Selection of optimal flocculant for effective harvesting of the fucoxanthin-rich marine microalga <Emphasis Type="Italic">Isochrysis galbana</Emphasis>

Flocculation harvesting of the fucoxanthin-rich marine microalga Isochrysis galbana has received little attention. Therefore, we attempted to screen for an optimal chemical flocculant and optimize flocculation conditions from five chemical flocculants—ferric chloride (FC), aluminum sulfate (AS), polyaluminum chloride (PAC), aluminum potassium sulfate (APS), and zinc sulfate (ZS)—for effective flocculation of I. galbana. The growth rate, photosynthetic performance, and fucoxanthin content were determined in re-suspended flocculated algal cells and in the flocculation supernatant cultured algal cells. The results showed that high growth rate and fucoxanthin accumulation were observed when FC was used as the flocculant in I. galbana cultures, which indicated that FC may cause less harm to I. galbana than the other aluminum-based flocculants. Furthermore, satisfactory flocculation efficiency was also observed when FC was used to flocculate I. galbana, and the FC dosage was less than that required for flocculation of I. galbana using PAC, APS, and AS. Thus, we selected FC as the optimal flocculant for harvesting I. galbana based on its flocculation efficiency together with algal physiological performance, growth rate, and fucoxanthin content.     

Flocculation of microalgae in brackish and sea waters

Flocculation is an essential step in the concentration and harvesting of microalgae from aquatic media. Salinity of brackish water and sea water requires high flocculant dosages and renders flocculation less effective than in freshwater algal media. Experiments with the marine microalgae Isochrysis galbana and Chlorella stigmatophora showed that effective alum or ferric chloride flocculation was obtained only with dosages which are 5 to 10 times higher than the dosages required for the flocculation of freshwater microalgae. The flocculant dosages required for removing over 90% of the algae from suspensions were found to increase linearly with salinity as expressed in ionic strength. High salinity was found to inhibit flocculation with polyelectrolytes which are quite effective in freshwater algae flocculation. This inhibition was diminished at reduced salinity levels and effective flocculation was attained at salinity levels of 5 g/liter and below, which is typical of desert brackish water. Two methods were found to induce flocculation in sea water: (a) combining polyelectrolytes with inorganic flocculants such as ferric chloride or alum, and (b) ozone oxidation pretreatment followed by flocculation with inorganic flocculants.     

Comparative evaluation of inorganic and organic amendments for their flocculation efficiency of selected microalgae

Cost-efficient harvesting of microalgae is a major challenge due to their small size and often low concentration in the culture medium. The flocculation efficacy of different inorganic and organic amendments was evaluated on various microalgae genera—one strain each belonging to Chlamydomonas, Chlorococcum, two of Botryococcus, and of Chlorella. An improvised medium comprising of commercial grade urea, single super phosphate, and muriate of potash was used to grow the microalgae for flocculation experiments. High pH induced increased flocculation efficiency (72–76 %) in selected microalgal strains. Ferric chloride was found to be the most efficient for most of the microalgal strains, while maize starch and rice starch proved superior for Chlorella sp. MCC6 and Botryococcus sp. MCC32. Although the highest flocculation efficiency was obtained with inorganic flocculant, i.e., ferric chloride (87.3 %) with Botryococcus MCC31, this was comparable with rice starch (86.8 %) for Botryococcus MCC32. This study showed that widely available cheaper biopolymers such as rice starch, maize, and potato starch can be promising flocculants due to their better harvesting efficiency (>80 %) and low price, thereby contributing to economical production of biodiesel from algae.     

Harvesting of microalgae using flocculation combined with dissolved air flotation

This study explored efficient methods of harvesting the Tetraselmis sp. KCTC12236BP using flocculation and dissolved air flotation. Concentration ranges of flocculation agents were optimized using jar tests (batch flocculation experiments) using inorganic (aluminum sulfate, ferric sulfate) and organic (chitosan) flocculants in a pH range of 4 ～ 10. The optimal dosage and pH level were 1.2 g/L and pH 5 ～ 6 for aluminum sulfate, 0.7 g/L and pH 4 ～ 8 for ferric sulfate, and 5.0 mg/mL and pH 7 ～ 8 for chitosan. The highest harvesting efficiency achieved with each of the four compounds was 85.6, 92.6, 93, and 91.3%, respectively.     

Evaluation of electro‐coagulation–flocculation for harvesting marine and freshwater microalgae

Although microalgae are considered as a promising feedstock for biofuels, the energy efficiency of the production process needs to be significantly improved. Due to their small size and low concentration in the culture medium, cost‐efficient harvesting of microalgae is a major challenge. In this study, the use of electro‐coagulation–flocculation (ECF) as a method for harvesting a freshwater (Chlorella vulgaris) and a marine (Phaeodactylum tricornutum) microalgal species is evaluated. ECF was shown to be more efficient using an aluminum anode than using an iron anode. Furthermore, it could be concluded that the efficiency of the ECF process can be substantially improved by reducing the initial pH and by increasing the turbulence in the microalgal suspension. Although higher current densities resulted in a more rapid flocculation of the microalgal suspension, power consumption, expressed per kg of microalgae harvested, and release of aluminum were lower when a lower current density was used. The aluminum content of the harvested microalgal biomass was less than 1% while the aluminum concentration in the process water was below 2 mg L⁻¹. Under optimal conditions, power consumption of the ECF process was around 2 kWh kg⁻¹ of microalgal biomass harvested for Chlorella vulgaris and ca. 0.3 kWh kg⁻¹ for Phaeodactylum tricornutum. Compared to centrifugation, ECF is thus more energy efficient. Because of the lower power consumption of ECF in seawater, ECF is a particularly attractive method for harvesting marine microalgae. Biotechnol. Bioeng. 2011;108: 2320–2329. © 2011 Wiley Periodicals, Inc.     

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.     

Harvesting the microalgae Phaeodactylum tricornutum with polyaluminum chloride, aluminium sulphate, chitosan and alkalinity-induced flocculation

The purpose of this study was to explore efficient methods of harvesting the microalga Phaeodactylum tricornutum. Natural sedimentation experiments, performed at different light and temperature conditions, did not yield significant improvements in efficiency even after 1?week. When alkalinity-induced flocculation was performed, both the flocculation efficiency and the concentration factor dramatically improved at pH?=?9.75 (0.5–0.7 units over the original pH of the culture) after 10?min settling time. Sedimentation rates are documented at pH ranging between pH?9.75 and 11.0. The results of the application of two conventional flocculants used in wastewater treatment, polyaluminium chloride and aluminium sulphate, are also presented. Chitosan was also used as a natural flocculating agent to improve possible contamination problems in the downstream process. pH was adjusted in order to determine optimum flocculation efficiency of chitosan in combination with a high concentration factor. Satisfactory results were found with chitosan at an adjusted pH of 9.9 using concentrations as low as 20?mg?L^?1, after testing a flocculant range of 5–200?mg?L^?1.     

Comparison of different artificial neural network architectures in modeling of Chlorella sp. flocculation

Biodiesel production from microalgae feedstock should be performed after growth and harvesting of the cells, and the most feasible method for harvesting and dewatering of microalgae is flocculation. Flocculation modeling can be used for evaluation and prediction of its performance under different affective parameters. However, the modeling of flocculation in microalgae is not simple and has not performed yet, under all experimental conditions, mostly due to different behaviors of microalgae cells during the process under different flocculation conditions. In the current study, the modeling of microalgae flocculation is studied with different neural network architectures. Microalgae species, Chlorella sp., was flocculated with ferric chloride under different conditions and then the experimental data modeled using artificial neural network. Neural network architectures of multilayer perceptron (MLP) and radial basis function architectures, failed to predict the targets successfully, though, modeling was effective with ensemble architecture of MLP networks. Comparison between the performances of the ensemble and each individual network explains the ability of the ensemble architecture in microalgae flocculation modeling.     

利用饵料微藻表达抗菌肽及其初步应用

为了解抗菌肽在饵料微藻中表达后的抗菌特性,构建海洋微拟球藻(Nannochloropsis oceanica)、湖泊微拟球藻(N.limnetica)和三角褐指藻(Phaeodactylum tricornutum)的抗菌肽(源自虹鳟,Cath-1a)表达质粒,分别转化相应的微藻,检测转化子中抗菌肽的表达量和体外抑菌效果,将藻株作为鱼饲料添加剂喂食斑马鱼,初步分析了抗菌肽及藻体自身的岩藻黄素和多不饱和脂肪酸对鱼免疫系统的影响。结果表明,外源抗菌肽在3种微藻中均可以成功表达,体外抑菌试验表明,仅三角褐指藻对水产领域常见致病菌爱德华氏菌(Edwardsiella tarda)有一定的抑菌效果,然而抗菌肽的表达并未使3种藻株的体外抑菌性增加。添加藻粉对斑马鱼的生长无明显影响,通过检测鱼体肝脏中与抗氧化和免疫相关基因的表达水平及丙二醛的含量,表明添加藻粉可增强斑马鱼的抗氧化和抗炎症能力,表达抗菌肽(PtC组)能进一步提高斑马鱼的免疫力。另外,添加Pt6(富含岩藻黄素)藻粉组比添加PtC的抗炎效果更显著,表明三角褐指藻中的岩藻黄素和二十碳五烯酸对增强鱼的抗病能力具有潜在作用。     

Growth kinetics and fucoxanthin production of <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Isochrysis galbana</Emphasis> cultures at different light and agitation conditions

Fucoxanthin is a carotenoid that exerts multiple beneficial effects on human health. However, reports comparing microalgae culture conditions and their effect on growth and fucoxanthin production are still limited. Isochrysis galbana and Phaeodactylum tricornutum cultures in different light (62.0, 25.9, 13.5, or 9.1 μmol photons m^-2 s^-1), mixing conditions (1 vvm aeration or 130 rpm agitation), and media compositions (F/2 and Conway medium) were studied for comparison of cellular growth and fucoxanthin production on F/2 medium. I. galbana showed a better adaptation to tested culture conditions in comparison with P. tricornutum, reaching 2.15?×?10⁷?±?4.07?×?10⁶ cells mL^-1 and a specific growth rate (μ) of 1.12?±?0.05 day^-1 under aerated conditions and 62.0 μmol photons m^-2 s^-1 light intensity. Fucoxanthin concentration was about 25 % higher in P. tricornutum cultures under 13.5 μmol photons m^-2 s^-1 light intensity and aerated conditions, but the highest fucoxanthin total production was higher in I. galbana, where 3.32 mg can be obtained from 1 L batch cultures at the 16th day under these conditions. Moreover, higher cell densities (~32.41 %), fucoxanthin concentration (~42.46 %), and total production (~50.68 %) were observed in I. galbana cultures grown in Conway medium, if compared with cultures grown in F/2 medium. The results show that the best growth conditions did not result in the best fucoxanthin production for either microalgae, implying that there is not a direct relationship between cellular growth and fucoxanthin production. Moreover, the results suggest that I. galbana cultures on Conway medium are strong candidates for fucoxanthin production, where 1.2 to 15 times higher fucoxanthin concentration are observed in comparison to macroalgal sources.     

Toxicity and bioaccumulation of iron in soil microalgae

Microalgae are extensively used in the remediation of heavy metals like iron. However, factors like toxicity, bioavailability and iron speciation play a major role in its removal by microalgae. Thus, in this study, toxicity of three different iron salts (FeSO₄, FeCl₃ and Fe(NO₃)₃) was evaluated towards three soil microalgal isolates, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11. Interestingly, all the three iron salts gave different EC50 concentrations; however, ferric nitrate was found to be significantly more toxic followed by ferrous sulphate and ferric chloride. The EC₅₀ analysis revealed that Chlorella sp. was significantly resistant to iron compared to other microalgae. However, almost 900 μg g^?1 iron was accumulated by Chlamydomonas sp. grown with 12 mg L^?1 ferric nitrate as an iron source when compared to other algae and iron salts. The time-course bioaccumulation confirmed that all the three microalgae adsorb the ferric salts such as ferric nitrate and ferric chloride more rapidly than ferrous salt, whereas intracellular accumulation was found to be rapid for ferrous salts. However, the amount of iron accumulated or adsorbed by algae, irrespective of species, from ferrous sulphate medium is comparatively lower than ferric chloride and ferric nitrate medium. The Fourier transform infrared spectroscopy (FTIR) analysis shows that the oxygen atom and P?=?O group of polysaccharides present in the cell wall of algae played a major role in the bioaccumulation of iron ions by algae.     

Harvesting of microalgae by bio-flocculation

The high-energy input for harvesting biomass makes current commercial microalgal biodiesel production economically unfeasible. A novel harvesting method is presented as a cost and energy efficient alternative: the bio-flocculation by using one flocculating microalga to concentrate the non-flocculating microalga of interest. Three flocculating microalgae, tested for harvesting of microalgae from different habitats, improved the sedimentation rate of the accompanying microalga and increased the recovery of biomass. The advantages of this method are that no addition of chemical flocculants is required and that similar cultivation conditions can be used for the flocculating microalgae as for the microalgae of interest that accumulate lipids. This method is as easy and effective as chemical flocculation which is applied at industrial scale, however in contrast it is sustainable and cost-effective as no costs are involved for pre-treatment of the biomass for oil extraction and for pre-treatment of the medium before it can be re-used.     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Antheraxanthin, a light harvesting carotenoid found in a chromophyte alga

The pigments of the chromophyte freshwater alga, Chrysophaera magna Belcher were analyzed by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) to reveal the presence of chlorophylls a and c, β-carotene, fucoxanthin, and antheraxanthin. The presence of antheraxanthin was verified by comparison of TLC R_F values, HPLC retention times, and absorption features to those of authentic, synthetic antheraxanthin. Antheraxanthin accounted for about 15% of the total carotenoid content of C. magna. The molar ratio of the major carotenoids was antheraxanthin:fucoxanthin:β-carotene, 1:2.3:3.3. The whole-cell absorption spectrum revealed a broad band between 470 and 520 nanometers which was attributed to fucoxanthin and antheraxanthin in vivo. Upon extraction in hydrocarbon, this broad absorption region was lost. The in vivo fluorescence excitation spectrum for 680 nm emission revealed the energy transfer activities and light harvesting roles of chlorophylls a and c, and fucoxanthin. In addition, an excitation band was resolved at 487 nanometers which could be attributed only to antheraxanthin. Comparison of whole-cell fluorescence excitation spectra of C. magna with the diatom Phaeodactylum tricornutum, which possesses fucoxanthin but not antheraxanthin, supports the assignment of the 487 nm band to antheraxanthin. This is the first report of a photosynthetic light harvesting function of the xanthophyll, antheraxanthin. This carotenoid broadens the absorption cross-section for photosynthesis in C. magna and extends light harvesting into the green portion of the spectrum.     

pH-induced flocculation,indirect electrocoagulation,and hollow fiber filtration techniques for harvesting the saltwater microalga Dunaliella

This research assessed the efficacy of three harvesting methods on a strain of Dunaliella viridis. While there is strong potential to use lipids from microalgae as a feedstock for biofuels to replace petroleum-based fuel, at present microalgal harvesting for biofuel production is not yet economically feasible or energy efficient. pH-induced flocculation (by adjusting the pH of exponentially growing cells), indirect electrocoagulation (applying aluminum hydroxide coagulant to culture), and hollow fiber filtration (separating biomass from medium using tangential flow) were compared as potential harvesting mechanisms for small-scale (3–10 L) and large-scale (30–150 L) volumes of D. viridis. Both pH-induced flocculation and electrocoagulation yielded significant biomass recovery (>95 %), but both methods required removal of added chemicals and/or coagulant before the medium could be reused. In contrast, hollow fiber filtration did not require added chemicals or coagulant, and as another advantage, the filtrate was successfully reused as culture medium without apparent detrimental effects on cell size, cell shape, or cell production. When high salinity stress was imposed on the concentrate produced from the filtration method, total fatty acids (FAs) did not increase. However, total FAs did significantly increase after hollow fiber filtration (49 %) in comparison to FA content before filtration (36 %). This research indicates that hollow fiber filtration as a commercial harvesting mechanism offers attractive advantages as a harvesting mechanism for microalgae such as Dunaliella, relative to pH-induced flocculation and indirect electrocoagulation.     

Xanthophyll synthesis in diatoms: quantification of putative intermediates and comparison of pigment conversion kinetics with rate constants derived from a model

Recently, we reported the presence of the violaxanthin-antheraxanthin-zeaxanthin cycle in diatoms, and showed that violaxanthin is the putative precursor of both diadinoxanthin and fucoxanthin in the diatom Phaeodactylum tricornutum Bohlin (M. Lohr and C. Wilhelm, 1999, Proc. Natl. Acad. Sci. USA 96: 8784–8789). In the present study, two possible intermediates in the synthesis of violaxanthin from β-carotene were identified in P. tricornutum, namely β-cryptoxanthin and β-cryptoxanthin epoxide. In low light, the latter pigment prevails, but in high light β-cryptoxanthin accumulates, probably as the result of an increased activity of the xantophyll-cycle de-epoxidase. The apparent kinetics of several xanthophyll conversion steps were determined for P. tricornutum and Cyclotella meneghiniana Kützing. The experimentally determined conversion rates were used to evaluate the hypothetical pathway of xanthophyll synthesis in diatoms. For this purpose a mathematical model was developed which allows the calculation of theoretical rates of pigment conversion for microalgae under steady-state growth conditions. A comparison between measured and calculated conversion rates agreed well with the proposal of a sequential synthesis of fucoxanthin via violaxanthin and diadinoxanthin. The postulation of zeaxanthin as an obligatory intermediate in the synthesis of violaxanthin, however, resulted in large discrepancies between the measured and calculated rates of its epoxidation. Instead of zeaxanthin, β-cryptoxanthin epoxide may be involved in the biosynthesis of violaxanthin in diatoms. Received: 16 March 2000 / Accepted: 30 June 2000     

Kinetic exploration of nitrate-accumulating microalgae for nutrient recovery

Within sustainable resource management, the recovery of nitrogen and phosphorus nutrients from waste streams is becoming increasingly important. Although the use of microalgae has been described extensively in environmental biotechnology, the potential of nitrate-accumulating microalgae for nutrient recovery has not been investigated yet. The ability of these marine microorganisms to concentrate environmental nitrate within their biomass is remarkable. The aim of this study was to investigate the application potential of nitrate-accumulating diatoms for nutrient recovery from marine wastewaters. The intracellular nitrate storage capacity was quantified for six marine diatom strains in synthetic wastewater. Amphora coffeaeformis and Phaeodactylum tricornutum stored the highest amount of nitrate with respectively 3.15 and 2.10 g N L^?1 of cell volume, which accounted for 17.3 and 4.6 %, respectively, of the total nitrogen content. The growth and nitrate and phosphate uptake of both diatoms were further analyzed and based on these features P. tricornutum showed the highest potential for nutrient recovery. A mathematical model was developed which included intracellular nitrate storage and the kinetic parameters were derived for P. tricornutum. Furthermore, a simulation study was performed to compare the performance of a proposed microalgal nutrient recovery unit with a conventional denitrification system for marine wastewater treatment. Overall, this study demonstrates the potential application of P. tricornutum for saline wastewater treatment with concurrent nitrogen and phosphorus recycling.     

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.     

A diatom light-harvesting pigment-protein complex : purification and characterization

A light-harvesting pigment-protein complex was isolated from the diatom Phaeodactylum tricornutum using the zwitterionic detergent CHAPS (3-[3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Detergent-solubilized membranes were fractionated by sucrose density gradient centrifugation into three components. The medium density fraction contained chlorophyll a, chlorophyll c, and fucoxanthin. This fraction was purified by DEAE-ion exchange chromatography, and contained chlorophyll a, chlorophyll c, and fucoxanthin in a molar ratio of 2.4:1.0:4.8. Fluorescence emission and excitation spectra of the isolated complex demonstrated that light energy absorbed by chlorophyll c and fucoxanthin was coupled to chlorophyll a fluorescence. Upon denaturation, the apoprotein yielded a polypeptide doublet at 17.5 to 18.0 kilodaltons which accounted for 30 to 40% of the toal membrane protein. These findings indicate that this pigment-protein complex is a major component of the diatom photosynthetic lammellae. The quantitative amino acid composition of the apoprotein was very similar to those reported for other membrane-bound pigment-protein complexes. Based on the protein to chlorophyll a ratio of 7700 grams protein per mole chlorophyll a for the complex, each apoprotein molecule contains, to the nearest integer, two chlorophyll a, one chlorophyll c, and five fucoxanthin molecules. Polyclonal antibodies raised against the 17.5 to 18.0 kilodaltons apoprotein showed a monospecific reaction with only the 17.5 to 18.0 protein zone from denatured P. tricornutum membranes as well as to the nondenatured pigment-protein complex. It appears that this complex is common to other diatom species.