Metabolic engineering and genome editing strategies for enhanced lipid production in microalgae

Depleting global petroleum reserves and skyrocketing prices coupled with succinct supply have been a grave concern,which needs alternative sources to conventional fuels.Oleaginous microalgae have been explored for enhanced lipid production,leading towards biodiesel production.These microalgae have short life cycles,require less labor,and space,and are easy to scale up.Triacylglycerol,the primary source of lipids needed to produce biodiesel,is accumulated by most microalgae.The article focuses on different types of oleaginous microalgae,which can be used as a feedstock to produce biodiesel.Lipid biosynthesis in microalgae occurs through fatty acid synthesis and TAG synthesis approaches.In-depth discussions are held regarding other efficient methods for enhancing fatty acid and TAG synthesis,regulating TAG biosynthesis bypass methods,blocking competing pathways,multigene approach,and genome editing.The most potential targets for gene transformation are hypothesized to be a malic enzyme and diacylglycerol acyltransferase while lowering phosphoenolpyruvate carboxylase activity is reported to be advantageous for lipid synthesis.

Fluorescence-assisted Image Analysis of Harmful Microalgae

In order to realize the detection and analysis of microalgae in sediment samples with complex scenes, the project takes advantage of the character of microalgae that they can auto-fluoresce when exposed to the illumination of certain exciation waves. The project takes grey-scale and fluorescent pictures of microalgae in the same field of view and uses the image processing technique to deal with the images, such as threshold segmentation, contour and texture analysis and pattern recognition. The results show that the fluorescent image can effectively elimate the yawp in the complex background and make the consequent image processing more effective and easy. Then the project comes to the conclusion that fluorescence-assisted image processing can realize the detection and analysis of microalgae in sediment samples containing complex scenes.

Preliminary screening of antimicrobial and antitumor activities from cultivated microalgaes

Hydrophilic and lipophilic extracts were prepared from 8 microalgal strains, and screened for antimicrobial and antitumor activities. Antimicrobial activity was determined by observing bacterial （ S. aureus, Bacillus subtilis and Escherichia coh~ and fungal（Aspergillus niger and Penicillium chrysogenum） growth inhibition. All the microalgae had different degrees of antimicrobial activity against one or more microbe - tested, and 56.47% of the extracts showing the anti-S.aureus activity exhibited the antibacterial activity against （MRSA）. Cytotoxic activities were measured in vitro against human cancer cell lines HeLa by the MTT assay. Most of these extracts showed potent activity against the growth of the tumor cells, especially the intracellular lipophilic extracts from Isochrysis galbana Parke 3011 and Isochrysis galbana Parke H29, which exhibited strong antitumor activity against HeLa cell lines. The overall results of this study indicate that the extracts from microalgae represent a potential sources of medicine for the treatment of infectious and cancer diseases.

Study on Tolerance and Biosorption of Various Microalgae Species to Arsenic

[Objective] This study aimed to select microalgae species which are capable to effectively remove arsenic contamination from water under natural conditions. [Method] Four microalgae species [Chlorella sp. （zfsaia）, Chlorella minata, Chlorella vulgaris and Selenastrum capricormulum] were used as experimental materials and cultured with six different concentrations of As （Ⅲ） （0.5, 1.0, 2.0, 5.0, 10.0, 20.0 mg/L）. Biomass, chlorophyll a content and other physiological indicators were determined to investigate the arsenic tolerance and biosorption of four microalgae species. [Result] Chlorella sp. is sensitive to arsenic toxicity, its growth was inhibited when arsenic concentration exceeded 10 mg/L, with an EC 50 of 17.32 mg/L; when the arsenic concentration was 0-20 mg/L, growth of S.c, ww1 and C.v was not affected, which showed relatively high tolerance to arsenic, with arsenic removal rates of 77.02% , 72.18% and 81.36% respectively after 24 h. [Conclusion] This study indicates that microalgae have good application prospects for processing arsenic wastewater and being indicator plants of arsenic wastewater.

Screening of the Gene for Chlorella Identification and Identification of oil-producing Microalgae

[Objective] The aim was to select suitable gene for Chlorella identification and to identify the oil-producing microalgae.[Method] Four candidate gene sequences,the nuclear genomic rDNA of the 18S rRNA gene,internal transcribed spacer（ITS）,internal transcribed spacer Ⅱ（ITS Ⅱ）and the chloroplast rbcL gene,were selected for Chlorella molecular identification.Through these four candidate genes,the genetic variability and distinguish ability between intra-species and inter-species was analyzed to choose the right genes for identification of the high oil-content Chlorella.On this basis,application of these gene segments were classified and identified for five fresh-water isolated Chlorella,which oil-content is more than 30%.[Result] ITS gene was a suitable gene because of its high variation and short fragment length,meanwhile its genetic distance intra-species（0.439 6±0.135 9）was larger than inter-species（0.045 7±0.084 3）.Its sequence length varied between different species whereas highly conserved in the same species.By the application of ITS sequences,respectively,five high oil-content stains were identified as one C.vulgaris,two strains of C.sorokiniana and two strains of algae Chlorella sp.[Conclusion] This study had provided reference for the establishment of identification gene pool of Chlorella.

Aqueous extracts of microalgae exhibit antioxidant and anticancer activities

Objective:To investigate the antioxidant and anticancer activities of aqueous extracts of nine microalgal species.Methods:Variable percentages of major secondary metabolites(total phenolic content,terpenoids and alkaloids) as well as phycobiliprotein pigments(phycocyanin, allophycocyanin and phycoerythrin) in the aqueous algal extracts were recorded.Antioxidant activity of the algal extracts was performed using 2,2 diphenyl-1-picrylhydrazyl(DPPH) test and 2,2'azino-bis(ethylbenzthiazoline-6-sulfonic acid(ABTS.) radical cation assay.Anticancer efficiency of the algal water extracts was investigated against Ehrlich Ascites Carcinoma cell(EACC) and Human hepatocellular cancer cell line(HepG2).Results:Antioxidant activity of the algal extracts was performed using DPPH test and ABTS.^+ radical cation assays which revealed 30.1-72.4%and 32.0-75.9%respectively.Anticancer efficiency of the algal water extracts was investigated against Ehdich Ascites Carcinoma Cell(EACC) and Human Hepatocellular cancer cell line(HepG2) with an activity ranged 87.25%and 89.4%respectively.Culturing the promising cyanobacteria species;Nostoc muscorum and Oscillatoria sp.under nitrogen stress conditions(increasing and decreasing nitrate content of the normal BG11 medium,1.5 g/L),increased nitrate concentration(3,6 and 9 g/L) led to a remarkable increase in phycobilin pigments followed by an increase in both antioxidant and anticancer activities in both cyanobacterial species.While the decreased nitrate concentration(0.75,0.37 and 0.0 g/L) induced an obvious decrease in phycobilin pigments with complete absence of allophycocyanin in case of Oscillatoria sp.Conclusions:Nitrogen starvation(0.00 g/L nitrate) induced an increase and comparable antioxidant and anticancer activities to those cultured in the highest nitrate content.

Research on KOH/La-Ba-Al_2O_3 catalysts for biodiesel production via transesterification from microalgae oil

Alumina supports modified by lanthanum （La） and barium （Ba） were prepared by peptization. Catalysts with different KOH contents supported on modified alumina were prepared by impregnation method. Various techniques, including N2 adsorption-desorption （Brunauer-Emmet-Teller method, BET）, X-ray diffraction （XRD）, scanning electron microscopy （SEM）, and fourier transform infrared absorption spectroscopy （FT-IR）. Catalytic activity for microalgae oil conversion to methyl ester via transesterification was evaluated and analyzed by GC-MS and GC. BET results showed that the support possessed high specific surface area, suitable pore volume and pore size distribution. Activity results indicated that the catalyst with 25 wt% KOH showed the best activity for microalgae oil conversion. XRD and SEM results revealed that Al-O-K compound was the active phase for microalgae oil conversion. The agglomeration and changing of pore structure should be the main reasons for the catalyst deactivation when KOH content was higher than 30 wt%.

Fatty acid methyl ester profiles and nutritive values of 20 marine microalgae in Korea

Objecive:To screen the fatty acid(FA) composition of 20 marine microalgae species,including seven Diophyceae,six Bacillariophyeae four Chlorophyceae,two Haptophyceae and one Raphidophyceae species.Methods:Microalgal cells cultured at the Korea Institute of Ocean Science & Technology were harvested during the late exponential growth phase and the FA composition analyzed.Results:The FA composition of microalgae was speciesspecific.For example,seven different species of Dinophyceae were composed primarily of C14:0,C16:0.C18:0.C20:4n-6.C20:5n-3 and C22:6n-3.while C14:0.C16:0,C16:1.C18:0.C20:5n-3 and C22:6n-3 were abundant FAs in six species of Bacillariophyceae.In addition,four Chlurophyceae,two Haptopkyeeae and one Raphidophyceae species all contained a high degree of C16:1 n-7[(9.2R-34.91)%and(34.48-35.04)%].C14:0[(13.34-25.96)%]and[(26.69-Z8.24)%],and C16:0[(5.89-29.15)%]and[(5.70-16.81)%].Several factors contribute to the nutritional value of microalgae.including the polyunsaturated FA content and n-3 to n-6 FA ratio,which could be used to assess the nutritional quality of microalgae.Conclusions:This study is the first comprehensive assessment of the FA composition and nutritional value of microalgae species in South Korea,and identifies the potential utility of FAs as species-specific biomarkers.

Experimental studies on dimethylsulfide (DMS) and dimethylsulfoniopropionate (DMSP) production by four marine microalgae

The production of dimethylsulfide （DMS） and dimethylsulfoniopropionate （DMSP） by marine microalgae was investigated to elucidate more on the role of marine phytoplankton in ocean-atmosphere interactions in the global biogeochemical sulfur cycle.Axenic laboratory cultures of four marine microalgae–Isochrysis galbana 8701,Pavlova viridis,Platymonas sp.and Chlorella were tested for DMSP production and conversion into DMS.Among these four microalgae,Isochrysis galbana 8701 and Pavlova viridis are two species of Haptophyta,while Chlorella and Platymonas sp.belong to Chlorophyta.The results demonstrate that the four algae can produce various amounts of DMS（P）,and their DMS（P） production was species specific.With similar cell size,more DMS was released by Haptophyta than that by Chlorophyta.DMS and dissolved DMSP （DMSPd） concentrations in algal cultures varied significantly during their life cycles.The highest release of DMS appeared in the senescent period for all the four algae.Variations in DMSP concentrations were in strong compliance with variations in algal cell densities during the growing period.A highly significant correlation was observed between the DMS and DMSPd concentrations in algal cultures,and there was a time lag for the variation trend of the DMS concentrations as compared with that of the DMSPd.The consistency of variation patterns of DMS and DMSPd implies that the DMSPd produced by phytoplankton cells has a marked effect on the production of DMS.In the present study,the authors’ results specify the significant contribution of the marine phytoplankton to DMS（P） production and the importance of biological control of DMS concentrations in oceanic water.

Food processing wastewater purification by microalgae cultivation associated with high value-added compounds production——A review

Microalgae have been considered as an efficient microorganism for wastewater treatment with simultaneously bioenergy and high value-added compounds production.However,the high energy cost associated with complicated biorefinery(e.g.microalgae cultivation,harvesting,drying,extraction,conversion,and purification)is a critical challenge that inhibits its large-scale application.Among different nutrition(e.g.carbon,nitrogen and phosphorous)sources,food processing wastewater is a relative safe and suitable one for microalgae cultivation due to its high organic content and low toxicity.In this review,the characteristic of different food wastewater is summarized and compared.The potential routes of value-added products(i.e.biofuel,pigment,polysaccharide,and amino acid)production along with wastewater purification are introduced.The existing challenges(e.g.biorefinery cost,efficiency and mechanism)of microalgal-based wastewater treatment are also discussed.The prospective of microalgae-based food processing wastewater treatment strategies(such as microalgae-bacteria consortium,poly-generation of bioenergy and value-added products)is forecasted.It can be observed that food wastewater treatment by microalgae could be a promising strategy to commercially realize waste source reduce,conversion and reutilization.

Microalgae:A potential alternative to health supplementation for humans

Microalgae has been consumed in human diet for thousands of years.It is an under-exploited crop for production of dietary foods.Microalgae cultivation does not compete with land and resources required for traditional crops and has a superior yield compared to terrestrial crops.Its high protein content has exhibited a huge potential to meet the dietary requirements of growing population.Apart from being a source of protein,presence of various bio-active components in microalgae provide an added health benefit.This review describes various microalgal sources of proteins and other bio-active components.One of the heavily studied group of bio-active components are pigments due to their anticarcenogenic,antioxidative and antihypertensive properties.Compared to various plant and floral species,microalgae contain higher amounts of pigments.Microalgal derived proteins have complete Essential Amino Acids(EAA)profiles and their protein content is higher than conventional sources such as meat,poultry and dairy products.However,microalgal based functional foods have not flooded the market.The lack of awareness coupled with scarce incentives for producers result in under-exploitation of microalgal potential.Application of microalgal derived components as dietary and nutraceutical supplements is discussed comprehensively.

Microalgae Tolerance to High Concentrations of Carbon Dioxide: A Review

The increasing concentration of carbon dioxide (CO2) in the atmosphere is considered to be one of the main causes of the global warming problem. Moreover, there is an international movement to reduce the emission of CO2 by imposing different measures such as carbon tax. Biological CO2 fixation has been extensively investigated as part of efforts to solve the global warming problem. Microalgae are fast growing systems that can consume high quantities of CO2 to produce different types of biomass. The efficiency of microalgae is highly related to the concentration of CO2 in the growth atmosphere and the higher the concentration of CO2 the better is the growth and hence productivity. The present review aimed at shedding some light upon microalgal capability to sustain their viability and propagate under high CO2 concentration.

Heavy Metal Bioaccumulation and Toxicity with Special Reference to Microalgae

The bioaccumulation and toxicity of heavy metals were reviewed with special reference to microalgae, the key compo- nent of the food web in aquatic ecosystems. Heavy metals enter algal cells either by means of active transport or by endocytosis through chelating proteins and affect various physiological and biochemical processes of the algae. The toxicity primarily results from their binding to the sulphydryl groups in proteins or disrupting protein structure or displacing essential elements. Metals can break the oxidative balance of the algae, inducing antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and ascorbate peroxidase (APX). The amount of oxidized proteins and lipids in the algal cells thus indicates the severity of the stress. Algal tolerance to heavy metal is highly dependent upon the defense response against the probable oxidative damages. Pro- duction of binding factors and proteins, exclusion of metals from cells by ion-selective transporters and excretion or compartmen- talization have been suggested with regard to reducing heavy metal toxicity. However, a comprehensive description on the mecha- nisms underlining metal toxicity of microalgae and gaining tolerance is yet to be elaborated.

A method of batch-purifying microalgae with multiple antibiotics at extremely high concentrations

Axenic microalgal strains are highly valued in diverse microalgal studies and applications. Antibiotics, alone or in combination, are often used to avoid bacterial contamination during microalgal isolation and culture. In our preliminary trials, we found that many microalgae ceased growing in antibiotics at extremely high concentrations but could resume growth quickly when returned to an antibiotics-free liquid medium and formed colonies when spread on a solid medium. We developed a simple and highly efficient method of obtaining axenic microalgal cultures based on this observation. First, microalgal strains of different species or strains were treated with a mixture of ampicillin, gentamycin sulfate, kanamycin, neomycin and streptomycin （each at a concentration of 600 mg/L） for 3 days; they were then transferred to antibiotics-free medium for 5 days; and finally they were spread on solid f/2 media to allow algal colonies to form. With this method, five strains ofNannochloropsis sp. （Eustigmatophyceae）, two strains of Cylindrotheca sp. （Bacillariophyceae）, two strains of Tetraselmis sp. （Chlorodendrophyceae） and one strain ofAmphikrikos sp. （Trebouxiophyceae） were purified successfully. The method shows promise for batch- purifying microalgal cultures.

Effect of dissolved phosphorus on alkaline phosphatase activity in marine microalgae

Alkaline phosphatase activity (APA) and dissolved phosphorus were monitored during the batch cultures of two bone microalgae. Results indicate that variation of APA was in the shape of 'S' curve. Different specs of dissolved phaphorus had different effects on APA. The concentrations of dis solved inorganic phosphorus (DIP) and and molecular dissolved organic phosphorus (SDOP) had a sig nificant effect on APA, while the concentration of large molecular dissolved organic phosphorus (LDOP) had a little effect on APA., and the increase of APA could accelerate the decomposing of LDOP in the medium. Results also show that algae species and abundance had why a little effect on APA.

Ingestion of Brachionus plicatilis under different microalgae conditions

The effects of four microalgae, Chlorella vulgaris, Platymonas helgolandicavar, Isochrysis galbana, and Nitzschia closterium on the grazing and filtering rates of the marine rotifer, Brachionus plicatilis, were evaluated under laboratory conditions. The grazing rates in separate cultures of the four microalga were as follows： C. vulgaris 〉 P. helgolandicavar 〉 L galbana 〉 N. closterium. However, the filtering rates occurred in the following order： P. helgolandicavar 〉 N. closterium 〉 C. vulgaris 〉 L galbana. A mixed diets experiment revealed that P. helgolandicavar was the preferred diet of B. plicatilis. In addition, the grazing rate of B. plicatilis increased gradually as the density of the microalgae increased, until concentrations of 2.5×10^6 cells mL^-1 for C. vulgaris and 1.5×10^6 cells mL^-1 for I. galbana were obtained. Furthermore, the filtering rate increased slightly when the density of the microalgae was low, after which it declined as the microalgal density increased. The grazing rates of B. plicatilis were as follows during the different growth phases： stationary phase 〉 exponential phase 〉 lag phase 〉 decline phase. Additionally, the filtering rates during the growth phases were： exponential phase 〉 lag phase 〉 stationary phase 〉 decline phase. The results of this study provide foundational information that can be used to explore the optimal culture conditions for rotifers and to promote the development of aquaculture.

Design of a Novel Photobioreactor for Culture of Microalgae

This paper presents the design of a novel photobioreactor for cultivation of microalgae. The body of the reactor with volume of about 40 L is parallelipipedic and divided in five compartments that can be put in series. The optical guides, plunged perpendicularly into the compartments, are upright Plexiglas plates on which side faces there are able to diffuse light laterally and ensure an even distribution of light in the medium. External airlifts through the side columns are used for mixing of culture medium. The external light source is a SON-T lamp mounted inside a projector that provides a conical light dispersion, it is interchangeable and may take different positions. The design offers the photobioreactor characteristics including mainly interchangeable light source, homogenous distribution of light, perfect mixing of suspension of algae, high ratio of illuminating surface to volume of reactor, compactness and absence of contamination. Schemes, view of the photobioractor and data of continuous culture forSpirulina maxima are presented.

Responses in growth, lipid accumulation,and fatty acid composition of four oleaginous microalgae to different nitrogen sources and concentrations

Nitrogen deficiency is an effective strategy for enhancing lipid production in microalgae. Close relationships exist among lipid production, microalgal species, and nitrogen sources. We report growth, lipid accumulation, and fatty acid composition in four microalgae （Chloroeoccum ellipsoideum UTEX972, Chlorococcum nivale LB2225, Chlorococcum tatrense UTEX2227, and Scenedesmus deserticola JNU19） under nitrate- and urea-nitrogen deficiencies. We found three patterns of response to nitrogen deficiency： Type-A （decrease in biomass and increase in lipid content）, Type-B （reduction in both biomass and lipid content）, and Type-C （enhancement of both biomass and lipid content）. Type-C microalgae are potential candidates for large-scale oil production. Chlorococcum ellipsoideum, for example, exhibited a neutral lipid production of up to 239.6 mg/（L＇d） under urea-nitrogen deficiency. In addition, nitrogen deficiency showed only a slight influence on lipid fractions and fatty acid composition. Our study provides useful information for further screening hyper-lipid microalgal strains for biofuel production.

Distribution characteristics of planktonic nano－and microalgae in the Prydz Bay and its adjacent Southern Indian Ocean，during austral summer

Nano- and microalgae samples were collected from 34 stations in the Prydz Bay and its adjacent Southern indian Ocean, Antarctica during a Period from December 1990 to January 1991. 122 taxa belonging to 40 genera 5 phyla were identified. Among them diatom occupy 73%,dinoflagellates 20. 5%, the other species 6. 5 %. The average cell abundance of nano- and microalgae was 2551×104 cells/m3 in surface waters. The concentration of nano- and microalgae occurred in the Prydz Bay and its adjacent continental station Ⅳ 7 and northern water area between West Ice Shelf and Shackleton Ice Shelf (Stations Ⅶ 3, Ⅷ 1, Ⅷ 2, Ⅷ 3, Ⅸ 1, Ⅺ 1); and minimal abundance occurred in northwestern area of the Prydz Bay (some stations of section Ⅲ and Station Ⅳ 1-5). The average cell abundance of planktonic nano- and microalgae from net sample was 811. 62×104 cells/m3, and the concentration area occurred in the adjacent continent waters of the Prydz Bay (Stations Ⅳ 2, Ⅳ 4, Ⅳ 5, Ⅳ 7, Ⅴ 4 and Ⅴ 5) and northern area of the West Ice Shelf(Stations Ⅶ 1 and Ⅶ 3); low abundance occurred in eastern area of the Shackleton Ice Shelf (at each station of sections Ⅹ and Ⅺ ). For the vertical distribution of planktonic nano- and microalgae, the maximum value was usually found at the surface and above 50 m depth, and gradually decreased with the water depth from 100 m to 200 m. The relationship between planktonic nano-and microalgal abundance and Euphausia superba density and nutrient (nitrate,phosphate and silicate) contents were negative significance.

Biodiesel Production from <i>Spirulina-Platensis</i>Microalgae by <i>In-Situ</i>Transesterification Process

This research investigates the effect of reaction variables that strongly affect the cost of biodiesel production from non-edible Spirulina-Platensis microalgae lipids, and use the acid-catalyzed in situ transesterification process. Experiments were designed to determine how variations in volume of reacting methanol, the concentration of an acid catalyst, time, temperature and stirring affected the biodiesel yield. The total lipid content of Spirulina-Platensis microalgae was obtained to be 0.1095g/g biomass. The weight of the by-product glycerol obtained was used to predict the percentage yield conversion of microalgae oil biodiesel. Best results (84.7%), a yield of fatty acid methyl ester (FAME), were obtained at 100% (wt./wt.oil) catalyst concentration, 80 ml methanol volumes, 8 h reaction time and 65℃ reaction temperature with continuous stirring at 650 rpm. Properties of the produced biodiesel were measured according to EN 14214 standards.