System-level network analysis of nitrogen starvation and recovery in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> reveals potential new targets for increased lipid accumulation

Background

Nitrogen starvation is known to cause drastic alterations in physiology and metabolism leading to the accumulation of lipid bodies in many microalgae, and it thus presents an important alternative for biofuel production. However, despite the importance of this process, the molecular mechanisms that mediate the metabolic remodeling induced by N starvation and especially by stress recovery are still poorly understood, and new candidates for bioengineering are needed to make this process useful for biofuel production.

Results

We have studied the molecular changes involved in the adaptive mechanisms to N starvation and full recovery of the vegetative cells in the microalga Chlamydomonas reinhardtii during a four-day time course.High throughput mass spectrometry was employed to integrate the proteome and the metabolome with physiological changes. N starvation led to an accumulation of oil bodies and reduced Fv/Fm.. Distinct enzymes potentially participating in the carbon-concentrating mechanism (CAH7, CAH8, PEPC1) are strongly accumulated. The membrane composition is changed, as indicated by quantitative lipid profiles. A reprogramming of protein biosynthesis was observed by increased levels of cytosolic ribosomes, while chloroplastidic were dramatically reduced. Readdition of N led to, the identification of early responsive proteins mediating stress recovery, indicating their key role in regaining and sustaining normal vegetative growth.Analysis of the data with multivariate correlation analysis, Granger causality, and sparse partial least square (sPLS) provided a functional network perspective of the molecular processes. Cell growth and N metabolism were clearly linked by the branched chain amino acids, suggesting an important role in this stress. Lipid accumulation was also tightly correlated to the COP II protein, involved in vesicle and lysosome coating, and a major lipid droplet protein. This protein, together with other key proteins mediating signal transduction and adaption (BRI1, snRKs), constitute a series of new metabolic and regulatory targets.

Conclusions

This work not only provides new insights and corrects previous models by analyzing a complex dataset, but also increases our biochemical understanding of the adaptive mechanisms to N starvation in Chlamydomonas, pointing to new bioengineering targets for increased lipid accumulation, a key step for a sustainable and profitable microalgae-based biofuel production.

     

High temperature enhances lipid accumulation in nitrogen-deprived <Emphasis Type="Italic">Scenedesmus obtusus</Emphasis> XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 °C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 °C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 °C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.     

Fatty Acid Characterization and Biodiesel Production by the Marine Microalga <Emphasis Type="Italic">Asteromonas gracilis</Emphasis>: Statistical Optimization of Medium for Biomass and Lipid Enhancement

Lipid production is an important indicator for evaluating microalgal species for biodiesel production. In this study, a new green microalga was isolated from a salt lake in Egypt and identified as Asteromonas gracilis. The main parameters such as biomass productivity, lipid content, and lipid productivity were evaluated in A. gracilis, cultivated in nutrient-starved (nitrogen, phosphorous), and salinity stress as a one-factor-at-a-time method. These parameters in general did not vary significantly from the standard nutrient growth media when these factors were utilized separately. Hence, response surface methodology (RSM) was assessed to study the combinatorial effect of different concentrations of the abovementioned factor conditions and to maximize the biomass productivity, lipid content, and lipid productivity of A. gracilis by determining optimal concentrations. RSM optimized media, including 1.36 M NaCl, 1 g/L nitrogen, and 0.0 g/L phosphorus recorded maximum biomass productivity, lipid content, and lipid productivity (40.6 mg/L/day, 39.3%, and 15.9 mg/L/day, respectively) which agreed well with the predicted values (40.1 mg/L/day, 43.6%, and 14.6 mg/L/day, respectively). Fatty acid profile of A. gracilis was composed of C16:0, C16:1, C18:0, C18:3, C18:2, C18:1, and C20:5, and the properties of fuel were also in agreement with international standards. These results suggest that A. gracilis is a promising feedstock for biodiesel production.     

Biochemical and physiological responses of <Emphasis Type="Italic">Selenastrum gracile</Emphasis> (Chlorophyceae) acclimated to different phosphorus concentrations

Algae are able to adjust their metabolism according to their environment, maximizing growth rate and production of biomolecules under adverse conditions such as pulses of excess of a contaminant or limitation of a nutrient. In order to evaluate the effects of phosphorus (P) availability on the biochemical composition of the freshwater microalga Selenastrum gracile, we acclimated the microalgae to different phosphorus concentrations. After acclimation, exponentially growing cells were inoculated and after 120 h, samples were processed for the determination of carbohydrate, lipid, fatty acid, chlorophyll, cell density, growth rate, and dry weight. Cell density, growth rate, and dry weight decreased with less P, while chlorophyll a, carbohydrates, lipids, and fatty acids per cell increased under P limitation. According to our lipid class and fatty acid results, algae alter their metabolism and membrane configuration to avoid more structural or metabolic damage under limitation, especially at 23 μmol P L^?1. The most sensitive parameters under P limitation were chlorophyll a, lipids, and poly- and monounsaturated fatty acids. The changes in fatty acids contributed to the fluorescence and photosynthesis changes under P limitation, and they occurred before changes were detected in other parameters, such as growth rate. Furthermore, we suggest that prior acclimation to different P affected microalgal physiology and metabolism.     

Role of autophagy in triacylglycerol biosynthesis in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> revealed by chemical inducer and inhibitors

Autophagy mediates degradation and recycling of cellular components and plays an important role in senescence and adaptive responses to biotic and abiotic stresses. Nutrient deprivation has been shown to trigger triacylglycerol (TAG) accumulation and also induces autophagy in various green algae. However, the functional relationship between TAG metabolism and autophagy remains unclear. To gain preliminary evidence supporting a role of autophagy in TAG synthesis, Chlamydomonas reinhardtii CC-2686 was grown in Tris-acetate phosphate medium with or without nitrogen and treated with an autophagy inducer (rapamycin) or inhibitors (wortmannin, 3-methyladenine, and bafilomycin A1). Fluorescence microscopic analysis of Nile red-stained cells following 72-h treatments showed that rapamycin induced accumulation of subcellular lipid droplets which are storage sites of TAG. Rapamycin treatment in combination with nitrogen starvation led to a greater abundance of lipid droplets. Wortmannin and bafilomycin A1, but not 3-methyladenine, inhibited lipid droplet accumulation in rapamycin-treated cells and to a less extent in nitrogen-depleted cells. These results suggested that autophagy contributes to TAG synthesis in C. reinhardtii, but is not a necessary process. Autophagy induction may also be used to further enhance TAG accumulation in microalgae under nutrient deprivation.     

Exploiting Phosphate-Starved cells of <Emphasis Type="Italic">Scenedesmus</Emphasis> sp. for the Treatment of Raw Sewage

Phosphate depletion is one of the favorable ways to enhance the sewage water treatment with the algae, however, detailed information is essential with respect to internal phosphate concentration and physiology of the algae. The growth rate of the phosphate-starved Scenedesmus cells was reduced drastically after 48 h. Indicating cells entered in the stationary phase of the growth cycle. Fourier Transform Infrared analysis of phosphate-starved Scenedesmus cells showed the reduction in internal phosphate concentration and an increase in carbohydrate/phosphate and carbohydrate/lipid ratio. The phosphate-starved Scenedesmus cells, with an initial cell density of, 1 × 10⁶ cells mL^?1 shows 87% phosphate and 100 % nitrogen removal in 24 h. The normal Scenedesmus cells need approximately 48 h to trim down the nutrients from wastewater up to this extent. Other microalgae, Ankistrodesmus, growth pattern was not affected due to phosphate starvation. The cells of Ankistrodesmus was able to reduce 71% phosphate and 73% nitrogen within 24 h, with an initial cell density of, 1 × 10⁶ cells mL^?1.     

Genotypic differences in phosphorus use physiology in producers (<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>) and consumers (<Emphasis Type="Italic">Daphnia pulex</Emphasis>) interact to alter primary and secondary production

By considering the relative abundance of elements in trophic interactions, ecological stoichiometry makes predictions about key ecological processes such as biomass production and consumer-driven nutrient recycling. Theoretical and empirical work has focused on interspecific variation in elemental composition, and stoichiometric imbalances between resources and consumers in determining productivity, particularly at the base of foodwebs. Recent work has found considerable intraspecific variation in elemental composition. We know little about the ecological relevance of such variation, and whether predictions of stoichiometric theory hold at the intraspecific level. Here, we used two genotypes of a primary producer Chlamydomonas reinhardtii, and two genotypes of a primary consumer Daphnia pulex, which are already known to vary considerably in their phosphorus (P) use physiology, under conditions of P abundance and limitation, to explore whether such intraspecific differences alter primary as well as secondary production. Specifically, we tested whether there are intraspecific differences in the carbon: phosphorus (C:P) stoichiometry of Chlamydomonas genotypes, whether such differences affect growth and abundance of the two Daphnia genotypes, and whether the two Daphnia genotypes had distinct effects on primary production and growth of the two Chlamydomonas genotypes. We found significant differences in C:P stoichiometry between the two Chlamydomonas genotypes in both P supply conditions. Such intraspecific differences altered the growth of Daphnia genotypes, and affected the outcome of genotypic competition. Finally, Daphnia genotype affected primary production, and interacted with P supply to distinctly affect the growth of the two Chlamydomonas genotypes. Together, our results highlight the potential ecological relevance of intraspecific differences in nutrient use physiology and elemental composition, and the utility of ecological stoichiometry in understanding such consequences.     

Simultaneous removal of inorganic nutrients and organic carbon by symbiotic co-culture of <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> and <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The co-culture system of photosynthetic microalgae Chlorella vulgaris and aerobic heterotrophic bacteria Pseudomonas putida was investigated as a possible combination of symbiotic mixed culture for the simultaneous removal of nutrients (ammonium and phosphate) and organic contaminants. Using synthetic municipal wastewater, the co-culture system exhibited symbiotic enhancement in the removal of nutrients and organic carbon compared to each of axenic cultures. The co-culture system performed successfully in removing both of ammonium and chemical oxygen demand (COD), showing around 80% removal for 4 days. Strategies of nitrogen and phosphorous starvation in C. vulgaris for two days prior to main treatment did not increase the performance of nutrients removal, indicating that the nutrient starvation as a pretreatment is unnecessary. Without alkalinity (as bicarbonate), nutrients and COD were not removed significantly, implying that the existence of alkalinity is essential for symbiotic treatment of both nutrients and organics. Results demonstrated that coculture system composed of C. vulgaris and P. putida can be a potential candidate of mixed culture system for the simultaneous removal of nutrients and organic carbon in wastewater treatment using a single reactor.     

Enhanced microbial lipid production by <Emphasis Type="Italic">Cryptococcus albidus</Emphasis> in the high-cell-density continuous cultivation with membrane cell recycling and two-stage nutrient limitation

As a potential feedstock for biofuel production, a high-cell-density continuous culture for the lipid production by Cryptococcus albidus was investigated in this study. The influences of dilution rates in the single-stage continuous cultures were explored first. To reach a high-cell-density culture, a single-stage continuous culture coupled with a membrane cell recycling system was carried out at a constant dilution rate of 0.36/h with varied bleeding ratios. The maximum lipid productivity of 0.69 g/L/h was achieved with the highest bleeding ratio of 0.4. To reach a better lipid yield and content, a two-stage continuous cultivation was performed by adjusting the C/N ratio in two different stages. Finally, a lipid yield of 0.32 g/g and lipid content of 56.4% were obtained. This two-stage continuous cultivation, which provided a higher lipid production performance, shows a great potential for an industrial-scale biotechnological production of microbial lipids and biofuel production.     

Isolation,phenotypic characterization and genome wide analysis of a <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels

Background

Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing.

Results

In this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation.

Conclusion

Our phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production.

     

<Emphasis Type="Italic">Scenedesmus</Emphasis> sp. cultivation using commercial-grade ammonium sources

The use of commercial-grade nutrients such as agricultural fertilizers is important for commercial microalgae cultivation, and this is particularly the case for biofuel production which is associated with low added value. Nitrogen is a very important macronutrient in microalgae cultivation, and ammonium sources are cheaper than nitrate sources. However, the growth response and cellular composition can be altered by the different nutrient sources. In the study reported here, we investigated the effects of different ammonium doses and commercial-grade macronutrients from agricultural fertilizers on the growth of Scenedesmus sp. BR003, a promising genus for biofuel production. Five growth media were developed using fertilizers and evaluated during Scenedesmus sp. cultivation under autotrophic conditions. The growth media differed in terms of their composition and concentration of macronutrients. We found that all commercial-grade media supported equal or higher cell concentrations, dry weight, water-soluble proteins, neutral carbohydrates, and total lipid production compared to the conventional BG11 medium. However, the commercial-grade growth medium with the highest ammonium content affected the coenobium pattern of Scenedesmus sp. BR003. Commercial-grade nutrient sources were a low-cost alternative to improve the growth of Scenedesmus sp. BR003. The different fertilizers also allowed for manipulation of microalgae chemical composition and phenotypic plasticity to target traits of commercial interest. Our results demonstrate the potential of using ammonium from agricultural fertilizers as a nitrogen source in combination with other commercial-grade macronutrients sources. In addition, this work demonstrates the ability of a robust Scenedesmus strain to grow in media of different compositions, even when a high dosage of ammonium was used.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Roles of nitrogen and phosphorus in growth responses and toxin production (using LC-MS/MS) of tropical <Emphasis Type="Italic">Microcystis ichthyoblabe</Emphasis> and <Emphasis Type="Italic">M. flos-aquae</Emphasis>

In experiments investigating nutrient effects on tropical Microcystis, increasing nitrogen and phosphorus concentrations were found to have a significant positive effect on maximum cell yields of two strains of Microcystis ichthyoblabe (from Lower Peirce and Tengeh Reservoirs) and one strain of Microcystis flos-aquae isolated (Lower Peirce Reservoir) from Singapore. However, only increasing nitrogen concentration had a positive effect on growth rates of M. ichthyoblabe and M. flos-aquae from Lower Peirce Reservoir. MC-RR and MC-LR were produced by all three strains with MC-RR being the dominant variant. Phosphorus played an important role in MC production with increases in phosphorus from medium to high concentrations leading to decreases in MC-RR cell quotas for all three strains at the two highest nitrogen levels tested. The different growth and toxin production responses between M. ichthyoblabe strains could be due to location-specific differences.     

Self-supporting artificial system of the green alga <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> and the ascomycetous fungus <Emphasis Type="Italic">Alternaria infectoria</Emphasis>

A long-living (of up to several years) bipartite system was constructed between the unicellular green alga Chlamydomonas reinhardtii and the ascomycetous fungus Alternaria infectoria. The metabolic cooperation between the two organisms was tested with infecting A. infectoria hyphae into nitrogen starving yellow C. reinhardtii culture. After the infection, a slow greening process of the algal cells was observed, which was studied by measuring the increasing chlorophyll content, the appearance of chlorophyll-protein complexes – using 77 K fluorescence spectroscopy, and the measurement of photosynthetic oxygen production. Transmission electron microscopy and laser scanning microscopy images showed that no direct physical contacts were formed between the algal cells and the hyphae in the long-living symbiosis but they were joint in a mucilaginous bed allowing diffusion processes for metabolic cooperation. The increased free amino acid content of the medium of the long-living bipartite cultures’ indicated possible nitrogen supply of hyphal origin, which allowed the re-greening of the algal cells. The results of this work underline the importance of symbiosis-like stable metabolic coexistence, which ensures survival under extreme environmental conditions.     

Comparative proteomic analysis between nitrogen supplemented and starved conditions in <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis>

Background

Fungi are constantly exposed to nitrogen limiting environments, and thus the efficient regulation of nitrogen metabolism is essential for their survival, growth, development and pathogenicity. To understand how the rice blast pathogen Magnaporthe oryzae copes with limited nitrogen availability, a global proteome analysis under nitrogen supplemented and nitrogen starved conditions was completed.

Methods

M. oryzae strain 70–15 was cultivated in liquid minimal media and transferred to media with nitrate or without a nitrogen source. Proteins were isolated and subjected to unfractionated gel-free based liquid chromatography-tandem mass spectrometry (LC-MS/MS). The subcellular localization and function of the identified proteins were predicted using bioinformatics tools.

Results

A total of 5498 M. oryzae proteins were identified. Comparative analysis of protein expression showed 363 proteins and 266 proteins significantly induced or uniquely expressed under nitrogen starved or nitrogen supplemented conditions, respectively. A functional analysis of differentially expressed proteins revealed that during nitrogen starvation nitrogen catabolite repression, melanin biosynthesis, protein degradation and protein translation pathways underwent extensive alterations. In addition, nitrogen starvation induced accumulation of various extracellular proteins including small extracellular proteins consistent with observations of a link between nitrogen starvation and the development of pathogenicity in M. oryzae.

Conclusion

The results from this study provide a comprehensive understanding of fungal responses to nitrogen availability.

     

Enrichment as a screening method for a high-growth-rate microalgal strain under continuous cultivation system

Microalgae are a promising feedstock for renewable biodiesel production. High productivity of biodiesel production from microalgae is directly related to growth rate as well as lipid content of cells. In the present study, an enrichment process in a continuous cultivation system was developed to screen a high-growth-rate microalga from a mixed culture of microalgal species; Chlorella vulgaris, Chlorella protothecoides, and Chlamydomonas reinhardtii were used as test organisms for our experiments. The time-dependent washout of mixed microalgal pool was executed to successfully enrich the C. reinhardtii, which exhibits the higher growth rate than C. vulgaris and C. protothecoides under turbidostat conditions within 75 h. The domination of C. reinhardtii in the mixed culture was validated by on-line monitoring of growth rate and flowcytometric analysis. For the time-efficient production of microalgal biomass, this screening process has a high potential to segregate the fast-growing microalgal strains from the pool of various uncharacterized microalgal species and random mutants.