High pH-induced flocculation of marine <Emphasis Type="Italic">Chlorella</Emphasis> sp. for biofuel production

Microalgae are being considered as a promising raw material for biofuel production. However, rapid, efficient, and economic technologies for harvesting microalgae are essential for successful applications. In this study, the high–pH-induced flocculation method was applied to harvest marine Chlorella sp. strains. These algae could be concentrated up to approximately 20-fold by increasing pH using NaOH, with a flocculation efficiency of 90 %. When NaOH dosage was low (1 or 3 mM), the flocculation efficiency decreased considerably with the increase of biomass concentration. At higher NaOH dosage tested (5 or 7 mM), flocculation occurred quickly and efficiently, which tended to be independent of biomass concentration. In larger volumes, all strains were flocculated with similar efficiencies (approximately 90 %) after adding 5 mM NaOH. After flocculation, the flocculated algae cells could be re-cultured as inoculum, and the growth yields in flocculated medium were slightly higher than those from fresh medium. Additionally, for each strain, there were no significant differences in lipid extraction yield and fatty acid composition according to different harvesting methods. These results showed that the high–pH-induced flocculation method could be used to harvest marine Chlorella sp. for biofuel production successfully.     

Oil production by six microalgae: impact of flocculants and drying on oil recovery from the biomass

Oil production in batch photoautotrophic cultures of the following microalgae is reported: the freshwater microalgae Chlorella vulgaris, Choricystis minor, and Neochloris sp.; the marine microalgae Nannochloropsis salina and Cylindrotheca fusiformis; and C. vulgaris grown in a full-strength seawater medium. In all cases, the solvent extraction of lipids from freeze-dried biomass is compared with extraction from the fresh biomass paste. For all algae, the oils could be extracted equally effectively from freeze-dried samples and the paste samples (67–88 % moisture by weight). Moisture content determinations of the biomass using the freeze-drying method and the high-temperature oven drying were found to be equivalent for all algae. The biomass recovered by flocculation with metal salts (aluminum sulfate, ferric chloride) followed by centrifugation had a certain amount of the flocculant irreversibly bound to it. Washing failed to remove the adsorbed flocculants. For all algae, the adsorbed flocculants did not interfere with oil recovery by solvent extraction. The solvent system of chloroform–methanol–water proved highly effective for quantitative extraction of the lipids from all algae.     

Why is algaculture still incipient in Brazil?

Macroalgae represent 26 % of the global production of cultivated organisms, with Gracilaria spp. representing 12 % of that production; Eucheuma spp. and Kappaphycus alvarezii account for 34 % of world’s algae production. Despite the potential for cultivating seaweed in Brazil, and with its more than 8000 km of coastline, there is neither marine algaculture nor detailed knowledge even among aquaculture farmers concerning the utility of algae in agriculture, industry, and gastronomy, with the result that algaculture represents only the smallest fraction of national aquaculture production. The main cultivated species of seaweed sold in Brazil include the exotic K. alvarezii and native species of Gracilaria that are grown on small scales and do not meet national industrial demands, which must be supplemented by imports. We discuss Brazilian algaculture here, pointing out some of the problems that restrict commercial production of algae in that country and offer solutions that could be shared with other nations.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Setae thickening in <Emphasis Type="Italic">Daphnia magna</Emphasis> alleviates the food stress caused by the filamentous cyanobacteria

It is assumed that daphnids adjust the filter screen morphology in order to minimize the interference with cyanobacterial filaments. The aim of this study was to investigate the impact of filamentous cyanobacteria (Aphanizomenon gracile Lemmermann, Cylindrospermopsis raciborskii Woloszynska Seenaya et Subba Raju) on the thickness and length of setae of the third pair of thoracic limbs of Daphnia magna. The second objective was to assess whether the setae modifications could improve the performance of daphnids in the presence of cyanobacteria. Three clones of Daphnia magna Straus were cultured with: green algae; green algae with filaments of Cylindrospermopsis; and green algae with filaments of Aphanizomenon. The size and age of animals in the first reproduction cycle as well as the number of offspring were recorded. Setae thickness and length were measured in the central part of each endopodite. Additionally, we analyzed how the changes in setae morphology affect the fitness of experimental animals using the intrinsic rate of population increase calculated with the Euler–Lotka equation. The results showed that the thickness and length of setae increased in the presence of filamentous cyanobacteria. Moreover, cyanobacteria-induced setae thickening was positively correlated to the fitness of daphnids, which may indicate setae thickening as a phenotypic adaptation to cope with food stress caused by filamentous cyanobacteria.     

Characterisation of 12 microsatellite loci in the Vietnamese commercial clam <Emphasis Type="Italic">Lutraria rhynchaena</Emphasis> Jonas 1844 (Heterodonta: Bivalvia: Mactridae) through next-generation sequencing

The marine clam Lutraria rhynchaena is gaining popularity as an aquaculture species in Asia. Lutraria populations are present in the wild throughout Vietnam and several stocks have been established and translocated for breeding and aquaculture grow-out purposes. In this study, we demonstrate the feasibility of utilising Illumina next-generation sequencing technology to streamline the identification and genotyping of microsatellite loci from this clam species. Based on an initial partial genome scan, 48 microsatellite markers with similar melting temperatures were identified and characterised. The 12 most suitable polymorphic loci were then genotyped using 51 individuals from a population in Quang Ninh Province, North Vietnam. Genetic variation was low (mean number of alleles per locus = 2.6; mean expected heterozygosity = 0.41). Two loci showed significant deviation from Hardy–Weinberg equilibrium (HWE) and the presence of null alleles, but there was no evidence of linkage disequilibrium among loci. Three additional populations were screened (n = 7–36) to test the geographic utility of the 12 loci, which revealed 100 % successful genotyping in two populations from central Vietnam (Nha Trang). However, a second population from north Vietnam (Co To) could not be successfully genotyped and morphological evidence and mitochondrial variation suggests that this population represents a cryptic species of Lutraria. Comparisons of the Qang Ninh and Nha Trang populations, excluding the 2 loci out of HWE, revealed statistically significant allelic variation at 4 loci. We reported the first microsatellite loci set for the marine clam Lutraria rhynchaena and demonstrated its potential in differentiating clam populations. Additionally, a cryptic species population of Lutraria rhynchaena was identified during initial loci development, underscoring the overlooked diversity of marine clam species in Vietnam and the need to genetically characterise population representatives prior to microsatellite development. The rapid identification and validation of microsatellite loci using next-generation sequencing technology warrant its integration into future microsatellite loci development for key aquaculture species in Vietnam and more generally, aquaculture countries in the South East Asia region.     

Field and laboratory methods to monitor lake aerosols for cyanobacteria and microcystins

This study tested field and laboratory methods for the collection of cyanobacteria and microcystins emitted from lake water. These methods feature a highly portable, on-lake system for collecting aerosols directly from the lake, as well as a laboratory system for measurement of aerosols from freshly collected water samples under controlled conditions. Membrane air filters (0.45 μm) collected small particles such as picoplankton (0.2–2.0 μm) from aerosolized lake water. Picocyanobacteria were distinguished from other photosynthetic cells with epifluorescence microscopy using excitation filters for chlorophyll a (435 nm) and for phycobilin pigments (572 nm), characteristic of cyanobacteria. Aerosolization of picocyanobacteria ranged from 8872 to 167,297 cells m ^?3 in the field and 23,764 to 365,011 cells m ^?3 in the laboratory. Microcystin levels from field air filters ranged (below detectable limits) <13–384 pg MC m ^?3 of air. The described methods could be used for monitoring aerosolized cyanobacteria for public health purposes.     

Blue light reduces photosynthetic efficiency of cyanobacteria through an imbalance between photosystems I and II

Several studies have described that cyanobacteria use blue light less efficiently for photosynthesis than most eukaryotic phototrophs, but comprehensive studies of this phenomenon are lacking. Here, we study the effect of blue (450 nm), orange (625 nm), and red (660 nm) light on growth of the model cyanobacterium Synechocystis sp. PCC 6803, the green alga Chlorella sorokiniana and other cyanobacteria containing phycocyanin or phycoerythrin. Our results demonstrate that specific growth rates of the cyanobacteria were similar in orange and red light, but much lower in blue light. Conversely, specific growth rates of the green alga C. sorokiniana were similar in blue and red light, but lower in orange light. Oxygen production rates of Synechocystis sp. PCC 6803 were five-fold lower in blue than in orange and red light at low light intensities but approached the same saturation level in all three colors at high light intensities. Measurements of 77 K fluorescence emission demonstrated a lower ratio of photosystem I to photosystem II (PSI:PSII ratio) and relatively more phycobilisomes associated with PSII (state 1) in blue light than in orange and red light. These results support the hypothesis that blue light, which is not absorbed by phycobilisomes, creates an imbalance between the two photosystems of cyanobacteria with an energy excess at PSI and a deficiency at the PSII-side of the photosynthetic electron transfer chain. Our results help to explain why phycobilisome-containing cyanobacteria use blue light less efficiently than species with chlorophyll-based light-harvesting antennae such as Prochlorococcus, green algae and terrestrial plants.     

The effects of phosphorus and temperature on the competitive success of an invasive cyanobacterium

Rising lake temperatures and changing nutrient inputs are believed to favour the spread of a toxic invasive cyanobacterium, Cylindrospermopsis raciborskii (Woloszynska) Seenayya and Subba Raju, in temperate lakes. However, most evidence for these hypotheses is observational or based on physiological measurements in monocultures. We lack clear experimental evidence relating temperature and nutrients to the competitive success of C. raciborskii. To address this, we performed a 2 × 2 factorial laboratory experiment to study the dynamics of mixed phytoplankton communities subjected to different levels of temperature and phosphorus over 51 days. We allowed C. raciborskii to compete with ten different species from major taxonomic groups (diatoms, green algae, cryptophytes, and cyanobacteria) typical of temperate lakes, under low and high summer temperatures (25 and 30 °C) at two levels of phosphorus supply (1 and 25 µmol L^?1). Cylindrospermopsis raciborskii dominated the communities and strongly decreased diversity under low-phosphorus conditions, consistent with the hypothesis that it is a good phosphorus competitor. In contrast, it remained extremely rare in high-phosphorus conditions, where fast-growing green algae dominated. Surprisingly, temperature played a negligible role in influencing community composition, suggesting that changes in summer temperature may not be important in determining C. raciborskii’s spread.     

Cloning,Expression, and Characterization of Siamese Crocodile (<Emphasis Type="Italic">Crocodylus siamensis</Emphasis>) Hemoglobin from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Recombinant Crocodylus siamensis hemoglobin (cHb) has been constructed and expressed using Escherichia coli as the expression system in conjunction with a trigger factor from the Cold-shock system as the fusion protein. While successful processing as soluble protein in E. coli was achieved, the net yields of active protein from downstream purification processes remained still unsatisfactory. In this study, cHb was constructed and expressed in the eukaryotic expression system Pichia pastoris. The results showed that cHb was excreted from P. pastoris as a soluble protein after 72 h at 25 °C. The amino acid sequence of recombinant cHb was confirmed using LC–MS/MS. Indeed, the characteristic of Hb was investigated by external heme incorporation. The UV–Vis profile showed a specific pattern of the absorption at 415 nm, indicating the recombinant cHb was formed complex with heme, resulting in active oxyhemoglobin (OxyHb). This result suggests that the heme molecules were fully combined with heme binding site of the recombinant cHb, thus producing characteristic red color for the OxyHb at 540 and 580 nm. The results revealed that the recombinant cHb was prosperously produced in P. pastoris and exhibited a property as protein–ligand binding. Thus, our work described herein offers a great potential to be applied for further studies of heme-containing protein expression. It represents further pleasing option for protein production and purification on a large scale, which is important for determination and characterization of the authenticity features of cHb proteins.     

Algicidal Activity of Thiazolidinedione Derivatives Against Harmful Algal Blooming Species

Thiazolidinedione (TD) derivatives exhibit algicidal activity against harmful algal blooming species such as Chattonella marina, Heterosigma akashiwo, and Cochlodinium polykrikoides, as reported previously. In this study, the efficacies and selectivities of TD derivatives were tested by analyzing the structure–activity relationships of various TD derivatives. To investigate structure–activity relationships for growth inhibition of harmful algae, we added a methylene group between the cyclohexyl ring and oxygen of 5-(3-chloro-4-hydroxybenzylidene)-TD, which decreased the inhibitory potency of compound 17. Interestingly, another addition of a methylene group significantly increased the inhibitory potency against C. polykrikoides. The addition of 1 μM compound 17 resulted in the cell rupture of harmful algae after less than 10 h incubation at 20 °C. Compound 17 was applied to both harmful and non-harmful algae and showed a drastic reduction in the efficiency of photosystem II, resulting in reduced photosynthetic oxygen evolution. Compound 17 at a 5 μM concentration destroyed all of the harmful algae, while algicidal activity against non-harmful algae did not exceed 30% of the control within the concentration range tested. In contrast, a herbicide, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, tested at a 5 μM concentration, exhibited 40–70% algicidal activity relative to that of the control against both harmful and non-harmful algae. Compound 17 is a promising lead compound for the development of algicides to control harmful algal blooming species.     

Extraction of sterols in brown macroalgae from Antarctica and their identification by liquid chromatography coupled with tandem mass spectrometry

This study reports for the first time the extraction and quantification of sterols in six species of brown macroalgae from Antarctica: Adenocystis utricularis, Ascoseira mirabilis, Cystosphaera jacquinotii, Desmarestia anceps, Desmarestia antarctica, and Himantothallus grandifolius. Ultrasound irradiation was used as a promotor to extract sterols from algal biomass. The extracts were analyzed by high-performance liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for sterol quantization (ergosterol, brassicasterol, fucosterol, β-sitosterol, campesterol, cholesterol, and stigmasterol). In general, fucosterol was the most abundant (6.60 to 48.13 mg kg^?1), followed by β-sitosterol (5.29 to 16.49 mg kg^?1), stigmasterol (2.69 to 14.84 mg kg^?1) in five of the six examined algae. The sterol campesterol was found in lower concentrations (0.07 to 0.15 mg kg^?1) in all studied samples.     

High-yield expression,purification, characterization,and structure determination of tag-free <Emphasis Type="Italic">Candida utilis</Emphasis> uricase

We report the successful high-yield expression of Candida utilis uricase in Escherichia coli and the establishment of an efficient three-step protein purification protocol. The purity of the recombinant protein, which was confirmed to be C. utilis uricase by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometer analysis, was >98% and the specific activity was 38.4 IU/mg. Crystals of C. utilis uricase were grown at 18°C using 25% polyethylene glycol 3350 as precipitant. Diffraction by the crystals extends to 1.93 Å resolution, and the crystals belong to the space group P2₁2₁2₁ with unit cell parameters a?=?69.16 Å, b?=?139.31 Å, c?=?256.33 Å, and α?=?β?=?γ?=?90°. The crystal structure of C. utilis uricase shares a high similarity with other reported structures of the homologous uricases from other species in protein database, demonstrating that the three-dimensional structure of the protein defines critically to the catalytic activities.     

Fate of radiocesium in freshwater aquatic plants and algae in the vicinity of the Fukushima Daiichi nuclear power plant

The behavior of radiocesium (¹³⁷Cs) in aquatic plants (five species) and algae (three genera) grown in either a river (one sampling point) or pond (four sampling points) in the vicinity of the Fukushima Daiichi nuclear power plant was investigated. The ¹³⁷Cs concentration of <0.45-μm fractions of water taken from the river and ponds was between 5.01 × 10^?1 and 2.98 Bq/L, while that of sediment was between 4.85 × 10³ and 5.72 × 10⁴ Bq/kg dry weight. The ratio of ¹³⁷Cs concentration of sediment/water in ponds was ~10⁴. The sediment-to-plant transfer factor (TF) [(¹³⁷Cs concentration Bq/kg dry weight_plant) × (¹³⁷Cs concentration Bq/kg dry weight_sediment)^?1] was also measured. For aquatic plants, the highest value was 5.55 for Potamogeton crispus from the river, while the lowest was 3.34 × 10^?2 for P. distinctus from a pond. There were significant differences in values between aquatic plants belonging to the same genus. The water-to-plant TF [(¹³⁷Cs concentration Bq/kg dry weight_plant) × (¹³⁷Cs concentration Bq/L_water)^?1] of filamentous algae (Spirogyra sp.) and cyanobacteria (coexisting Anabaena sp. and Microcystis sp.) were 2.39 × 10³ and 1.26 × 10³, respectively. The ¹³⁷Cs concentration of cyanobacteria in pond water was 4.87 × 10^?1 Bq/L, which was the same order of magnitude as the ¹³⁷Cs concentration of pond water. Enrichment of ¹³⁷Cs in cyanobacteria was not observed.     

Algal phospholipids by 31P NMR: Comparing isopropanol pretreatment with simple chloroform/methanol extraction

• 1.1. A modified Folch procedure [potassium (ethylenedinitrilo)-tetraacetic acid at pH 6 substituted for KC1] is suitable for the extraction of marine algae.
• 2.2. The quantitative ³¹P nuclear magnetic resonance phospholipid profiles of four marine algae, Gracilaria verrucosa, Bryothamnion triquetrum, Padina gymnospora, and Caulerpa sertularioides, were obtained from Folch and Nichols extractions of both fresh and dried algae, and essentially identical results were obtained using either extraction procedure.
• 3.3. Extracts of air-dried algae are statistically different when compared to extracts of living algae, suggesting that tissue handling is a critical factor in phospholipid extractions.

     

Optimization of environmental parameters for <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> growth and lipid content using the response surface method and invading organisms

Algae biofuel has the potential to replace fossil fuels. However, cultivation and productivity of target algae need improvement, while controlling undesired organisms that can lower the efficiency of production systems. A central composite design and response surface model were utilized to predict cultivation optima of marine microalga, Nannochloropsis salina, under a suite of environmental parameters. The effects of salinity, pH, and temperature and their interactions were studied on maximum sustainable yield (MSY, a measure for biomass productivity), lipid content of N. salina, and invading organisms. Five different levels of each environmental predictor variable were tested. The environmental factors were kept within ranges that had previously been determined to allow positive N. salina growth (14.5–45.5 PSU; pH 6.3–9.7; 11–29 °C). The models created for this experiment showed that N. salina’s MSY and lipid content are not strongly affected over the broad range of salinity and temperature values. Calculated optima levels were 28 PSU/20 °C for MSY and 14.5 PSU/20 °C for lipid accumulation, but neither value significantly influenced the model. However, pH was the most important factor to influence algae productivity, and pH optimum was estimated around 8. Both MSY and lipid content were strongly reduced when pH deviated from the optimum. Occurrence of invading organisms seemed stochastic, and none of the environmental factors studied significantly influenced abundance. In conclusion, pH should be kept around 8 for maximum productivity of N. salina. Temperature and salinity should be kept around 20 °C and 28 PSU; however, moderate variations are not too much of a concern and might enhance lipid content of N. salina.     

Butyric acid production from red algae by a newly isolated <Emphasis Type="Italic">Clostridium</Emphasis> sp. S1

Objective

To produce butyric acid from red algae such as Gelidium amansii in which galactose is a main carbohydrate, microorganisms utilizing galactose and tolerating inhibitors in hydrolysis including levulinic acid and 5-hydroxymethylfurfural (HMF) are required.

Results

A newly isolated bacterium, Clostridium sp. S1 produced butyric acid not only from galactose as the sole carbon source but also from a mixture of galactose and glucose through simultaneous utilization. Notably, Clostridium sp. S1 produced butyric acid and a small amount of acetic acid with the butyrate:acetate ratio of 45.4:1 and it even converted acetate to butyric acid. Clostridium sp. S1 tolerated 0.5–2 g levulinic acid/l and recovered from HMF inhibition at 0.6–2.5 g/l, resulting in 85–92 % butyric acid concentration of the control culture. When acid-pretreated G. amansii hydrolysate was used, Clostridium sp. S1 produced 4.83 g butyric acid/l from 10 g galactose/l and 1 g glucose/l.

Conclusion

Clostridium sp. S1 produces butyric acid from red algae due to its characteristics in sugar utilization and tolerance to inhibitors, demonstrating its advantage as a red algae-utilizing microorganism.

     

Detection of microcystin producing cyanobacteria in Spirulina dietary supplements using multiplex HRM quantitative PCR

Arthrospira species, under the name ‘Spirulina’, are used as food supplement for its protein, vitamins, and minerals which have several health benefits. Cyanobacterial toxins including microcystins can possibly contaminate these dietary supplements causing hepatotoxicity, tumour formation, and other disorders. The safe use of dietary supplements necessitates the need to assess such toxins in the algal food supplement. The methods which evaluate these dietary supplements should be highly sensitive, cost-effective, and rapid. In this study, multiplex HRM qPCR analysis was used to detect microcystin (MC)-producing cyanobacteria in Spirulina dietary supplements. The multiplex HRM qPCR detection limit was found to be 25 ag of mcyB spiked in a standard concentration of pcb (25 pg). Two distinct melt curves characteristic of pcb (Tm 82.8 ± 0.07 °C) and mcyB (Tm 77.9 ± 0.05 °C) were observed. Microcystin contamination was detected only in the fish food supplements and not in human dietary supplements of Spirulina. Liquid chromatography–high-resolution mass spectrometry analysis further confirmed the presence of the congeners of microcystin in the identified positive samples.