Les principales voies enzymatiques de l'assimilation de l'azote chez les microalgues eucaryotes

Nitrogen utilization by eukaryotic microalgae involves 1) the uptake of the nutrient nitrogen, 2) the intracellular processing of the nitrogen to convert it to ammonia if necessary, 3) the assimilation of the ammonia into small organic molecules (amino acids) and 4) the synthesis of larger organic macromolecules. The ammonia is assimilated into the intracellular amino acid pool of eukaryotic microalgae primarily by either the GS/GOGAT pathway or the reaction of GDH. Here we review our current knowledge on the characteristics of these enzymes and on their putative functions in ammonia assimilation. It seems to be generally accepted that the GS/GOGAT pathway is the main pathway for the ammonia assimilation in eukaryotic microalgae. There is, however, evidence for the role of GDH in ammonia assimilation in some species. In other species the role of this enzyme is thought to be in catalysing the conversion of glutamate to ammonia. The distribution of the isoforms of glutamate dehydrogenase that use NADH or 97 NADPH as reductant make any generalisation difficult.     

Microalgae as multi-functional options in modern agriculture: current trends,prospects and challenges

Algae are a group of ubiquitous photosynthetic organisms comprising eukaryotic green algae and Gram-negative prokaryotic cyanobacteria, which have immense potential as a bioresource for various industries related to biofuels, pharmaceuticals, nutraceuticals and feed. This fascinating group of organisms also has applications in modern agriculture through facilitating increased nutrient availability, maintaining the organic carbon and fertility of soil, and enhancing plant growth and crop yields, as a result of stimulation of soil microbial activity. Several cyanobacteria provide nitrogen fertilization through biological nitrogen fixation and through enzymatic activities related to interconversions and mobilization of different forms of nitrogen. Both green algae and cyanobacteria are involved in the production of metabolites such as growth hormones, polysaccharides, antimicrobial compounds, etc., which play an important role in the colonization of plants and proliferation of microbial and eukaryotic communities in soil. Currently, the development of consortia of cyanobacteria with bacteria or fungi or microalgae or their biofilms has widened their scope of utilization. Development of integrated wastewater treatment and biomass production systems is an emerging technology, which exploits the nutrient sequestering potential of microalgae and its valorisation. This review focuses on prospects and challenges of application of microalgae in various areas of agriculture, including crop production, protection and natural resource management. An overview of the recent advances, novel technologies developed, their commercialization status and future directions are also included.     

Mechanisms of response to salinity in halotolerant microalgae

Summary A limited number of organic solutes are used by microalgae to adjust their internal osmotic pressure in response to changing external salinities. Glycerol and proline are used by the most extremely halotolerant algae. Only glycerol allows growth at salinities approaching saturation. In addition to organic osmoregulatory solutes, inorganic ions also play an important role in osmoregulation. The ability of microalgae to maintain intracellular ions at levels compatible with metabolic functions may set upper limits for their salt tolerance. Requirements for NaCl in the external medium for nutrient transport may define the lower salinity limits for growth observed for some euryhaline algae.Osmotic upshocks generally cause severe temporary inhibition of photosynthesis in euryhaline microalgae. Extensive osmotic downshocks have little effect on photosynthesis in microalgae with strong cell walls, while wall-less species appear to be more sensitive. Rapid glycerol synthesis takes place in response to increased external salinity inChlamydomonas pulsatilla both in light and dark. Starch supplies carbon for glycerol synthesis in the dark and also during the initial periods of inhibition of photosynthesis in the light. Turnover of osmoregulatory solutes such as glycerol and isofloridoside may be an important aspect of the osmoregulatory mechanism.At salinities beyond the growth limit for the green flagellateChlamydomonas pulsatilla, resting spores are formed that enable this alga to survive extreme salinities.     

Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation

Microalgae are regarded as a potential biomass source for biofuel purposes. With regard to bioethanol production, microalgae seem to overcome traditional substrate drawbacks. Enzymatic activities are responsible for carbon allocation and hence for carbohydrate profiles. Enzyme activities may be manipulated by metabolic engineering; however, this goal may also be achieved by controlling environmental conditions of the culture system. We outline the key-enzymes as well as the main operational conditions applied to microalgae growth (inorganic nutrient supplementation, irradiance and temperature) that affect carbohydrate synthesis on microalgae and cyanobacteria. Normally, harsh conditions are needed for such a goal and thus, arrested microalgae growth may occur. Potential strategies to avoid arrested growth, while enhancing carbohydrate accumulation, were also pointed out in this review.     

Borrowing genes from Chlamydomonas reinhardtii for free fatty acid production in engineered cyanobacteria

Photosynthetically derived fuels, such as those produced by microalgae, are touted as a future renewable energy source and a means for achieving energy independence. Realization of these claims, however, will require fuel production rates beyond the native capabilities of these microorganisms. The development of a metabolic engineering toolkit for microalgae will be key for reaching the production rates necessary for fuel production. This work advances the toolkit for cyanobacterial fuels by exploring the use of eukaryotic algal gene sources for free fatty acid biosynthesis rather than the traditional bacterial and plant sources. Many species of eukaryotic algae naturally accumulate high levels of triacylglycerol, a compound requiring three fatty acid side chains. Triacylglycerol accumulation implies that eukaryotic algae have naturally efficient enzymes for free fatty acid production, representing an unexplored resource for metabolic engineering targets. In this work, the model cyanobacterium, Synechococcus elongatus PCC7942, was engineered for free fatty acid production by targeting three main rate-limiting steps: (1) fatty acid release, catalyzed by a thioesterase, (2) fixation of carbon by ribulose-1,5-bisphosphate carboxylase/oxygenase, and (3) the first committed step in fatty acid biosynthesis, acetyl-CoA carboxylase. The recombinant acyl-ACP thioesterase and acetyl-CoA carboxylase were derived from the model green alga, Chlamydomonas reinhardtii CC-503. By targeting these proposed rate-determining steps, free fatty acid production was improved on a cell weight basis; however, the overall concentration of excreted free fatty acid did not increase. Recombinant gene expression was optimized by using native promoters, and while expression improved, the free fatty acid yield did not likewise increase. From physiological measurements, it was determined that free fatty acid production in S. elongatus PCC7942 is ultimately limited by the negative physiological effects associated with free fatty acid synthesis rather than bottlenecks within the metabolic pathway. This work demonstrates the successful expression of algal genes in a cyanobacterial host, but further improvement in free fatty acid yields will only be possible when the negative effects of free fatty acid production are mitigated.     

A Critical Review of Genome Editing and Synthetic Biology Applications in Metabolic Engineering of Microalgae and Cyanobacteria

Being the green gold of the future, microalgae and cyanobacteria have recently attracted considerable interest worldwide, for their metabolites such as lipids, protein, pigments, and bioactive compounds have immense potential for sustainable energy and pharmaceutical production capabilities. In the last decades, the efforts attended to enhance the usage of microalgae and cyanobacteria by genetic manipulation, synthetic and metabolic engineering. However, the development of photoautotrophic cell factories have rarely compared to the heterotrophic counterparts due to limited tools, bioinformatics, and multi‐omics database. Therefore, recent advances of their genome editing techniques by clustered regularly interspaced short palindromic repeats (CRISPR) technology, and potential applications of their metabolic engineering and regulation approaches are examined in this review. Moreover, the contemporary achievements of synthetic biology approaches of microalgae and cyanobacteria in carbon fixation and sequestration, lipid and triacylglycerol (TAG), and sustainable production of high value‐added chemicals, such as carotenoids and docosahexaenoic acid (DHA), have been also discussed. From recent genomic study to trends in metabolic regulation of microalgae and cyanobacteria and a comprehensive assessment of the current challenges and opportunities for microalgae and cyanobacteria is also conducted.     

Allelopathic effects of Ulva lactuca on selected species of harmful bloom-forming microalgae in laboratory cultures

Allelopathic effects of the green macroalgae Ulva lactuca on the growth of three species of red tide microalgae, Heterosigma akashiwo, Alexandrium tamarense, and Skeletonema costatum were tested in laboratory co-cultures precluding the nutrient and light limitation and the effect of high pH. The growth of all three species of microalgae was significantly (p < 0.01) inhibited by fresh U. lactuca. In nutrient replete semicontinuous co-cultures with U. lactuca, H. akashiwo was completely dead in 12 days, and the growth of A. tamarense and S. costatum was reduced by 48 and 46%, respectively by U. lactuca within 12 days. The U. lactuca culture filtrate exhibited a significant (p < 0.05) inhibitory effect on the microalgae in the first 1 or 2 days, but growth resumed in the following days, and S. costatum growth was slightly (p > 0.05) promoted from day 3. The results suggested that the allelopathic compounds are quickly degradable and a long-term inhibition might need the continuous addition of compounds originated from macroalgae. Dried U. lactuca also exhibited inhibitory effects on the microalgae, and the normalized mean growth rates of microalgae decreased with the biomass of dried U. lactuca. The dependent relationships were y = −2.1208x² + 1.0159x + 0.9752 for H. akashiwo, y = 0.7133x² − 3.5813x + 1.1665 for A. tamarense, and y = −0.2114x² − 1.063x + 1.0873 for S. costatum, respectively. The potential feasibility of utilization of dried U. lactuca against red tide microalgae was 2.0 g dry wt L⁻¹. The present study shows that U. lactuca exhibits negative allelopathic effects on harmful bloom-forming microalgae.     

Acclimation of Chlamydomonas reinhardtii to its nutrient environment

To cope with low nutrient availability in nature, organisms have evolved inducible systems that enable them to scavenge and efficiently utilize the limiting nutrient. Furthermore, organisms must have the capacity to adjust their rate of metabolism and make specific alterations in metabolic pathways that favor survival when the potential for cell growth and division is reduced. In this article I will focus on the acclimation of Chlamydomonas reinhardtii, a unicellular, eukaryotic green alga to conditions of nitrogen, sulfur and phosphorus deprivation. This organism has a distinguished history as a model for classical genetic analyses, but it has recently been developed for exploitation using an array of molecular and genomic tools. The application of these tools to the analyses of nutrient limitation responses (and other biological processes) is revealing mechanisms that enable Chlamydomonas to survive harsh environmental conditions and establishing relationships between the responses of this morphologically simple, photosynthetic eukaryote and those of both nonphotosynthetic organisms and vascular plants.     

基于环境DNA技术的辽东湾真核微藻群落结构特征

以18S rDNA V4区作为目标基因,利用自行设计的真核微藻鉴定引物V4（F/R）,结合高通量测序技术,对辽东湾2014年四季海水中真核微藻多样性进行了检测。结果发现,辽东湾海域注释到种的真核微藻有136种,41%的种类在中国海域未见报道,其中自养型占60%、异养型占10%、混合营养型占30%。研究对丰富中国海域微藻名录和外来海洋微藻背景数据库具有较大意义。     

光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶活力的影响

单细胞真核绿藻在中国水螅(Hydra sinensis)内胚层皮肌细胞中共生是有较高科研价值的特殊生物学现象。水螅宿主细胞为共生藻提供CO2、氮源及矿物质,而共生藻通过光合作用可能为宿主提供碳水化合物等有机物营养,因此水螅与共生藻间代谢流是以共生藻光合作用为中心,但基于代谢流二者间的互作机制目前尚未阐明。水螅通过营养积累进行出芽生殖,从母体脱落的芽体数量间接反映水螅营养积累的相对量。而光暴露时长能影响共生绿藻光合作用,如果共生藻的确能向水螅细胞转移光合作用产物,那光暴露时长应该能间接影响水螅的营养积累、从而进一步影响水螅无性出芽生殖。为证实该假说,本研究应用种群累积培养法,观察了光周期对中国水螅种群增长、无性出芽生殖及抗氧化酶(SOD和CAT)活力的影响。结果显示,光周期对中国水螅种群增长具有明显的影响。培养15 d后,所有实验组水螅的种群密度均为正增长,其中8L∶16D(在一个24h周期内光暴露8 h、黑暗16 h)实验组种群密度最大、而0L∶24D(持续黑暗)实验组种群密度最小。另外,随着光暴露时长的增加,中国水螅SOD及CAT活力整体均呈下降趋势。结果表明,从光周期对中国水螅无性出芽生殖及两种抗氧化酶活力的影响来看,中国水螅对光周期的生理学响应较为敏感,这个现象可能源于共生绿藻能通过向宿主细胞转移光合作用产物的方式为水螅提供营养补充。     

An alternative strategy for enhancing lipid accumulation in chlorophycean microalgae for biodiesel production

Oleaginous microalgae are considered as important feedstocks for production of biodiesel. Under nutrient stress conditions, microalgae have the ability to accumulate higher amount of lipids, which can be transesterified for the production of biodiesel. In the present investigation, four different phosphate application strategies were examined in five green microalgae (Tetradesmus obliquus, Tetradesmus lagerheimii, Chlorella vulgaris, Chlorella minutissima, and Chlamydomonas sp.) to achieve higher lipid productivity. Effects of those strategies such as phosphate-sufficient (Control), phosphate-starved approach (PSA), biphasic phosphate-starved approach (BPSA), and sequential phosphate addition (SPA) were studied under batch culture mode. The BPSA emerging as the best in terms of lipid productivity consisted of two biomass harvesting phases, which would lead to an increase in the overall cost of biodiesel production. On the other hand, the SPA with a 1/200th dose of N 11 medium, i.e., 0.4 mg L^?1 of phosphate application in 3-day intervals, also resulted into higher lipid productivity which was equal to BPSA. Fatty acid composition of the biodiesel obtained from the microalgae was analyzed and the fuel characteristics were also evaluated. A profound (~14-fold) reduction in phosphorus requirements under the SPA mode with higher lipid productivity ensured qualitative biodiesel production and a lesser amount of phosphorus release, thus making the process eco-friendly.     

New horizons in culture and valorization of red microalgae

Research on marine microalgae has been abundantly published and patented these last years leading to the production and/or the characterization of some biomolecules such as pigments, proteins, enzymes, biofuels, polyunsaturated fatty acids, enzymes and hydrocolloids. This literature focusing on metabolic pathways, structural characterization of biomolecules, taxonomy, optimization of culture conditions, biorefinery and downstream process is often optimistic considering the valorization of these biocompounds. However, the accumulation of knowledge associated with the development of processes and technologies for biomass production and its treatment has sometimes led to success in the commercial arena. In the history of the microalgae market, red marine microalgae are well positioned particularly for applications in the field of high value pigment and hydrocolloid productions. This review aims to establish the state of the art of the diversity of red marine microalgae, the advances in characterization of their metabolites and the developments of bioprocesses to produce this biomass.     

Is axenicity crucial to cryopreserve microalgae?

Large culture collections of microalgae and cyanobacteria such as the Coimbra Collection of Algae (ACOI) hold unialgal cultures consisting of a population of cells/colonies of a certain species. These cultures are usually non-axenic, as other organisms such as bacteria and microfungi are also present in culture due to co-isolation. Attention has been recently given to partner organisms since studies indicate that some bacteria are important for nutrient uptake of the algal cells, acting as simbionts. Despite this benign effect in the actively growing cultures, when cryopreservation is applied for inactive-stage storage, these organisms may recover faster than the algae, thus affecting their recovery and the viability assessments. In this study, a set of mucilaginous ACOI microalgae were selected, cell features known for their relevance in cryopreservation success were recorded and simple two-step cryopreservation tests were applied. Thawed samples were transferred to fresh culture medium for recovery. Viability was assessed and partner organism proliferation (pop) was recorded. Results were analyzed by t-tests. Statistical models allowed us to support the known tendency for small, unicellular algae with no outer structures to be successfully cryopreserved and the negative effect of vacuoles in the cell prior to cryopreservation. On average cryopreservation with MeOH or Me₂SO led to the recovery of nearly half the cells. It was found that the cryoprotection step with MeOH is when pop is triggered and that the use of Me₂SO can prevent this effect. Progress on understanding the cultured consortia will assist the improvement of cryopreservation and research using microalgal cultures.     

长山群岛海域真核微藻粒级结构及扇贝摄食选择性

采用高通量测序-分子鉴定分级技术于2019年对长山群岛全海域真核微藻粒级结构进行了研究。结果发现,春季以中（47%）、小粒级（41%）为主,夏季以小（39%）、大粒级（38%）为主,秋季以大粒级（60%）为主,春、夏、秋季小、中、大粒级微藻比例为42：47：11、39：23：38、22：18：60。小粒级微藻优势种为细小微胞藻（Micromonas pusilla）、融合微胞藻（Micromonas commoda）和金牛微球藻（Ostreococcus tauri）,中粒级微藻优势种为剧毒卡尔藻（Karlodinium veneficum）、大粒级微藻优势种为柔弱几内亚藻（Guinardia delicatula）、平野亚历山大藻（Alexandrium hiranoi）、多纹膝沟藻（Gonyaulax polygramma）,综合整个真核微藻群落,春季由中粒径的剧毒卡尔藻占据优势（23.9%）,夏季由大粒径的平野亚历山大藻占据优势（29.4%）,秋季由大粒径的多纹膝沟藻占据优势（66.8%）,有毒甲藻在该海域中占有绝对优势,贝毒累积风险较高,小粒径微藻中金牛微球藻和抑食金球藻曾在渤海引发褐潮,潜在威胁该海域贝类养殖业。虾夷扇贝对小粒级和大粒级微藻的选择性较低,对中粒级微藻的选择性较高,尤其对水体中优势种剧毒卡尔藻一直表现出主动选择。光学需氧量、无机氮、溶解氧、石油类及部分重金属Cd、As、Hg影响着整个长山群岛海域真核微藻粒级结构时空演变。     

In vitro culture and conservation of microalgae: Applications for aquaculture, biotechnology and environmental research

Summary Microalgae are a highly diverse group of unicellular organisms comprising the eukaryotic protists and the prokaryotic cyanobacteria or blue-green algae. The microalgae have a unique environmental status; being virtually ubiquitous in euphotic aquatic niches, they can occupy extreme habitats ranging from tropical coral reefs to the polar regions, and they contribute to half of the globe’s photosynthetic activity. Furthermore, they form the basis of the food chain for more than 70% of the world’s biomass. Microalgae are a valuable environmental and biotechnological resource, and the aim of this review is to explore the use of in vitro technologies in the conservation and sustainable exploitation of this remarkable group of organisms. The first part of the review evaluates the importance of in vitro methods in the maintenance and conservation of microalgae and describes the central role of culture collections in applied algal research. The second part explores the application of microalgal in vitro technologies, particularly in the context of the aquaculture and biotechnology industries. Emphasis is placed upon the exploitation of economically important algal products including aquaculture feed, biomass production for the health care sector, green fertilizers, pigments, vitamins, antioxidants, and antimicrobial agents. The contribution that microalgae can make to environmental research is also appraised; for example, they have an important role as indicator organisms in environmental impact assessments. Similarly, designated culture collection strains of microalgae are used for ecotoxicity testing. Throughout the review, emphasis is placed on the application of in vitro techniques for the continued advancement of microalgal research. The paper concludes by assessing future perspectives for the novel application of microalgae and their products.     

Commercial production of microalgae: ponds, tanks, tubes and fermenters

The commercial culture of microalgae is now over 30 years old with the main microalgal species grown being Chlorella and Spirulina for health food, Dunaliella salina for β-carotene, Haematococcus pluvialis for astaxanthin and several species for aquaculture. The culture systems currently used to grow these algae are generally fairly unsophisticated. For example, Dunaliella salina is cultured in large (up to approx. 250 ha) shallow open-air ponds with no artificial mixing. Similarly, Chlorella and Spirulina also are grown outdoors in either paddle-wheel mixed ponds or circular ponds with a rotating mixing arm of up to about 1 ha in area per pond. The production of microalgae for aquaculture is generally on a much smaller scale, and in many cases is carried out indoors in 20–40 l carboys or in large plastic bags of up to approximately 1000 l in volume. More recently, a helical tubular photobioreactor system, the BIOCOIL™, has been developed which allows these algae to be grown reliably outdoors at high cell densities in semi-continuous culture. Other closed photobioreactors such as flat panels are also being developed. The main problem facing the commercialisation of new microalgae and microalgal products is the need for closed culture systems and the fact that these are very capital intensive. The high cost of microalgal culture systems relates to the need for light and the relatively slow growth rate of the algae. Although this problem has been avoided in some instances by growing the algae heterotrophically, not all algae or algal products can be produced this way.     

Thermal constraints on growth,stoichiometry and lipid content of different groups of microalgae with bioenergy potential

Temperature has a range of effects on population demographic rates and physiology of microalgae for potential bioenergy application. The changes in microalgal growth, stoichiometry and cellular lipid contents of 14 different microalgae species with high bioenergy potentials in response to different temperature between 10 and 35 °C were investigated. For most of the chosen microalgae, the specific growth rate increased with temperature over most of the range. On the other hand, the biomass density of most of the selected species declined with temperature at stationary phase. Certain cyanobacteria failed to grow below 20 °C, while eukaryotes had a wider temperature range. Lipid content, by contrast, showed different responses to temperature among the 14 species. Nine of the species showed a positive response with increased temperature, while others showed neural or unimodal response. Cellular C:N and C:P ratios were mostly unimodally related to temperature. Some eukaryotic species showed the lowest total lipid contents but the highest C:N and C:P ratios, the fastest growth, and the highest biomass density at their stationary phases. The results indicated that the responses of the life history and cellular stoichiometry to temperature were more consistent across microalgae than that of total lipid content.     

Progress in physicochemical parameters of microalgae cultivation for biofuel production

Microalgae have been exploited for biofuel generation in the current era due to its enormous energy content, fast cellular growth rate, inexpensive culture approaches, accumulation of inorganic compounds, and CO₂ sequestration. Currently, research is ongoing towards the advancement of the microalgae cultivation parameters to enhance the biomass yield. The main objective of this study was to delineate the progress of physicochemical parameters for microalgae cultivation such as gaseous transfer, mixing, light demand, temperature, pH, nutrients and the culture period. This review demonstrates the latest research trends on mass transfer coefficient of different microalgae culturing reactors, gas velocity optimization, light intensity, retention time, and radiance effects on microalgae cellular growth, temperature impact on chlorophyll production, and nutrient dosage ratios for cellulosic metabolism to avoid nutrient deprivation. Besides that, cultivation approaches for microalgae associated with mathematical modeling for different parameters, mechanisms of microalgal growth rate and doubling time have been elaborately described. Along with that, this review also documents potential lipid-carbohydrate-protein enriched microalgae candidates for biofuel, biomass productivity, and different cultivation conditions including open-pond cultivation, closed-loop cultivation, and photobioreactors. Various photobioreactor types, the microalgae strain, productivity, advantages, and limitations were tabulated. In line with microalgae cultivation, this study also outlines in detail numerous biofuels from microalgae.     

微藻废水生物处理技术研究进展

微藻因生长速率快、细胞脂质含量高及具有生物隔离二氧化碳能力,已作为新一代生物质能源受到广泛关注.然而,投入大量淡水资源并需在生长期间持续提供营养物质已成为规模化培育微藻的主要障碍.将微藻培育系统与废水处理相结合是经济可行的污水资源化方案.基于微藻生长期间对氮磷等营养物质的利用机制,本文综述了微藻在各类废水生物处理过程中的应用情况,着重分析了其对废水中有机与无机化合物、重金属以及病原体的去除或抑制能力.同时,考察了废水初始营养物浓度、光照、温度、pH与盐度以及气体交换量等环境因素对微藻生长代谢的影响.此外,结合微藻规模化应用所面临的问题,对微藻废水处理技术的应用前景及发展方向进行了展望,旨在为水生态系统的建设与管理提供参考.