Bacterial diketopiperazines stimulate diatom growth and lipid accumulation

Diatoms are photosynthetic microalgae that fix a significant fraction of the world’s carbon. Because of their photosynthetic efficiency and high-lipid content, diatoms are priority candidates for biofuel production. Here, we report that sporulating Bacillus thuringiensis and other members of the Bacillus cereus group, when in co-culture with the marine diatom Phaeodactylum tricornutum, significantly increase diatom cell count. Bioassay-guided purification of the mother cell lysate of B. thuringiensis led to the identification of two diketopiperazines (DKPs) that stimulate both P. tricornutum growth and increase its lipid content. These findings may be exploited to enhance P. tricornutum growth and microalgae-based biofuel production. As increasing numbers of DKPs are isolated from marine microbes, the work gives potential clues to bacterial-produced growth factors for marine microalgae.     

Nutrient resupplementation arrests bio-oil accumulation in Phaeodactylum tricornutum

Phaeodactylum tricornutum is a marine diatom in the class Bacillariophyceae and is important ecologically and industrially with regards to ocean primary production and lipid accumulation for biofuel production, respectively. Triacylglyceride (TAG) accumulation has been reported in P. tricornutum under different nutrient stresses, and our results show that lipid accumulation can occur with nitrate or phosphate depletion. However, greater lipid accumulation was observed when both nutrients were depleted as observed using a Nile Red assay and fatty acid methyl ester (FAME) profiles. Nitrate depletion had a greater effect on lipid accumulation than phosphate depletion. Lipid accumulation in P. tricornutum was arrested upon resupplementation with the depleted nutrient. Cells depleted of nitrogen showed a distinct shift from a lipid accumulation mode to cellular growth post-resupplementation with nitrate, as observed through increased cell numbers and consumption of accumulated lipid. Phosphate depletion caused lipid accumulation that was arrested upon phosphate resupplementation. The cessation of lipid accumulation was followed by lipid consumption without an increase in cell numbers. Cells depleted in both nitrate and phosphate displayed cell growth upon the addition of both nitrate and phosphate and had the largest observed lipid consumption upon resupplementation. These results indicate that phosphate resupplementation can shut down lipid accumulation but does not cause cells to shift into cellular growth, unlike nitrate resupplementation. These data suggest that nutrient resupplementation will arrest lipid accumulation and that switching between cellular growth and lipid accumulation can be regulated upon the availability of nitrogen and phosphorus.     

Effects of fertilizer-based culture media on the production of exocellular polysaccharides and cellular superoxide dismutase by <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> (Bohlin)

Microalgae cultures are receiving attention because of increasing biotechnological and biomedical production of active biomolecules. We evaluated various fertilizer-based culture media to scale up production of the marine microalga Phaeodactylum tricornutum for production of exocellular polysaccharides (EPS), soluble proteins, and cellular superoxide dismutase (SOD). The standard source of sodium nitrate was the same as that used in the synthetic f/2 culture medium and ammonium nitrate, urea, ammonium sulfate, and calcium nitrate as alternative sources of nitrogen. The maximum production of EPS was achieved in microalgae cells grown in the culture media containing 63 and 23% nitrogen from ammonium sulfate, and also in microalgae cells grown in the culture media containing 3% nitrogen from ammonium nitrate. The maximum production of cellular SOD was achieved in microalgae cells grown in the culture media containing 35 and 26% nitrogen from ammonium sulfate, and in the culture media containing 17% nitrogen from urea. The results suggest that it is possible to use a source of nitrogen, other than sodium nitrate, to scale up growth of P. tricornutum for production of EPS and SOD at reduced costs.     

Detection of intracellular neutral lipid content in the marine microalgae Prorocentrum micans and Phaeodactylum tricornutum using Nile red and BODIPY 505/515

Diatoms and dinoflagellates not only have extensive distribution and a huge biomass in marine ecosystems, but also have high lipid accumulation in nature or after physiological and genetic modification, which indicates that these organisms may be optimal candidate algal strains for biodiesel production. In this study, we determined the content of intracellular neutral lipids (triacylglycerol [TAG]) in the dinoflagellate Prorocentrum micans and in the diatom Phaeodactylum tricornutum using NR and BODIPY 505/515 staining. The freshwater green alga Scenedesmus obliquus was used as a control. Optimum concentrations of 1.000 and 1.500 μg mL^?1 were determined for neutral lipid Nile red (NR) staining in P. micans and P. tricornutum. Unlike NR staining, the optimal concentrations of BODIPY 505/515 staining in P. micans and P. tricornutum were lower, at 0.100 and 0.075 μg mL^?1, respectively. High correlation coefficients of R ²?=?0.990 and R ²?=?0.989 were obtained for P. micans and P. tricornutum intracellular neutral lipid content and the relative fluorescence intensity with NR staining, while the reference alga, S. obliquus, had a relatively low correlation coefficient of R ²?=?0.908 when stained with NR. The neutral lipid content determined by thin-layer chromatography-flame ionization detector matched the analytical data from fluorescence measurements. These results indicated that NR and BODIPY 505/515 staining can be used as an excellent high-throughput approach to screen marine diatoms and dinoflagellates.     

High predictability of direct competition between marine diatoms under different temperatures and nutrient states

The distribution of marine phytoplankton will shift alongside changes in marine environments, leading to altered species frequencies and community composition. An understanding of the response of mixed populations to abiotic changes is required to adequately predict how environmental change may affect the future composition of phytoplankton communities. This study investigated the growth and competitive ability of two marine diatoms, Phaeodactylum tricornutum and Thalassiosira pseudonana, along a temperature gradient (9–35°C) spanning the thermal niches of both species under both high‐nitrogen nutrient‐replete and low‐nitrogen nutrient‐limited conditions. Across this temperature gradient, the competitive outcome under both nutrient conditions at any assay temperature, and the critical temperature at which competitive advantage shifted from one species to the other, was well predicted by the temperature dependencies of the growth rates of the two species measured in monocultures. The temperature at which the competitive advantage switched from P. tricornutum to T. pseudonana increased from 18.8°C under replete conditions to 25.3°C under nutrient‐limited conditions. Thus, P. tricornutum was a better competitor over a wider temperature range in a low N environment. Being able to determine the competitive outcomes from physiological responses of single species to environmental changes has the potential to significantly improve the predictive power of phytoplankton spatial distribution and community composition models.     

Nutrient removal from high strength nitrate containing industrial wastewater using <Emphasis Type="Italic">Chlorella</Emphasis> sp. strain ACUF_802

the research aim of this study was to characterize an isolated native strain of Chlorella sp. ACUF_802, well adapted to a high nitrate concentration environment and to investigate its potential to nitrate and phosphate removal from industrial wastewaters with the minimal addition of chemical reagents and energy. The isolated strain was identified and evaluated for its capability to support biomass growth and nutrient removal from synthetic wastewater in batch tests using different concentrations of carbon and nitrogen, different carbon sources and N:P ratios. The strain was isolated via the plating method from the settler of a pilot scale moving bed biofilm reactor performing a nitrification process. The strain was identified using molecular analysis with rDNA primers. Using sodium bicarbonate as carbon source, the batch productivity (71.43 mg L^?1 day^?1) of the strain Chlorella sp. ACUF_802 was calculated with a logistic model and compared to the values reported in the literature. Assays on the effect of the N:P ratio indicated that the productivity was increased 36% when the N:P ratio was close to 1 (111.96 mg L^?1 day^?1), but for a complete phosphorus removal a 5:1 N:P ratio with nitrate concentrations ≤125 mg?L^?1 is recommended. The isolated microalgae strain Chlorella sp. ACUF_802 showed versatility to grow in the synthetic industrial wastewaters tested and can be considered as an appropriate organism for nitrogen removal from industrial wastewaters in the presence of an organic or inorganic carbon source.     

The effect of varying LED light sources and influent carbon/nitrogen ratios on treatment of synthetic sanitary sewage using Chlorella vulgaris

Sanitary sewage can create serious environmental problems if discharged directly into natural waters without appropriate treatment. This study showed that red light is the optimum light wavelength for growing microalgae Chlorella vulgaris in microalgae biological wastewater treatment systems, given a harvest time of 144 h. Only moderate light intensities (1,000, 1,500, 2,000, and 2,500 μmol m^?2 s^?1) were able to remove nutrients from synthetic sanitary sewage, but higher light intensity led to better nutrient removal effects. Because of economic considerations, the optimum light intensity range for efficient nutrient removal was determined to be between 1,500 and 2,000 μmol m^?2 s^?1. Furthermore, nutrient removal efficiency was significantly affected by light wavelength, light intensity, the interaction of these two factors, and the interaction among light wavelength, light intensity, and influent carbon/nitrogen (C/N) ratios. Total nitrogen and total phosphorus removal efficiency was also significantly affected by influent C/N ratios. Appropriate control of carbon and nitrogen source concentrations enabled optimal nutrient removal. The optimal influent C/N ratio was determined to be 6:1.     

Optimization of CO2 concentration and light intensity for biodiesel production by Chlorella vulgaris FACHB-1072 under nitrogen deficiency with phosphorus luxury uptake

Microalgal biodiesel is an alternative bioenergy for the future. Nitrogen deprivation is usually used to increase lipid content in microalgae, however, it also lowers biomass production, resulting in not much increase of lipid productivity. Our previous study found that phosphorus played an important role in enhancing biodiesel productivity of C. vulgaris FACHB-1072 under nitrogen deficient condition. The aim of this study was to optimize two significant parameters of CO₂ concentration (0.03, 4, 6, 12 %) and light intensity (40, 120, 200 μmol photons m^-2 s^-1) with respect to biodiesel productivity and P uptake rate of C. vulgaris FACHB-1072. It was found that the optimized conditions were 4 % CO₂ concentration and 200 μmol photons m^-2 s^-1 light intensity. The maximum biodiesel productivity was 34.56 mg L^-1 day^-1; 2.7 times higher than the control (nutrient sufficient condition). Phosphorus was accumulated as polyphosphate and its maximum uptake rate was 2.08 mg L^-1 day^-1; twice that of the control. After optimization, the performances under nitrogen deficiency were significantly better compared with those under nitrogen sufficiency, which were rarely reported in literature. Our findings suggest a great potential to combine phosphorus removal from wastewater with biodiesel production via microalgae.     

Changes in fatty acid profiles of thermo-intolerant and thermo-tolerant marine diatoms during temperature stress

Fatty acid composition and degree of fatty acid saturation during temperature stress in thermo-intolerant (Phaeodactylum tricornutum) and thermo-tolerant (Chaetoceros muelleri) marine diatoms were investigated. A greater number of fatty acids were observed in C. muelleri than in P. tricornutum regardless of treatment. The major fatty acids detected were 14:0, 16:0, 16:1, 16:2, 16:3, 18:0, 18:1(n-9)c, 18:2(n-6) and 20:5(n-3) with additional fatty acids 18:1(n-9)t and 20:4(n-6) detected in C. muelleri. Short duration (2 h) temperature increase above optimal growth temperature had a greater effect on fatty acid composition in C. muelleri than in P. tricornutum and the degree of fatty acid saturation was affected more by temperature in C. muelleri than in P. tricornutum during both short and long duration (24 h) treatments. Total protein assay results suggest that P. tricornutum, but not C. muelleri, was undergoing stress under our growing conditions although lipids in both diatoms were affected by increased temperature. Immunodetection of proteins with anti-rubisco indicates that the rubisco large subunit was undergoing greater turnover in C. muelleri than in P. tricornutum. However, the integrity of rubisco as a suitable indicator of lipid status needs further study. This work supports the hypothesis that a particular temperature, and not treatment duration, has the greater effect on changes in fatty acid composition. Furthermore, changes in fatty acid composition and degree of fatty acid saturation occurred more quickly in the diatoms in response to increased temperature than previously observed in nutrient starvation studies. Since diatom lipids represent an important resource for growth and reproduction of marine animals, the rapid alteration of their lipid composition under temperatures normally encountered in marine environments warrants further study.     

Integration of consumer-targeted microalgal production with marine fish effluent biofiltration – a strategy for mariculture sustainability

EU regulations recommend effluent treatment and nutrient recycling for aquaculture sustainability, so a study was undertaken to provide base-line data for the integration of commercial fish-farm effluents with the production of microalgae. The project relates to a specific bivalve consumer (Tapes decussatus) and biofiltration. Effluent inorganic nutrient composition was assessed and evaluated as culture media for Phaeodactylum tricornutum, Tetraselmis suecica and Tetraselmis sp. Optimization of the microalgal compartment included studies on preparation of a simple medium, nutrient or dilution rate manipulation and nutrient removal. Cell harvest was increased chiefly by N correction (6-fold for Tetraselmis sp.) and semi-continuous or continuous operation (by a factor of 3 to 11). Nutrient removal efficiency was high for ammonium and nitrite-nitrogen (80–100%), depending on species, nutrient ratio (Si correction for P. tricornutum) and culture regime for nitrate (41–100%) or phosphorus (21–99%). Data obtained under cyclostat cultivation (yields of 1.38 and 0.50×10⁶ P. tricornutum or Tetraselmis sp. cells mL⁻¹ d⁻¹ and nutrient uptake rates of 2.32 mg N L⁻¹ d⁻¹ and 0.96 mg P L⁻¹ d⁻¹) were used to show clam production and simultaneous wastewater treatment possibilities through the proposed fish-microalgae-clam integrated aquaculture system.     

Microalgae-based agro-industrial wastewater treatment: a preliminary screening of biodegradability

The potential of algal–bacterial symbiosis for the removal of carbon, nitrogen and phosphorus from five agro-industrial wastewaters was investigated in enclosed batch biodegradation tests using a mixed microalgae consortium and activated sludge as model microorganisms. The target wastewaters were obtained from potato processing (PW), fish processing (FW), animal feed production (MW), coffee manufacturing (CW) and yeast production (YW). The initial C/N/P ratio of the agro-industrial wastewater was correlated with its biodegradability. Thus, the highest removals of total organic carbon (TOC) and nitrogen were recorded in two fold diluted FW (64?±?2 % and 85?±?1 %, respectively), while the maximum P-PO₄ ^3? removal achieved was 89?±?1 % in undiluted PW. The biodegradable TOC was in most cases the limiting component in the treatment of the wastewaters evaluated. This study confirmed the potential of coupling carbon and nutrient recovery from agro-industrial effluents with the production of a valuable algal–bacterial biomass, despite their poor biodegradability.     

Removal of nitrogen and phosphorus from wastewater using microalgae immobilized on twin layers: an experimental study

Removal of nitrogen and phosphorus from wastewater by two green microalgae (Chlorella vulgaris and Scenedesmus rubescens) was investigated using a novel method of algal cell immobilization, the twin-layer system. In the twin-layer system, microalgae are immobilized by self-adhesion on a wet, microporous, ultrathin substrate (the substrate layer). Subtending the substrate layer, a second layer, consisting of a macroporous fibrous tissue (the source layer), provides the growth medium. Twin-layers effectively separate microalgae from the bulk of their growth medium, yet allow diffusion of nutrients. In the twin-layer system, algae remain 100% immobilized, which compares favourably with gel entrapment methods for cell immobilization. Both microalgae removed nitrate efficiently from municipal wastewater. Using secondary, synthetic wastewater, the two algae also removed phosphate, ammonium and nitrate to less than 10% of their initial concentration within 9 days. It is concluded that immobilization of C. vulgaris and S. rubescens on twin-layers is an effective means to reduce nitrogen and phosphorus levels in wastewater.     

Temperature-influenced variations in speciation and chemical composition of marine phytoplankton in outdoor mass cultures

Between September, 1976 and July, 1977 Phaeodactylum tricornutum Bohlin was replaced as the dominant species by Skeletonema costatum (Grev) Cleve as temperatures fell below 10°C in the fall in an outdoor pond supplied with a mixture of waste water and sea water. Phaeodactylum tricornutum returned in the spring as the major species when temperatures rose above 10°C. In an adjacent pond in which only nitrogen and phosphorus were added in excess, however, P. tricornutum dominated throughout the entire study period even through the temperature varied between 0 and 25°C. We suspected that the difference inspecies dominance in the two ponds occurred because Skeletonema costatum requires silicon, which was present in sufficient quantities only in the waste-water-enriched pond. whereas Phaeodatylum tricornutum does not have a specififc requirement for this nutrient. The cellular chemical composition of P. tricornutum varied in a U-shaped fashion with changing temperature: minimum values for the cellular carbon, nitrogen, and chlorophyll contents were displayed at 15–20°C and maximum values at 3 and 15°C. Both the cellular carbon: nitrogen and carbon: chlorophyll ratios by weight were invariant with changing temperatures at ≈6: 1 and 50: 1 respectively, indicating nutrient saturation. Only under conditionsof nutrient saturation, which can be established in various ways, can the influence of temperature on phytoplankton physiology be separated from nutrient-related factors.     

Interactive Effects of Ocean Acidification and Nitrogen-Limitation on the Diatom Phaeodactylum tricornutum

Climate change is expected to bring about alterations in the marine physical and chemical environment that will induce changes in the concentration of dissolved CO₂ and in nutrient availability. These in turn are expected to affect the physiological performance of phytoplankton. In order to learn how phytoplankton respond to the predicted scenario of increased CO₂ and decreased nitrogen in the surface mixed layer, we investigated the diatom Phaeodactylum tricornutum as a model organism. The cells were cultured in both low CO₂ (390 μatm) and high CO₂ (1000 μatm) conditions at limiting (10 μmol L⁻¹) or enriched (110 μmol L⁻¹) nitrate concentrations. Our study shows that nitrogen limitation resulted in significant decreases in cell size, pigmentation, growth rate and effective quantum yield of Phaeodactylum tricornutum, but these parameters were not affected by enhanced dissolved CO₂ and lowered pH. However, increased CO₂ concentration induced higher rETR_max and higher dark respiration rates and decreased the CO₂ or dissolved inorganic carbon (DIC) affinity for electron transfer (shown by higher values for K_{1/2 DIC} or K_{1/2 CO2}). Furthermore, the elemental stoichiometry (carbon to nitrogen ratio) was raised under high CO₂ conditions in both nitrogen limited and nitrogen replete conditions, with the ratio in the high CO₂ and low nitrate grown cells being higher by 45% compared to that in the low CO₂ and nitrate replete grown ones. Our results suggest that while nitrogen limitation had a greater effect than ocean acidification, the combined effects of both factors could act synergistically to affect marine diatoms and related biogeochemical cycles in future oceans.     

Effects of nitrogen,phosphorus, iron and silicon on growth of five species of marine benthic diatoms

The effects of nitrogen, phosphorus, iron and silicon on growth of five species of marine benthic diatoms, namely Navicula patrickae, Nitzschia panduriformis, Navicula thienemannii, Nitzschia longissima and Navicula atomus were studied by single factor experiments and the optimal concentration ratios of the four nutrient elements beneficial for diatoms growth were screened out separately using the L₉ (3⁴) orthogonal design. The results highlighted that nitrogen, phosphorus, iron and silicon all had highly significant effects on growth of five diatoms while the diatoms growth rates reached the highest averagely in the 2nd to the 6th culture day. In addition, the optimal concentrations (mg/L) of four nutrients suitable for diatoms growth were found higher than that in f/2 medium except that Nitzschia longissima had the same concentration of nitrogen as that in f/2 medium which is optimal for growth. Moreover, the optimal growth concentrations of four elements for five diatoms varied in the range of 12.36–74.16 mg/L for nitrogen, 1.70–3.98 mg/L for phosphorus, 2.00–4.00 mg/L for iron, 23.01–69.03 mg/L for silicon, respectively. By means of the orthogonal test of four nutrients for five benthic diatoms, the optimal concentration ratios N:P:Fe:Si (mg/L) were obtained as follows: 74.16:2.27:3.33:23.01 for N. patrickae; 37.08:3.98:4.00:11.50 for N. panduriformis; 49.44:3.98:3.33:34.51 for N. thienemannii; 12.36:1.70:4.00:11.50 for N. longissima; 74.16:2.27:4.00:69.03 for N. atomus.     

Mixed Wastewater Coupled with CO2 for Microalgae Culturing and Nutrient Removal

Biomass, nutrient removal capacity, lipid productivity and morphological changes of Chlorella sorokiniana and Desmodesmus communis were investigated in mixed wastewaters with different CO₂ concentrations. Under optimal condition, which was 1:3 ratio of swine wastewater to second treated municipal wastewater with 5% CO₂, the maximum biomass concentrations were 1.22 g L^-1 and 0.84 g L^-1 for C. sorokiniana and D. communis, respectively. Almost all of the ammonia and phosphorus were removed, the removal rates of total nitrogen were 88.05% for C. sorokiniana and 83.18% for D. communis. Lipid content reached 17.04% for C. sorokiniana and 20.37% for D. communis after 10 days culture. CO₂ aeration increased intracellular particle numbers of both microalgae and made D. communis tend to be solitary. The research suggested the aeration of CO₂ improve the tolerance of microalgae to high concentration of NH₄-N, and nutrient excess stress could induce lipid accumulation of microalgae.     

Effects of nitrogen and phosphorus availabilities on growth, pigment, and protein contents in Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta)

The selection of seaweed species for their use as biofilters should be based on the knowledge of their nutrient requirements and tolerance to wide variations of nutrient concentrations. Therefore, tolerance and the physiological capabilities of Hypnea cervicornis J. Agardh (Gigartinales, Rhodophyta) to growth under nitrate, ammonium, and phosphate variations and to assimilate them into soluble proteins and photosynthetic pigments were evaluated in laboratory conditions. Treatments were composed of sterilized seawater enriched with 25 % von Stosch solution (without nitrogen and phosphorus), and nitrate or ammonium and phosphate were added in combination of 100:1 and 10:1 nitrogen/phosphorus (N/P). Nitrate concentrations varied from 0 to 500 μM, and ammonium concentrations varied from 0 to 50 μM. Growth rates of H. cervicornis increased linearly with addition of ammonium, but with nitrate addition, growth varied following a saturation kinetic, and the highest growth rate (14.45 % d^?1) was observed in 200 μM of N/P ratio of 10:1. An excess of nutrients was accumulated as proteins and phycobiliproteins (mainly as allophycocyanin and phycoerythrin) at higher phosphate availability (N/P ratio of 10:1), and H. cervicornis tolerated the highest ammonium and nitrate concentrations (50 and 500 μM, respectively). These physiological responses suggest that this species could be used as biofilter for nutrient removal in eutrophicated seawater and could be cultivated in integrated multitrophic aquaculture systems.     

Effect of nitrogen and phosphorus concentration on their removal kinetic in treated urban wastewater by Chlorella vulgaris

This study evaluates the feasibility of removing nutrients by the microalgae Chlorella vulgaris, using urban wastewater as culture medium, namely the effluent subjected to secondary biological treatment in a wastewater treatment plant (WWTP). For this, laboratory experiments were performed in batch cultures to study the effect of initial nitrogen and phosphorus concentrations on growth and reduction of nutrient performance of C. vulgaris. The microalga was cultivated in enriched wastewater containing different phosphorus (1.3-143.5 mg x L(-1) P.PO4(3-)), ammonium (5.8-226.8 mg x L(-1) N-NH4+) and nitrate (1.5-198.3 mg x L(-1) N-NO3-) concentrations. The nutrient removal and growth kinetics have been studied: maximum productivity of 0.95 g SS x L(-1) x day(-1), minimum yield factor for cells on substrate (Y) of 11.51 g cells x g nitrogen(-1) and 0.04 g cells x g phosphorus(-1) were observed. The results suggested that C. vulgaris has a high potential to reduce nutrients in secondary WWTP effluents.