Electricity Production by Geobacter sulfurreducens Attached to Electrodes

Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (E^o′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m² of electrode surface) or poised-potential (163 to 1,143 mA/m²) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.     

Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1

Previous studies have suggested that the conductive pili of Geobacter sulfurreducens are essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain of G. sulfurreducens reduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of the c-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport through G. sulfurreducens biofilms is more effective via pili.     

Red tide blooms of Cochlodinium polykrikoides in a coastal cove

Successive blooms of the dinoflagellate Cochlodinium polykrikoides occurred in Pettaquamscutt Cove, RI, persisting from September through December 1980 and again from April through October 1981. Cell densities varied from <100 cells L⁻¹ at the onset of the bloom and reached a maximum density exceeding 3.4 × 10⁶ cells L⁻¹ during the summer of 1981. The bloom was mainly restricted to the mid to inner region of this shallow cove with greatest concentrations localized in surface waters of the southwestern region during summer/fall periods of both years. Highly motile cells consisting of single, double and multiple cell zooids were found as chains of 4 and 8 cells restricted to the late August/September periods. The highest cell densities occurred during periods when annual temperatures were between 19 and 28 °C and salinities between 25 and 30. A major nutrient source for the cove was Crying Brook, located at the innermost region at the head of the cove. Inorganic nitrogen (NH₃ and NO₂ + NO₃) from the brook was continually detectable throughout the study with maximum values of 57.5 and 82.5 μmol L⁻¹, respectively. Phosphate (PO₄-P) was always present in the source waters and rarely <0.5 μmol L⁻¹; silicate always exceeded 30 μmol L⁻¹ with maximum concentrations reaching 226 μmol L⁻¹. Chlorophyll a and ATP concentrations during the blooms varied directly with cell densities. Maximum Chl a levels were 218 mg m⁻³ and ATP-carbon was >20 g C m⁻³. Primary production by the dinoflagellate-dominated community during the bloom varied between 4.3 and 0.07 g C m⁻³ d⁻¹. Percent carbon turnover calculated from primary production values and ATP-carbon varied from 6 to 129% d⁻¹. The dinoflagellates dominated the entire summer period; other flagellates and diatoms were present in lesser amounts. A combination of low washout rate due to the cove dynamics, active growth, and life cycles involving cysts allowed C. polykrikoides to maintain recurrent bloom populations in this area.     

Root development and competitive ability of the invasive species Melaleuca quinquenervia (Cav.) S.T. Blake in the South Florida flatwoods

Melaleuca quinquenervia (Cav.) S.T. Blake is an aggressive, invasive species in sub-tropical Florida that is considered a serious threat to the existing biological integrity of many subtropical ecosystems in south Florida. It prevents other species from thriving through its high rate of seed production/germination and the formation of a dense tree canopy. However, its ability to take over a site between initial seedling establishment and crown closure is not well understood. The objective of this study was to determine (i) the nature of root development with time and soil depth, and (ii) the ability of M. quinquenervia to invade and absorb nutrients from soil already occupied by native vegetation. The working hypotheses were that M. quinquenervia captures a site either by (i) tolerating competition by prolifically growing roots into soil already occupied by native plants, or (ii) avoiding competition by rooting to depths where inter-root competition is less and water supply during a drought is available. Soil trenches and in-growth trays were used to measure root distribution and growth. Root number (# m^–2), root length density (m root m^–3 soil volume), and root biomass (g root m^–3 soil) were determined. This study demonstrated that M. quinquenervia (1) is a prolific rooter with or without the presence of competing vegetation; (2) can develop root densities higher than many mature native species at an early age; (3) can develop roots in the soil surface during soil drying periods, even while competitive grasses are dying out; (4) can develop a deep root system at an early age; and (5) is an effective rooter in both moist and dry water regimes in this fluctuating water table soil. The data suggested that this species is a strong competitor through the use of both competition avoidance and tolerance mechanisms and that the rooting habit of M. quinquenervia should be an important consideration when evaluating its ability as an invasive species.     

Enhancing electrical outputs of the fuel cells with Geobacter sulferreducens by overexpressing nanowire proteins

Protein nanowires are critical electroactive components for electron transfer of Geobacter sulfurreducens biofilm. To determine the applicability of the nanowire proteins in improving bioelectricity production, their genes including pilA, omcZ, omcS and omcT were overexpressed in G. sulfurreducens. The voltage outputs of the constructed strains were higher than that of the control strain with the empty vector (0.470–0.578 vs. 0.355 V) in microbial fuel cells (MFCs). As a result, the power density of the constructed strains (i.e. 1.39–1.58 W m⁻²) also increased by 2.62- to 2.97-fold as compared to that of the control strain. Overexpression of nanowire proteins also improved biofilm formation on electrodes with increased protein amount and thickness of biofilms. The normalized power outputs of the constructed strains were 0.18–0.20 W g⁻¹ that increased by 74% to 93% from that of the control strain. Bioelectrochemical analyses further revealed that the biofilms and MFCs with the constructed strains had stronger electroactivity and smaller internal resistance, respectively. Collectively, these results demonstrate for the first time that overexpression of nanowire proteins increases the biomass and electroactivity of anode-attached microbial biofilms. Moreover, this study provides a new way for enhancing the electrical outputs of MFCs.     

Response of cylindrospermopsin production and release in Aphanizomenon flos-aquae (Cyanobacteria) to varying light and temperature conditions

The influence of light and temperature on the cylindrospermopsin (CYN) production of two Aphanizomenon flos-aquae strains, isolated from North-eastern German lakes, was investigated with semi-continuously growing cultures. A light gradient from 10 to 60 μE m⁻² s⁻¹ in combination with temperatures of 16, 20, and 25 °C was tested.CYN concentrations varied by a maximum factor of 2.7 in strain 10E9 with a significant decrease with increasing temperature. Strain 22D11 showed less pronounced changes, i.e. by a factor of 1.6, and without clear relationship to temperature.Reaction patterns of CYN production to changing light intensities are different at different temperatures. In both strains CYN concentrations increase significantly at 20 °C between 10 and 60 μE m⁻² s⁻¹, whereas they decrease significantly at 25 °C in the same light gradient. The amount of synthesised CYN is not reflected by growth rates of the strains in a uniform manner. Nonetheless several temperature–light combinations which constitute physiological stress seem to trigger CYN production and particularly CYN release from cells. The lowest growth rate observed at 16 °C and 60 μE m⁻² s⁻¹ of strain 22D11 may reflect photoinhibition due to the lower temperature and related limited CO₂-fixation. Under these conditions, extracellular CYN concentrations increased to 58% of total CYN, while the share of extracellular CYN of all other light and temperature regimes was 11–26%. From the results and the experimental design we conclude an active release of the toxin into medium to be more likely than mere leakage from cells.     

Primary production and nutrient budgets of Sarcocornia perennis ssp. alpini (Lag.) Castroviejo in the salt marsh of the Palmones River estuary (Southern Spain)

Biomass, primary production and nutrient budgets associated to Sarcocornia perennis subspecies (ssp.) alpini were studied in the Palmones River estuary salt marsh (Southern Spain) to evaluate the nutrient sequestration capacity of the low marsh. Above- and belowground living and dead biomass, as well as carbon, nitrogen and phosphorus content were monitored during 1 year. Additionally, the fate of aboveground detritus was evaluated in an experiment on litter decomposition. The detritus production of S. perennis ssp. alpini was almost equivalent to its annual primary production indicating a rapid turnover of biomass. We calculated that only 12% of the aboveground detritus was exported out of the low marsh while the rest was decomposed in the sediment with a rate of 0.8 year⁻¹. Changes in concentrations of total carbon, nitrogen and phosphorus in the sediment showed patterns related to S. perennis ssp. alpini belowground biomass. Our results suggested that the sediment functions as a net sink for nutrients accumulating 550 g C m⁻² year⁻¹, 55 g N m⁻² year⁻¹, and 13 g P m⁻² year⁻¹.     

Investigation of biomass and lipids production with Neochloris oleoabundans in photobioreactor

The fresh water microalga Neochloris oleoabundans was investigated for its ability to accumulate lipids and especially triacylglycerols (TAG). A systematic study was conducted, from the determination of the growth medium to its characterization in an airlift photobioreactor. Without nutrient limitation, a maximal biomass areal productivity of 16.5 g m⁻² day⁻¹ was found. Effects of nitrogen starvation to induce lipids accumulation was next investigated. Due to initial N. oleoabundans total lipids high content (23% of dry weight), highest productivity was obtained without mineral limitation with a maximal total lipids productivity of 3.8 g m⁻² day⁻¹. Regarding TAG, an almost similar productivity was found whatever the protocol was: continuous production without mineral limitation (0.5 g m⁻² day⁻¹) or batch production with either sudden or progressive nitrogen deprivation (0.7 g m⁻² day⁻¹). The decrease in growth rate reduces the benefit of the important lipids and TAG accumulation as obtained in nitrogen starvation (37% and 18% of dry weight, respectively).     

Growth and carrageenan quality of <Emphasis Type="Italic">Kappaphycus striatum</Emphasis> var. <Emphasis Type="Italic">sacol</Emphasis> grown at different stocking densities,duration of culture and depth

Kappaphycus striatum var. sacol was grown in two separate studies: (1) at two stocking densities, and (2) at four different depths, each for three different durations of culture (30, 45 and 60 days) in order to determine the growth rate of the seaweed and evaluate the carrageenan content and its molecular weight. The results demonstrated that stocking density, duration of culture and depth significantly (P < 0.01) affected the growth rate, carrageenan content and molecular weight of K. striatum var. sacol. Decreasing growth rate was observed at both stocking densities and at four depths as duration of culture increased. A lower stocking density (500 g m⁻¹line⁻¹) showed a higher growth rate for the shortest durations, i.e. 30 days, as compared to those grown at a higher density. Likewise, decreasing growth rate was observed as depth increased, except at 50 cm after 60 days of culture. A 45-day culture period produced the highest molecular weight at both stocking densities (500 g m⁻¹line⁻¹ = 1,079.5 ± 31.8 kDa, 1,000 g m⁻¹line⁻¹ = 1,167 ± 270.6 kDa). ‘Sacol’ grown for 30 days at 50 cm (1,178 kDa) to 100 cm (1,200 kDa) depth showed the highest values of molecular weight of carrageenan extracted. The results suggested that K. striatum var. sacol is best grown at a stocking density of 500 g m⁻¹line⁻¹, at a depth of 50–100 cm, and for a duration of 30 days in order to provide the highest growth rate, carrageenan content and molecular weight.     

Hydrogen Production by Geobacter Species and a Mixed Consortium in a Microbial Electrolysis Cell

A hydrogen utilizing exoelectrogenic bacterium (Geobacter sulfurreducens) was compared to both a nonhydrogen oxidizer (Geobacter metallireducens) and a mixed consortium in order to compare the hydrogen production rates and hydrogen recoveries of pure and mixed cultures in microbial electrolysis cells (MECs). At an applied voltage of 0.7 V, both G. sulfurreducens and the mixed culture generated similar current densities (ca. 160 A/m³), resulting in hydrogen production rates of ca. 1.9 m³ H₂/m³/day, whereas G. metallireducens exhibited lower current densities and production rates of 110 ± 7 A/m³ and 1.3 ± 0.1 m³ H₂/m³/day, respectively. Before methane was detected in the mixed-culture MEC, the mixed consortium achieved the highest overall energy recovery (relative to both electricity and substrate energy inputs) of 82% ± 8% compared to G. sulfurreducens (77% ± 2%) and G. metallireducens (78% ± 5%), due to the higher coulombic efficiency of the mixed consortium. At an applied voltage of 0.4 V, methane production increased in the mixed-culture MEC and, as a result, the hydrogen recovery decreased and the overall energy recovery dropped to 38% ± 16% compared to 80% ± 5% for G. sulfurreducens and 76% ± 0% for G. metallireducens. Internal hydrogen recycling was confirmed since the mixed culture generated a stable current density of 31 ± 0 A/m³ when fed hydrogen gas, whereas G. sulfurreducens exhibited a steady decrease in current production. Community analysis suggested that G. sulfurreducens was predominant in the mixed-culture MEC (72% of clones) despite its relative absence in the mixed-culture inoculum obtained from a microbial fuel cell reactor (2% of clones). These results demonstrate that Geobacter species are capable of obtaining similar hydrogen production rates and energy recoveries as mixed cultures in an MEC and that high coulombic efficiencies in mixed culture MECs can be attributed in part to the recycling of hydrogen into current.Electrohydrogenesis is an efficient method for generating hydrogen gas from organic matter in reactors known as microbial electrolysis cells (MECs) (17, 18, 26). MECs differ from air-cathode microbial fuel cells (MFCs) in that the cathode remains anaerobic, and voltage is added in order to generate hydrogen at the cathode. Under the biological conditions in MECs, hydrogen evolution is not a thermodynamically favorable reaction. However, combining the hydrogen formation reaction potential of −0.41 V at the cathode (E_CAT) with the anode potential (E_AN) typically obtained in MFCs with an E_AN of −0.30 V (1 g of acetate/liter) results in a minimum required voltage of only 0.14 V. Applied voltages (E_AP) of 0.2 V (0.45 kWh/m³ H₂) or larger are needed in practice to produce measurable quantities of hydrogen, but this input is substantially less than the average of 2.3 V (5.1 kWh/m³ H₂) required for water electrolysis (13).Recent improvements in designs and materials have substantially improved hydrogen yields, production rates, and energy recoveries (3, 18, 27-29, 33). Hydrogen recoveries using typical dead-end fermentation end products such as acetate and butyrate have reached 80 to 100%, whereas other complex substrates such as glucose and cellulose have yielded recoveries of ca. 70% (5). Production rates larger than 6 m³ H₂/m³/day have been obtained using MECs (32), which are similar to an average rate of 2.5 m³ H₂/m³/day obtained for hydrogen production by biological fermentation (10). Energy recoveries relative to the electrical energy input as high as 680% have already been shown (5), and overall energy recoveries that include the energy of the substrate have reached 85% (2, 5).Hydrogen losses can occur using a mixed culture in an MEC, reducing hydrogen yields, production rates, and recoveries (3, 11, 16, 32). Hydrogen recoveries can drop significantly at lower applied voltages in membraneless MECs because of methanogenic consumption of hydrogen (2, 8, 11, 34). Using a membraneless MEC, Call and Logan (2) found that the overall hydrogen recovery of 90% at an E_AP of 0.6 V was reduced to 18% at an E_AP of 0.2 V and that methane concentrations increased from 0.9 to 28% in the product gas. Reducing solution pH can help inhibit methanogens, but a methane concentration of 22% was observed in a membrane free MEC at pH 5.8 (11). When hydrogen is the intended product of an MEC, methane production is detrimental to the process. However, biologically produced methane is a renewable energy source, and membraneless MECs can be used to generate methane instead of hydrogen, although energy recoveries are lower (8). Hydrogen can also be consumed by chemolithotrophic bacteria in mixed-culture MECs. These bacteria may transfer the associated electrons to a suitable electron acceptor, such as carbon dioxide, and in some cases, the anode. In the latter scenario, the electrons from hydrogen would be recycled internally, causing an increase in coulombic efficiency (16). Hydrogen losses reduce hydrogen and energy recoveries, and alternative methods for generating methane-free and high hydrogen content gas are needed.Pure culture MECs are one method to avoid losses to methanogens, but production rates and efficiencies with pure cultures can be low compared to those with mixed cultures. Using a pure culture of Shewanella oneidensis MR-1 and lactate, Hu et al. obtained a hydrogen production rate of 0.025 m³ H₂/m³/day at an E_AP of 0.6 V (11). However, production rates at this same applied voltage using mixed cultures have reached 1 to 2 m³ H₂/m³/day (2, 5). In MFCs, S. oneidensis has produced low coulombic efficiencies (<10%) (24, 25) and maximum current densities of ca. 50 mA/m² (15) with lactate, compared to ca. 9,900 mA/m² (9) for mixed cultures.Several Geobacter species are commonly found in mixed culture MFCs, and tests with pure cultures of Geobacter sulfurreducens have demonstrated power and current densities close to or equal to those achieved with mixed cultures. In an air cathode MFC, G. sulfurreducens produced a lower power density (461 mW/m², 1.5 A/m²) than a mixed culture (576 mW/m², 1.3 A/m²) (12). The reduced performance of G. sulfurreducens in the air cathode MFC may have been due to oxygen intrusion across the cathode. Using an MFC with a ferricyanide cathode, Nevin et al. (23) reported a power density of 1.9 W/m² (4.6 A/m²) for G. sulfurreducens compared to 1.6 W/m² (3.2 A/m²) for a mixed consortium. When the authors placed the G. sulfurreducens MFC in an anaerobic chamber, the coulombic efficiency improved from 55% to ca. 100%, confirming the importance of strictly anaerobic conditions for G. sulfurreducens. This suggests that the anaerobic environment of MECs may provide excellent conditions for obtaining current densities comparable to those of mixed cultures with pure cultures of Geobacter species, while at the same time eliminating methane gas production.In order to investigate the performance of Geobacter species in MECs, we selected two Geobacter species based on their differences in hydrogen utilization. G. sulfurreducens was selected because it is capable of producing high current densities in MFCs, and it can utilize hydrogen. G. metallireducens, which does not oxidize hydrogen, was examined to determine whether higher hydrogen recoveries were possible with a bacterium that cannot oxidize hydrogen. Both of these cultures were compared to a mixed culture under identical conditions in order to further examine the role of internal hydrogen recycling in MECs and to show that methane-free gas can be produced in MECs at rates comparable to those obtained with mixed cultures.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

Isolation of a novel high erythritol-producing <Emphasis Type="Italic">Pseudozyma tsukubaensis</Emphasis> and scale-up of erythritol fermentation to industrial level

This study isolated a novel erythritol-producing yeast strain, which is capable of growth at high osmolarity. Characteristics of the strain include asexual reproduction by multilateral budding, absence of extracellular starch-like compounds, and a negative Diazonium blue B color reaction. Phylogenetic analysis based on the 26S rDNA sequence and physiological analysis indicated that the strain belongs to the species Pseudozyma tsukubaensis and has been named P. tsukubaensis KN75. When P. tsukubaensis KN75 was cultured aerobically in a fed-batch culture with glucose as a carbon source, it produced 245 g/L of erythritol, corresponding to 2.86 g/L/h productivity and 61% yield, the highest erythritol yield ever reported by an erythritol-producing microorganism. Erythritol production was scaled up from a laboratory scale (7 L fermenter) to pilot (300 L) and plant (50,000 L) scales using the dissolved oxygen as a scale-up parameter. Erythritol production at the pilot and plant scales was similar to that at the laboratory scale, indicating that the production of erythritol by P. tsukubaensis KN75 holds commercial potential.     

Nitrogen utilization by the raphidophyte Heterosigma akashiwo: Growth and uptake kinetics in laboratory cultures

The nitrogen uptake and growth capabilities of the potentially harmful, raphidophycean flagellate Heterosigma akashiwo (Hada) Sournia were examined in unialgal batch cultures (strain CCMP 1912). Growth rates as a function of three nitrogen substrates (ammonium, nitrate and urea) were determined at saturating and sub-saturating photosynthetic photon flux densities (PPFDs). At saturating PPFD (110 μE m⁻² s⁻¹), the growth rate of H. akashiwo was slightly greater for cells grown on NH₄⁺ (0.89 d⁻¹) compared to cells grown on NO₃⁻ or urea, which had identical growth rates (0.82 d⁻¹). At sub-saturating PPFD (40 μE m⁻² s⁻¹), both urea- and NH₄⁺-grown cells grew faster than NO₃⁻-grown cells (0.61, 0.57 and 0.46 d⁻¹, respectively). The N uptake kinetic parameters were investigated using exponentially growing batch cultures of H. akashiwo and the ¹⁵N-tracer technique. Maximum specific uptake rates (V_max) for unialgal cultures grown at 15 °C and saturating PPFD (110 μE m⁻² s⁻¹) were 28.0, 18.0 and 2.89 × 10⁻³ h⁻¹ for NH₄⁺, NO₃⁻ and urea, respectively. The traditional measure of nutrient affinity—the half saturation constants (K_s) were similar for NH₄⁺ and NO₃⁻ (1.44 and 1.47 μg-at N L⁻¹), but substantially lower for urea (0.42 μg-at N L⁻¹). Whereas the α parameter (α = V_max/K_s), which is considered a more robust indicator for substrate affinity when substrate concentrations are low (<K_s), were 19.4, 12.2 and 6.88 × 10⁻³ h⁻¹/(μg-at N L⁻¹) for NH₄⁺, NO₃⁻ and urea, respectively. These laboratory results demonstrate that at both saturating and sub-saturating N concentrations, N uptake preference follows the order: NH₄⁺ > NO₃⁻ > urea, and suggests that natural blooms of H. akashiwo may be initiated or maintained by any of the three nitrogen substrates examined.     

Effects of dwarf bamboo (Fargesia denudata) density on biomass, carbon and nutrient distribution pattern

Dense dwarf bamboo population is a structurally and functionally important component in many subalpine forest systems. To characterize the effects of stem density on biomass, carbon and majority nutrients (N, P, K, Ca and Mg) distribution pattern, three dwarf bamboo (Fargesia denudata) populations with different stem densities (Dh with 220 ± 11 stems m^?2, Dm with 140 ± 7 stems m^?2, and Dl with 80 ± 4 stems m^?2, respectively) were selected beneath a bamboo-fir (Picea purpurea) forest in Wanglang National Nature Reserve, Sichuan, China. Leaf, branch, rhizome, root and total biomass of dwarf bamboo increased with the increase of stem density, while carbon and nutrient concentrations in bamboo components decreased. Percentages of below-ground biomass and element stocks to total biomass and stocks decreased with the increase of stem density, whereas above-ground biomass and element stocks exhibited the opposite tendency. Moreover, more above-ground biomass and elements were allocated to higher part in the higher density population. In addition, percentages of culm biomass, above-ground biomass and element stocks below 100 cm culm height (H₁₀₀) increased with the increase of stem density, while percentages of branch and leaf biomass below H₁₀₀ decreased. Pearson’s correlation analyses revealed that root biomass, above-ground biomass, below-ground biomass and total biomass significantly correlated to leaf biomass in H_100?200 and total leaf biomass within high density population, while they significantly correlated to leaf biomass in H_50?150 within low density population. The results suggested that dwarf bamboo performed an efficient adaptive strategy to favor limited resources by altering biomass, carbon and nutrients distribution pattern in the dense population.     

Growth of dinoflagellates, Ceratium furca and Ceratium fusus in Sagami Bay, Japan: The role of temperature, light intensity and photoperiod

Seasonal changes of field populations and growth rates of two dinoflagellates, Ceratium furca and Ceratium fusus, were examined in the temperate coastal water of Sagami Bay, Japan. Weekly field sampling was conducted from August 2002 to August 2003, and laboratory experiments were also carried out to investigate effects of temperature, irradiance and photoperiod on the growth rates of these two Ceratium species. In the field, the abundances of both species increased significantly from April to August 2003, were gradually decreased from November 2002 and were not observed in January 2003. C. fusus was able to increase at lower temperatures in February 2003 compared to C. furca. In the laboratory, the two species did not grow at <10 °C or >32 °C. The highest specific growth rate of C. furca was 0.72 d⁻¹ at 24 °C and 600 μmol m⁻² s⁻¹. Optimum growth rates (>0.4 d⁻¹) of C. furca were observed at temperatures from 18 to 28 °C and at irradiances from 216 to 796 μmol m⁻² s⁻¹. The highest growth rate of C. fusus was 0.56 d⁻¹ at 26 °C and 216 μmol m⁻² s⁻¹. Optimum growth rates of C. fusus were observed at the same irradiance rage of C. furca, whereas optimum temperature range was narrower (26–28 °C). The growth curves of both species indicated saturation of the growth rates when light intensity was above 216 μmol m⁻² s⁻¹, and did not show photoinhibition at irradiances up to 796 μmol m⁻² s⁻¹. The specific growth rates of both Ceratium species were clearly decreased at L:D = 10:14 relative to those at L:D = 14:10 and L:D = 12:12. The present study indicates the two Ceratium species can adapt to a wide range of temperature and irradiance.     

Secondary production of Rhyacophila minora,Ameletus sp., and Isonychia bicolor from streams of low and circumneutral pH in the Appalachian Mountains of West Virginia

We estimated the secondary production of Rhyacophila minora, Ameletus sp., and Isonychia bicolor in three acidic streams and one circumneutral stream in Randolph County, West Virginia. Quantitative benthic samples were collected monthly from these second-order streams from November 1990 to October 1991. Mean pH values in the acidic streams were 4.5, 4.8, and 4.8, and mean pH in the circumneutral stream was 6.7. Production estimates for Rhyacophia minora in the acidic streams were 49.6, 19.2, and 15.8 mg m^–2 y^–1. Production of R. minora in the circumneutral stream was 1.0 mg m^–2 y^–1. Ameletus sp. production estimates for the acidic streams were 144.8, 176.8, and 208.3 mg m^–2 y^–1. Ameletus sp. production in the circumneutral stream was 7.4 mg m^–2 y^–1. Secondary production of I. bicolor in the circumneutral stream was 116.6 mg m^–2 y^–1. No Isonychia were collected from the acidic streams. The higher production of R. minora and Ameletus sp. in the acidic streams may be associated with differences in macroinvertebrate community structure.     

On the distribution patterns and population dynamics of sublittoral and profundal oligochaeta fauna from Lake Banyoles (Catalonia,NE Spain)

Oligochaeta assemblages from Lake Banyoles were composed of one species of Naididae and eight species of Tubificidae, Potamothrix heuscheri (8–1350 ind. m^–2) being the most abundant and widely distributed species, followed by Limnodrilus hoffmeisteri (25–858 ind. m^–2) and Psammoryctides barbatus (12–792 ind. m^–2). The remaining species were Potamothrix hammoniensis, Potamothrix bavaricus, Limnodrilus claparedeanus, Branchiura sowerbyi, Aulodrilus pigueti and Dero digitata, all of which occurred at lower densities and with restricted distributions.Regardless of sampling depth, maximum densities of oligochaetes were found in winter (up to 5,142 ind. m^–2 in December) and secondarily in June. Immature Tubificidae with hair setae and mature P. heuscheri were present and dominant throughout the year. Cestode parasites were recorded infesting L. hoffmeisteri and P. heuscheri populations.Oligochaeta densities increased with depth, but low oxygen concentrations (less than 1 mg l^–1) during a long period (4 months) in some basins of the lake acted as a key factor in reducing the density of worms and the species richness to one species, Potamothrix heuscheri.     

Change in colony morphology and kinetics of tylosin production after UV and gamma irradiation mutagenesis of Streptomyces fradiae NRRL-2702

Tylosin is a macrolide antibiotic used as veterinary drug and growth promoter. Attempts were made for hyper production of tylosin by a strain of Streptomyces fradiae NRRL-2702 through irradiation mutagenesis. Ultraviolet (UV) irradiation of wild-type strain caused development of six morphologically altered colony types on agar plates. After screening using Bacillus subtilis bioassay only morphological mutants indicated the production of tylosin. An increase of 2.7±0.22-fold in tylosin production (1500 mg/l) in case of mutant UV-2 in complex medium was achieved as compared to wild-type strain (550 mg/l). Gamma irradiation of mutant UV-2 using ⁶⁰Co gave one morphologically altered colony type γ-1, which gave 2500 mg/l tylosin yield in complex medium. Chemically defined media promoted tylosin production upto 3800 mg/l. Maximum value of q_p (3.34 mg/gh) was observed by mutant γ-1 as compared to wild strain (0.81 mg/gh). Moreover, UV irradiation associated changes were unstable with loss of tylosin activity whereas mutant γ-1 displayed high stability on subsequent culturing.     

Nitrogen source effects on the growth and toxicity of two strains of the ciguatera-causing dinoflagellate Gambierdiscus toxicus

Two Caribbean strains (1651 and 1655) of the ciguatera-causing dinoflagellate Gambierdiscus toxicus were grown in xenic, batch culture under defined, measured nutrient conditions with nitrate, ammonium, urea, a mix of free amino acids (FAA), or putrescine as the nitrogen source. Cultures were maintained at 27 °C, salinity 35, 110 μmol m⁻² s⁻¹ (12 h:12 h light:dark cycle) on L2 medium at an initial nitrogen concentration of 50 μM N. Toxicity was determined using a ouabain/veratridine-dependent cytotoxicity assay (N2A assay) standardized to a ciguatoxin standard. Nitrate, ammonium, FAA, and putrescine supported growth, but urea did not. The appearance of ammonium in the organic nitrogen cultures indicated that G. toxicus and/or associated bacteria remineralized the available organic nitrogen. Both strains were exposed to nitrogen-limiting conditions as evidenced by chlorophyll a content per cell, nitrogen content, and nitrogen (N) to phosphorus (P) (N:P) ratio significantly declining once nitrogen was no longer available in the medium and cells entered stationary phase. Strain 1651 grew significantly faster than strain 1655 when nitrate, FAA, and putrescine was the nitrogen source, but not ammonium. Nitrogen source had no effect on growth rate (0.14 d⁻¹) in strain 1651. The growth rate of strain 1655 (0.10–0.13 d⁻¹) was significantly faster on ammonium than the other nitrogen sources. Strain 1655 was significantly more toxic (10-fold) than strain 1651 except when growing on ammonium at exponential phase. Toxicity ranged from 1.3 to 8.7 fg C-CTX1-Eq cell⁻¹ in strain 1651 and from 30.7 to 54.3 fg C-CTX1-Eq cell⁻¹ in strain 1655. Nitrogen source had no significant affect on toxicity. Toxicity was greater in stationary versus exponential phase cells for strain 1651 when grown on nitrate and strain 1655 regardless of nitrogen source. The difference in toxicity between growth phases may result from an increase in ciguatoxin and/or maitotoxin. Our results suggest that some strains of G. toxicus when associated with bacteria are able to take advantage of organic as well as inorganic nitrogen sources on short time scales to support future growth. The uncoupling of total nitrogen and phosphorus pools from conditions in the water column suggest that instantaneous growth rates can be supported by nutrients acquired hours to days earlier.