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.

Experimental Study of Microalgae Cultivation under Selective Illumination by Ag/CoSO_(4)for Bioelectrode Materials Preparation

Microalgae biomass is an ideal precursor to prepare renewable carbon materials,which has broad application.The bioaccumulation efficiency(lipids,proteins,carbohydrates)and biomass productivity of microalgae are influenced by spectroscopy during the culture process.In this study,a bilayer plate-type photobioreactor was designed to cultivate Chlorella protothecoides with spectral selectivity by nanofluids.Compared to culture without spectral selectivity,the spectral selectivity of Ag/CoSO_(4)nanofluids increased microalgae biomass by 5.76%,and the spectral selectivity of CoSO_(4)solution increased by 17.14%.In addition,the spectral selectivity of Ag/CoSO_(4)nanofluids was more conducive to the accumulation of nutrients(29.46%lipids,50.66%proteins,and 17.86%carbohydrates)in microalgae.Further cultured chlorella was utilized to prepare bioelectrode materials,it was found that algal based biochar had a good pore structure(micro specific surface area:1627.5314 m^(2)/g,average pore size:0.21294 nm).As the current density was 1 A/g,the specific capacitance reached 230 F/g,appearing good electrochemical performance.

A Novel Hybrid Model Based on Machine and Deep Learning Techniques for the Classification of Microalgae

Classification and monitoring of microalgae species in aquatic ecosystems are important for understanding population dynamics.However,manual classification of algae is a time-consuming method and requires a lot of effort with expertise due to the large number of families and genera in its classification.The recognition of microalgae species has become an increasingly important research area in image recognition in recent years.In this study,machine learning and deep learning methods were proposed to classify images of 12 different microalgae species in order to successfully classify algae cells.8 Different novel models(MobileNetV3Small-Lr,MobileNetV3Small-Rf,MobileNetV3Small-Xg,MobileNetV3Large-Lr,MobileNetV3Large-Rf,MobileNetV3Large-Xg,Mobile-NetV3Small-Improved and MobileNetV3Large-Improved)have been proposed to classify these microalgae species.Among these proposed model structures,the best classification accuracy rate was 92.22%and the loss rate was 0.72,obtained from the MobileNetV3Large-Improved model structure.In addition,as a result of the experimental results obtained,metrics such as the confusion matrix,which can meet the experts in the correct diagnosis of microalgae species,were also evaluated.This research may in the future open a new avenue for the development of a cost-effective,highly sensitive computer-based system for the use of image analysis and deep learning techniques for the identification and classification of different microalgae.

Impacts of Sulfur and Microalgae Co-fertilization on Saline-alkaline Soil of Sunflower Field in Hetao Irrigation District

[Objectives]To evaluate the impacts of the elemental sulfur(S 0)and micro-algae(MA)co-fertilization on saline-alkaline soil of sunflower field in the Hetao Irrigation District(HID).[Methods]The greenhouse pot experiment was conducted with four treatments:control(CK),single S 0 fertilization(S),single MA fertilization(A),and S 0 and MA co-fertilization(SA)for comparing the selected soil properties and sunflower plant heights and weights in different treatments.[Results]The results showed that the mean soil organic matter(SOM)under the SA(25.08 g/kg)was significantly higher than that for the CK(20.59 g/kg),S(22.47 g/kg),and A(22.95 g/kg).The mean pH under the SA(7.75)was significantly lower than that for the CK(8.14),S(7.82),and A(7.96).The mean soil exchangeable Na+concentration under the SA was significantly lower than that for the S.The mean soil electrical conductivity(EC)under the SA was 9.76%lower than that for the S.The means of Cl-(1.22 g/kg)and SO 2-4(1.90 g/kg)in soil under the SA were lower than that for the S(1.30,2.06 g/kg)and A(1.31,1.97 g/kg),respectively.For plant height 3 at the late stage of plant growth,the mean plant height 3 under the SA(89.00 cm)was higher than that of the CK(69.60 cm)and A(74.33 cm).The total weights of the fresh sunflower heads,fresh stems,and dry seeds under the SA were higher than that for the CK,S,and A.[Conclusions]In conclusion,the S 0 and MA co-fertilization had positive effects on improving saline-alkaline soils,the soil under the S 0 and MA co-fertilization could be better conditions for promoting sunflower growth than that for the S,Z,and CK,and thereby the S 0 and MA co-fertilization could be a new idea to improve saline-alkaline soil in the cold and arid regions.

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.

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.

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%.

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.

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.

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.

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.

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.

Effects of carbon anhydrase on utilization of bicarbonate in microalgae:a case study in Lake Hongfeng

A bidirectional labeling method was established to distinguish the proportions of HCO3- and CO2 utiliza- tion pathways of microalgae in Lake Hongfeng. The method was based on microalgae cultured in a medium by adding equal concentrations of NaH13CO3 with different 613C values simultaneously. The inorganic carbon sources were quantified according to the stable carbon isotope composition in the treated microalgae. The effects of extracellular carbonic anhydrase （CAex） on the HCO3 and CO2 utilization pathways were distinguished using acetazolamide, a potent membrane-impermeable carbonic anhydrase inhibitor. The results show utilization of the added HCO3- was only 8% of the total carbon sources in karst lake. The proportion of the HCO3- utilization path- way was 52% of total inorganic carbon assimilation. Therefore, in the natural water of the karst area, the microalgae used less bicarbonate that preexisted in the aqueous medium than CO2 derived from the atmosphere. CAex increased the utilization of inorganic carbon from the atmosphere. The microalgae with CAex had greater carbon sequestration capacity in this karst area.

A sustainable integration of removing CO_(2)/NO_(x) and producing biomass with high content of lipid/protein by microalgae

Due to the boost of CO_(2)/NO_(x)emissions which cause environmental pollution,processes that remove such pollutants from flue gas have attracted increasing attention in recent years.Among these technologies,biological CO_(2)/NO_(x)emission reduction has received more interest.Microalgae,a kind of photosynthetic microorganism,offer great promise to convert CO_(2)/NO_(x)to biomass with high content of lipid and protein,which can be used as feedstock for various products such as biodiesel,health products,feedstuff and biomaterials.In this paper,biological CO_(2)/NO_(x)removing technologies by microalgae,together with the products(such as biofuel and protein)and their economic viability are discussed.Although commercial applications of microalgae for biodiesel and protein products are hampered by the high production cost of biomass,the use of CO_(2)/NO_(x)from flue gas as carbon and nitrogen sources can reduce the cost of biomass production,which makes these technologies more competent for real-life applications.Moreover,it is projected that the increasing in CO_(2)allowances will lead to further reduction in the cost of biomass production,which especially favors related products with lower values such as biodiesel.Furthermore,by combining various process optimization and integration,biorefinery is proposed and considered as the crucial component for the sustainable and economically feasible bulk applications of microalgae biomass.

Co-pyrolysis of South African bituminous coal and Scenedesmus microalgae:Kinetics and synergistic effects study

In this work,the co-pyrolysis of coal and algae is explored with special emphasis on decomposition kinetics and the possibility of the existence of synergistic effects.Modelling and kinetics analysis based approaches were used for the investigation of the existence of synergistic effects.The co-pyrolysis kinetics was studied using the model-free,Coats-Redfern integral method.The kinetics were evaluated for 1st and 2nd order reaction models.Results reveal that Scenedesmus microalgae is characterised by a two stage decomposition process that occurs at temperature ranges of 200-400℃and 500-700℃with activation energy of 145.5 and 127.3 kJ/mol,respectively.Bituminous coal has a two stage,slow decomposition process that occurs at temperature ranges of 400-700℃and above 750℃with an activation energy of 81.8 and 649.3 kJ/mol,respectively.Furthermore,co-pyrolysis of coal and microalgae is characterised by three stages whose kinetics are dominated by the pyrolysis of the individual materials.For the studied range of coal/algae ratios,the three pyrolysis stages occur in the approximate temperature ranges of 200-400℃,430-650℃and above 750℃,with activation energies in the ranges of 131-138,72-78 and 864.5-1235 kJ/mol,respectively.Modelling and kinetics study showed that there is strong evidence of interactions between coal and microalgae that manifest as synergistic effects especially in the second and third stages of decomposition.