determined from particulate carbon (ψ_c= 0.55 m · d^?1) and nitrogen (ψ_N= 0.64 m · d^?1). Field measurements indicated that variations in sinking may be associated with changes in irradiance and nitrate concentrations. Because these factors do not directly affect water density, they must be inducing physiological changes in the cell which affect buoyancy. Although a direct response to a single environmental variable was not always evident, sinking rates were positively correlated with growth rates in the marginal ice zone, further indicating a connection to physiological processes. Estimats of carbon flux at stations with vertically mixed euphotic zones indicated that approximately 30% of the daily primary production sank from the euphotic zone in the form of small particulates. Calculated carbon flux tended to increase with primary productivity.     

CHEMICAL COMPOSITION AND PHOTOSYNTHETIC CHARACTERISTICS OF ETHMODISCUS REX (BACILLARIOPHYCEAE): EVIDENCE FOR VERTICAL MIGRATION1

Ethmodiscus rex (Rattray) Wiseman and Hendey cells from near surface net tows in the Southwest Atlantic Ocean and Caribbean Sea were examined for chemical composition, internal nutrient pool concentrations, and oxygen evolution characteristics. Elemental ratios indicated nitrogen limitation with C:N:P ratios of 125:9:1 (atoms), and carbon: chlorophyll (chl) ratios of 129:1 (weight). However, internal nitrate pools (1.4–27.1 mM) suggested that cells were not N-limited. Intracellular NO₃^? accounted for up to 54% (range = 3–54%) of the total N quota in some samples. Photosynthetic parameters were consistent with a high-light-adapted population and suggested an instantaneous maximum chl-specific photosynthetic rate (P^B_max) of 4.8–12.4 nmol O₂·μg chl^?1· h^?1. Respiration rates varied ten-fold and were inversely related to P^B_max Ethmodiscus chemical composition and buoyancy characteristics are similar to vertically migrating Rhizosolenia mats and the non-motile dinoflagellate Pyrocystis noctiluca Murray (Schuett). The presence of internal NO₃^? pools in Ethmodiscus suggests that this genus is also vertically migrating to exploit sub-surface nitrogen pools. Such behavior may be widespread in large, non-motile oceanic phytoplankton. Based on ascent rate data, chemical composition, and photosynthetic rates, we estimate that the entire division–migration cycle for Ethmodiscus requires at least 7–12 days.     

KINETICS OF SILICON-LIMITED GROWTH IN THE FRESHWATER DIATOM ASTERIONELLA FORMOSA1,2

Growth rates of two clones of the freshwater planktonic diatom Asterionella formosa Hass. were measured under conditions in which external silicon concentrations controlled growth. Clone AfOH2 from Lake Ohrid, Yugoslavia, had a higher maximum growth rate (μ_max= 1.11 doublings/day) and apparent half-saturation constant (K_si] + Si_o= 1.93 μM Si) than clone L262 from Lake Windermere, England. (μ_max= 0.61 doublings/day; K_si+ Si_o= 1.09 μM Si). K_lim, the silicon concentration at μ= 0.9 μ_max, is 13.8 μM Si for clone AfOH2 and 6.5 μM Si for clone L262. These values agree well with published field observations showing A. formosa populations decreasing below 0.5 mg/l SiO₂ (= 8.4 μM Si). Calculations of yield gave a range of 0.5–1.5 μM Si/10⁶ cells for clone AfOH2 and 0.6–1.9 μM Si/10⁶ cells for clone L262.     

EFFECT OF THE SINKING RATE OF TWO DIATOMS (THALASSIOSIRA SPP.) ON UPTAKE FROM LOW CONCENTRATIONS OF PHOSPHATE1

The effect of the sinking rate, or rate of medium flow (φ) on the rate of phosphate incorporation (V) by the planktonic diatoms Thalassiosira fluviatilis Hust. and T. pseudonana Hasle & Heimdal in batch and chemostat cultures was determined by passing medium at defined flow rates (0.5–25.0 mm·min^?1) over algae on membrane filters. At concentrations from 1 to 100 μg phosphorus·l^?1 V, increases with increasing velocity of flow, approaching a maximum value (V_m) as described by the empirical relationship: where K_φ is the sinking rate value when V = 1/2 V_m+ V_o and V_o is the uptake at 0 rate of flow. By comparing uptake at controlled flow with uptake in a vigorously stirred medium, the phosphate concentration in the cell boundary layer can be determined. The sinking rate that reduces the phosphate concentration in the boundary layer to half of nominal concentration in the medium is much lower for the larger T. fluviatilis than for T. pseudonana. For both diatoms, it is inversely related to the nominal concentration.     

HOT WATER EXTRACTABLE PHOSPHORUS—AN INDICATOR OF NUTRITIONAL STATUS OF PERIDINIUM CINCTUM (DINOPHYCEAE) FROM LAKE KINNERET (ISRAEL)?1

The intracellular levels of hot water extractable and total phosphorus were determined in the dinoflagellate Peridinium cinctum. f. westii (Lemm.) Lef. for natural samples from the bloom in Lake Kinneret and from laboratory cultures. Amounts of phosphorus (P) in the hot water fraction, relative to total cellular phosphorus, were similar in lake Peridinium and in cells grown in high ambient orthophosphate (P_i) media (3–6 mg P · l^?1). The absolute amounts of hot water extractable P in natural cell and those cultured at lower P_i concentrations (0.02–0.05 mg P · 1^?1) were similar, although average P_i in lake water were 4 μg · l^?1. Under most growth conditions the hot water extract contained approximately equal amounts of molybdate reactive phosphorus (MRP) and non-MRP. Short chain (6–9 units) polyphosphates (mol wt 630–950) probably constituted the bulk of the non-MRP pool, which was hydrolysable by alkaline phosphatase and may serve as a precursor for a more permanent P store. Intracellular P levels and distribution were not directly dependent on external P_i concentrations but may be determined by the N:P atomic ratio or overall external ionic milieu. Peridinium grown in low ambient P_i released significant amounts of non-MRP compounds. In Lake Kinneret, for at least most of the bloom period, Peridinium does not appear to be limited by P supply.     

EFFECTS OF COPPER TOXICITY ON SILICIC ACID UPTAKE AND GROWTH IN THALASSIOSIRA PSEUDONANA11

The toxicity of Cu to Thalassiosira pseudonana (Hustedt) Hasle and Heimdal was investigated by examining both short and long term effects of Cu on cellular processes. Toxic levels of Cu (pCu* < 13) were found to inhibit short term Si(OH)₄ uptake rates with kinetics characteristic of irreversible inhibition at a hypothetical Cu-sensitive Si(OH)₄ transport site. Residual Si(OH)₄ concentrations (those below which no uptake could occur) were found to increase with increasing levels of Cu, and the toxic effects of Cu could be reversed by increasing the concentrations of Si(OH)₄ in the medium. The actual uptake of Cu by the cells was found to vary inversely with the ambient Si(OH)₄ concentration. Copper did not inhibit the uptake of NO₃^? or PO₄^3-. The long term inhibition of growth rate by Cu in this species was shown not to be a result of Si deficiency caused by the inhibition of Si(OH)₄ uptake. Cu inhibited cells were found to have higher Si cell quotas (including a sizable soluble pool) than the control cultures. They were, however, observed to have aberrant frustules, significantly larger than the control cells, suggesting interference with the silification process as a possible mechanism for inhibition of growth by Cu. A conceptual model is proposed for the Cu-Si(OH)₄-growth relationship. It includes a Cu sensitive Si(OH)₄ transport site that may also serve to transport Cu into the cell, and growth inhibition mediated by intracellular Cu concentrations which may block cell division or cause general cellular disfunction.     

EFFECT OF SEASONAL SEA ICE BREAKOUT ON THE PHOTOSYNTHESIS OF BENTHIC DIATOM MATS AT CASEY,ANTARCTICA1

Photosynthesis of marine benthic diatom mats was examined before and after sea ice breakout at a coastal site in eastern Antarctica (Casey). Before ice breakout the maximum under‐ice irradiance was between 2.5 and 8.2 μmol photons·m^?2·s^?1 and the benthic microalgal community was characterized by low E_k (12.1–32.3 μmol photons·m^?2·s^?1), low relETR_max (9.2–32.9), and high alpha (0.69–1.1). After breakout, 20 days later, the maximum irradiance had increased to between 293 and 840 μmol photons·m^?2·s^?1, E_k had increased by more than an order of magnitude (to 301–395 μmol photons·m^?2·s^?1), relETR_max had increased by more than five times (to 104–251), and alpha decreased by approximately 50% (to 0.42–0.68). During the same time interval the species composition of the mats changed, with a decline in the abundance of Trachyneis aspera (Karsten) Hustedt, Gyrosigma subsalsum Van Heurck, and Thalassiosira gracilis (Karsten) Hustedt and an increase in the abundance of Navicula glaciei Van Heurck. The benthic microalgal mats at Casey showed that species composition and photophysiology changed in response to the sudden natural increase in irradiance. This occurred through both succession shifts in the species composition of the mats and also an ability of individual cells to photoacclimate to the higher irradiances.     

THE GLUCOSE TRANSPORT SYSTEM OF AMPHORA COFFEAEFORMIS (BACILLARIOPHYCEAE)1

Amphora coffeaeformis (Ag.) Kütz. var. perpusilla (Grun.) Cleve took up glucose by an inducible transport system. The system was induced by d -fructose, d -mannose, as well as glucose. Some d -pentoses also induced a glucose uptake system but it may not be the same one as that induced by hexose. d -fructose, d -mannose and 2-deoxy-d -glucose inhibited 2 mM glucose uptake at equimolar concentration, but d -pentoses did not. The uptake system decayed in ca. 5 h in the absence of glucose. The half-saturation constant for uptake, K₈ was ca. 0.1 mM glucose with a maximum uptake rate, V_max= 0.4 nmol/10⁶ cells-min^?1.     

Radioisotopic Method for Measuring Cell Division Rates of Individual Species of Diatoms from Natural Populations

Silicon is an essential element for diatom frustule synthesis and is usually taken up only by dividing cells. With ⁶⁸Ge, a radioactive analog of Si, the cell cycle marker event of frustule formation was identified for individual species of diatom. The frequency of cells within a population undergoing this division event was estimated, and the cell division rate was calculated. In laboratory cultures, these rates of cell division and those calculated from changes in cell numbers were similar. By dual labeling with ⁶⁸Ge(OH)₄ and NaH¹⁴CO₃, rates of cell division and photosynthesis were coincidently measured for diatoms both in laboratory cultures and when isolated from natural populations in estuarine, offshore, and polar environments. These techniques permit the coupling between photosynthesis and cell division to be examined in situ for individual species of diatom.     

The relationships between gonad development and sex steroid levels at different ages in Acipenser schrenckii

The histological developments of the gonad and the associated sex steroid levels were determined in the breeding stocks of Acipenser schrenckii (age classes 1 to 5) maintained under natural temperature regimes (December 4°C; August 26°C). Early sex differentiation was observed in 1‐year‐old fish, while testosterone (T) and 17β‐estradiol (E₂) levels ranged from T 1.1 to 3.4 nmol l^?1 (average 1.8 nmol l^?1), and E₂ varied from 24 to 85 pmol l^?1 (av. 50.3 pmol l^?1). Gonadal status of 2‐year‐old males was in stage II while ovaries were at stage I, exhibiting T levels from 1.2 to 4.4 nmol l^?1 (av. 2.2 nmol l^?1), and E₂ concentrations from 10 to 97 pmol l^?1 (av. 38.9 pmol l^?1). At the age of 3 years, the testes in males were at developmental stage III while the ovaries remained in stage I, with T levels ranging from 1.3 to 21.7 nmol l^?1 (av. 9.6 nmol l^?1), and E₂ concentrations ranging from 17 to 108 pmol l^?1 (av. 44.8 pmol l^?1). At the age of 4 years, testes in males were at developmental stage III while ovaries in females had reached stage II, with T concentrations ranging from 7.3 to 52.6 nmol l^?1 (av. 26.3 nmol l^?1), and E₂ levels between 13 and 86 pmol l^?1 (average 55.3 pmmol l^?1). In 5‐year‐old fish, the testes reached maturity stage while the ovaries were mostly in stage III, with T values from 5.7 to 44.2 nmol l^?1 (av. 13.9 nmol l^?1), and E₂ concentrations from 21 to 453 pmol l^?1 (av. 137.7 pmol l^?1). Data demonstrated large differences in sex steroid levels among immature Amur sturgeon, and testicular maturation occurred earlier than ovarian maturation.