Functional Genomic Analyses Reveal an Open Pan-genome for the Chloroviruses and a Potential for Genetic Innovation in New Isolates.

Chloroviruses (family Phycodnaviridae) are large double-stranded DNA (dsDNA) viruses that infect unicellular green algae present in inland waters. These viruses have been isolated using three main chlorella-like green algal host cells, traditionally called NC64A, SAG, and Pbi, revealing extensive genetic diversity. In this study, we performed a functional genomic analysis on 36 chloroviruses that infected the three different hosts. Phylogenetic reconstruction based on the DNA polymerase B family gene clustered the chloroviruses into three distinct clades. The viral pan-genome consists of 1,345 clusters of orthologous groups of genes (COGs), with 126 COGs conserved in all viruses. Totals of 368, 268, and 265 COGs are found exclusively in viruses that infect NC64A, SAG, and Pbi algal hosts, respectively. Two-thirds of the COGs have no known function, constituting the "dark pan-genome" of chloroviruses, and further studies focusing on these genes may identify important novelties. The proportions of functionally characterized COGs composing the pan-genome and the core-genome are similar, but those related to transcription and RNA processing, protein metabolism, and virion morphogenesis are at least 4-fold more represented in the core genome. Bipartite network construction evidencing the COG sharing among host-specific viruses identified 270 COGs shared by at least one virus from each of the different host groups. Finally, our results reveal an open pan-genome for chloroviruses and a well-established core genome, indicating that the isolation of new chloroviruses can be a valuable source of genetic discovery. IMPORTANCE Chloroviruses are large dsDNA viruses that infect unicellular green algae distributed worldwide in freshwater environments. They comprise a genetically diverse group of viruses; however, a comprehensive investigation of the genomic evolution of these viruses is still missing. Here, we performed a functional pan-genome analysis comprising 36 chloroviruses associated with three different algal hosts in the family Chlorellaceae, referred to as zoochlorellae because of their endosymbiotic lifestyle. We identified a set of 126 highly conserved genes, most of which are related to essential functions in the viral replicative cycle. Several genes are unique to distinct isolates, resulting in an open pan-genome for chloroviruses. This profile is associated with generalist organisms, and new insights into the evolution and ecology of chloroviruses are presented. Ultimately, our results highlight the potential for genetic diversity in new isolates.

Juggling Lightning: How Chlorella ohadii handles extreme energy inputs without damage.

The green alga Chlorella ohadii was isolated from a desert biological soil crust, one of the harshest environments on Earth. When grown under optimal laboratory settings it shows the fastest growth rate ever reported for a photosynthetic eukaryote and a complete resistance to photodamage even under unnaturally high light intensities. Here we examined the energy distribution along the photosynthetic pathway under four light and carbon regimes. This was performed using various methodologies such as membrane inlet mass spectrometer with stable O2 isotopes, variable fluorescence, electrochromic shift and fluorescence assessment of NADPH level, as well as the use of specific inhibitors. We show that the preceding illumination and CO2 level during growth strongly affect the energy dissipation strategies employed by the cell. For example, plastid terminal oxidase (PTOX) plays an important role in energy dissipation, particularly in high light- and low-CO2-grown cells. Of particular note is the reliance on PSII cyclic electron flow as an effective and flexible dissipation mechanism in all conditions tested. The energy management observed here may be unique to C. ohadii, as it is the only known organism to cope with such conditions. However, the strategies demonstrated may provide an insight into the processes necessary for photosynthesis under high-light conditions.

Enhancement of Lipid Production under Heterotrophic Conditions by Overexpression of an Endogenous bZIP Transcription Factor in Chlorella sp. HS2.

Transcription factor engineering to regulate multiple genes has shown promise in the field of microalgae genetic engineering. Here, we report the first use of transcription factor engineering in Chlorella sp. HS2, thought to have potential for producing biofuels and bioproducts. We identified seven endogenous bZIP transcription factors in Chlorella sp. HS2 and named them HSbZIP1 through HSbZIP7. We overexpressed HSbZIP1, a C-type bZIP transcription factor, in Chlorella sp. HS2 with the goal of enhancing lipid production. Phenotype screening under heterotrophic conditions showed that all transformants exhibited increased fatty acid production. In particular, HSbZIP1 37 and 58 showed fatty acid methyl ester (FAME) yields of 859 and 1,052 mg/l, respectively, at day 10 of growth under heterotrophic conditions, and these yields were 74% and 113% higher, respectively, than that of WT. To elucidate the mechanism underlying the improved phenotypes, we identified candidate HSbZIP1-regulated genes via transcription factor binding site analysis. We then selected three genes involved in fatty acid synthesis and investigated mRNA expression levels of the genes by qRTPCR. The result revealed that the possible HSbZIP1-regulated genes involved in fatty acid synthesis were upregulated in the HSbZIP1 transformants. Taken together, our results demonstrate that HSbZIP1 can be utilized to improve lipid production in Chlorella sp. HS2 under heterotrophic conditions.

A first view on the unsuspected intragenus diversity of N-glycans in Chlorella microalgae.

Chlorella microalgae are increasingly used for various purposes such as fatty acid production, wastewater processing, or as health-promoting food supplements. A mass spectrometry-based survey of N-glycan structures of strain collection specimens and 80 commercial Chlorella products revealed a hitherto unseen intragenus diversity of N-glycan structures. Differing numbers of methyl groups, pentoses, deoxyhexoses, and N-acetylglucosamine culminated in c. 100 different glycan masses. Thirteen clearly discernible glycan-type groups were identified. Unexpected features included the occurrence of arabinose, of different and rare types of monosaccharide methylation (e.g. 4-O-methyl-N-acetylglucosamine), and substitution of the second N-acetylglucosamine. Analysis of barcode ITS1-5.8S-ITS2 rDNA sequences established a phylogenetic tree that essentially went hand in hand with the grouping obtained by glycan patterns. This brief prelude to microalgal N-glycans revealed a fabulous wealth of undescribed structural features that finely differentiated Chlorella-like microalgae, which are notoriously poor in morphological attributes. In light of the almost identical N-glycan structural features that exist within vertebrates or land plants, the herein discovered diversity is astonishing and argues for a selection pressure only explicable by a fundamental functional role of these glycans.

Selenium Incorporation to Amino Acids in Chlorella Cultures Grown in Phototrophic and Heterotrophic Regimes.

Microalgae accumulate bioavailable selenium-containing amino acids (Se-AAs), and these are useful as a food supplement. While this accumulation has been studied in phototrophic algal cultures, little data exists for heterotrophic cultures. We have determined the Se-AAs content, selenium/sulfur (Se/S) substitution rates, and overall Se accumulation balance in photo- and heterotrophic Chlorella cultures. Laboratory trials revealed that heterotrophic cultures tolerate Se doses ∼8-fold higher compared to phototrophic cultures, resulting in a ∼2-3-fold higher Se-AAs content. In large-scale experiments, both cultivation regimes provided comparable Se-AAs content. Outdoor phototrophic cultures accumulated up to 400 µg g-1 of total Se-AAs and exhibited a high level of Se/S substitution (5-10%) with 30-60% organic/total Se embedded in the biomass. A slightly higher content of Se-AAs and ratio of Se/S substitution was obtained for a heterotrophic culture in pilot-scale fermentors. The data presented here shows that heterotrophic Chlorella cultures provide an alternative for Se-enriched biomass production and provides information on Se-AAs content and speciation in different cultivation regimes.

Sequencing and comparative analysis of three Chlorella genomes provide insights into strain-specific adaptation to wastewater.

Microalgal Chlorella has been demonstrated to process wastewater efficiently from piggery industry, yet optimization through genetic engineering of such a bio-treatment is currently challenging, largely due to the limited data and knowledge in genomics. In this study, we first investigated the differential growth rates among three wastewater-processing Chlorella strains: Chlorella sorokiniana BD09, Chlorella sorokiniana BD08 and Chlorella sp. Dachan, and the previously published Chlorella sorokiniana UTEX 1602, showing us that BD09 maintains the best tolerance in synthetic wastewater. We then performed genome sequencing and analysis, resulting in a high-quality assembly for each genome with scaffold N50 > 2 Mb and genomic completeness ≥91%, as well as genome annotation with 9,668, 10,240, 9,821 high-confidence gene models predicted for BD09, BD08, and Dachan, respectively. Comparative genomics study unravels that metabolic pathways, which are involved in nitrogen and phosphorus assimilation, were enriched in the faster-growing strains. We found that gene structural variation and genomic rearrangement might contribute to differential capabilities in wastewater tolerance among the strains, as indicated by gene copy number variation, domain reshuffling of orthologs involved, as well as a ~1 Mb-length chromosomal inversion we observed in BD08 and Dachan. In addition, we speculated that an associated bacterium, Microbacterium chocolatum, which was identified within Dachan, play a possible role in synergizing nutrient removal. Our three newly sequenced Chlorella genomes provide a fundamental foundation to understand the molecular basis of abiotic stress tolerance in wastewater treatment, which is essential for future genetic engineering and strain improvement.

Lipid production from indigenous Greek microalgae: a possible biodiesel source.

OBJECTIVE: Microalgae gained interest for potential use as biodiesel producers, since they synthesize and accumulate significant quantities of lipids. The aim of this work was to isolate indigenous microalgae strains from Greek habitats, study their physicochemical growth conditions and finally select the best ones with respect to overall lipid production and profile. RESULTS: Two sampling sites of marine aquatic ecosystems were selected in Attica prefecture, Greece in order to screen for novel wild type strains with lipid production capacity. Microalgae isolates (59) were obtained from the selected areas and were morphologically and molecularly characterized. Fatty acids were estimated through Flow Cytometry combined with BODIPY staining method. Four isolates were selected for their lipid production properties and were cultivated in 15 L tank cultures. The four isolates were also identified by 18S rDNA gene sequencing. Two of them, Chlorella sp. ΑCΑ9 and ACA17, exhibited both maximum biomass and lipid productivity. Optimization of growth conditions with respect to pH and initial NaNO3 concentration was performed for the two microalgae in 15 L cultures. Finally, 20 L fed batch cultures were set up using the optimum culture conditions. Lipid profiles were stabilized for both strains at dry biomass levels over 1 g L-1 and lipid content of 25% (w/w). CONCLUSIONS: Two Chlorella strains (ACA9 and ACA17) were promising candidates for biodiesel production as they were easily grown in sea water in fed batch systems and produce lipids suitable for biodiesel-especially Chlorella sp. ACA9.

Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium "Candidatus Holospora parva".

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.

Combining and Comparing Coalescent, Distance and Character-Based Approaches for Barcoding Microalgaes: A Test with Chlorella-Like Species (Chlorophyta).

Several different barcoding methods of distinguishing species have been advanced, but which method is the best is still controversial. Chlorella is becoming particularly promising in the development of second-generation biofuels. However, the taxonomy of Chlorella-like organisms is easily confused. Here we report a comprehensive barcoding analysis of Chlorella-like species from Chlorella, Chloroidium, Dictyosphaerium and Actinastrum based on rbcL, ITS, tufA and 16S sequences to test the efficiency of traditional barcoding, GMYC, ABGD, PTP, P ID and character-based barcoding methods. First of all, the barcoding results gave new insights into the taxonomic assessment of Chlorella-like organisms studied, including the clear species discrimination and resolution of potentially cryptic species complexes in C. sorokiniana, D. ehrenbergianum and C. Vulgaris. The tufA proved to be the most efficient barcoding locus, which thus could be as potential "specific barcode" for Chlorella-like species. The 16S failed in discriminating most closely related species. The resolution of GMYC, PTP, P ID, ABGD and character-based barcoding methods were variable among rbcL, ITS and tufA genes. The best resolution for species differentiation appeared in tufA analysis where GMYC, PTP, ABGD and character-based approaches produced consistent groups while the PTP method over-split the taxa. The character analysis of rbcL, ITS and tufA sequences could clearly distinguish all taxonomic groups respectively, including the potentially cryptic lineages, with many character attributes. Thus, the character-based barcoding provides an attractive complement to coalescent and distance-based barcoding. Our study represents the test that proves the efficiency of multiple DNA barcoding in species discrimination of microalgaes.

Use of De Novo Transcriptome Libraries to Characterize a Novel Oleaginous Marine Chlorella Species during the Accumulation of Triacylglycerols.

Marine chlorophytes of the genus Chlorella are unicellular algae capable of accumulating a high proportion of cellular lipids that can be used for biodiesel production. In this study, we examined the broad physiological capabilities of a subtropical strain (C596) of Chlorella sp. "SAG-211-18" including its heterotrophic growth and tolerance to low salt. We found that the alga replicates more slowly at diluted salt concentrations and can grow on a wide range of carbon substrates in the dark. We then sequenced the RNA of Chlorella strain C596 to elucidate key metabolic genes and investigate the transcriptomic response of the organism when transitioning from a nutrient-replete to a nutrient-deficient condition when neutral lipids accumulate. Specific transcripts encoding for enzymes involved in both starch and lipid biosynthesis, among others, were up-regulated as the cultures transitioned into a lipid-accumulating state whereas photosynthesis-related genes were down-regulated. Transcripts encoding for two of the up-regulated enzymes-a galactoglycerolipid lipase and a diacylglyceride acyltransferase-were also monitored by reverse transcription quantitative polymerase chain reaction assays. The results of these assays confirmed the transcriptome-sequencing data. The present transcriptomic study will assist in the greater understanding, more effective application, and efficient design of Chlorella-based biofuel production systems.

Complete sequence and characterization of mitochondrial and chloroplast genome of Chlorella variabilis NC64A.

The complete nucleotide sequences of the mitochondrial (mtDNA) and chloroplast (cpDNA) genomes of Chlorella variabilis NC64A (Trebouxiophyceae) have been determined in this study (GenBank accession no. KP271968 and KP271969, respectively). The mt genome assembles as a circle of 78,500 bp and contains 62 genes, including 32 protein-coding, 27 tRNA and 3 rRNA genes. The overall GC content is 28.2%, while the coding sequence is 34%. The cp genome forms a circle of 124,793 bp, containing 114 genes, including 79 protein-coding, 32 tRNA and 3 rRNA genes. The overall GC content is 33,9%, while the coding sequence is 50%.

Interactive Effects of Temperature and UV Radiation on Photosynthesis of Chlorella Strains from Polar, Temperate and Tropical Environments: Differential Impacts on Damage and Repair.

Global warming and ozone depletion, and the resulting increase of ultraviolet radiation (UVR), have far-reaching impacts on biota, especially affecting the algae that form the basis of the food webs in aquatic ecosystems. The aim of the present study was to investigate the interactive effects of temperature and UVR by comparing the photosynthetic responses of similar taxa of Chlorella from Antarctic (Chlorella UMACC 237), temperate (Chlorella vulgaris UMACC 248) and tropical (Chlorella vulgaris UMACC 001) environments. The cultures were exposed to three different treatments: photosynthetically active radiation (PAR; 400-700 nm), PAR plus ultraviolet-A (320-400 nm) radiation (PAR + UV-A) and PAR plus UV-A and ultraviolet-B (280-320 nm) radiation (PAR + UV-A + UV-B) for one hour in incubators set at different temperatures. The Antarctic Chlorella was exposed to 4, 14 and 20°C. The temperate Chlorella was exposed to 11, 18 and 25°C while the tropical Chlorella was exposed to 24, 28 and 30°C. A pulse-amplitude modulated (PAM) fluorometer was used to assess the photosynthetic response of microalgae. Parameters such as the photoadaptive index (Ek) and light harvesting efficiency (α) were determined from rapid light curves. The damage (k) and repair (r) rates were calculated from the decrease in ΦPSIIeff over time during exposure response curves where cells were exposed to the various combinations of PAR and UVR, and fitting the data to the Kok model. The results showed that UV-A caused much lower inhibition than UV-B in photosynthesis in all Chlorella isolates. The three isolates of Chlorella from different regions showed different trends in their photosynthesis responses under the combined effects of UVR (PAR + UV-A + UV-B) and temperature. In accordance with the noted strain-specific characteristics, we can conclude that the repair (r) mechanisms at higher temperatures were not sufficient to overcome damage caused by UVR in the Antarctic Chlorella strain, suggesting negative effects of global climate change on microalgae inhabiting (circum-) polar regions. For temperate and tropical strains of Chlorella, damage from UVR was independent of temperature but the repair constant increased with increasing temperature, implying an improved ability of these strains to recover from UVR stress under global warming.

Ca(2+)-regulated cyclic electron flow supplies ATP for nitrogen starvation-induced lipid biosynthesis in green alga.

We previously showed that both the linear photosynthetic electron transportation rate and the respiration rate dropped significantly during N starvation-induced neutral lipid accumulation in an oil-producing microalga, Chlorella sorokiniana, and proposed a possible role for cyclic electron flow (CEF) in ATP supply. In this study, we further exploited this hypothesis in both Chlorella sorokiniana C3 and the model green alga Chlamydomonas. We found that both the rate of CEF around photosystem I and the activity of thylakoid membrane-located ATP synthetase increased significantly during N starvation to drive ATP production. Furthermore, we demonstrated that the Chlamydomonas mutant pgrl1, which is deficient in PGRL1-mediated CEF, accumulated less neutral lipids and had reduced rates of CEF under N starvation. Further analysis revealed that Ca(2+) signaling regulates N starvation-induced neutral lipid biosynthesis in Chlamydomonas by increasing calmodulin activity and boosting the expression of the calcium sensor protein that regulates Pgrl1-mediated CEF. Thus, Ca(2+)-regulated CEF supplies ATP for N starvation-induced lipid biosynthesis in green alga. The increased CEF may re-equilibrate the ATP/NADPH balance and recycle excess light energy in photosystems to prevent photooxidative damage, suggesting Ca(2+)-regulated CEF also played a key role in protecting and sustaining photosystems.

[Isolation, Identification and Characteristic Analysis of an Oil-producing Chlorella sp. Tolerant to High-strength Anaerobic Digestion Effluent].

A Chlorella strain tolerant to high-strength anaerobic digestion effluent was isolated from the anaerobic digestion effluent with a long-term exposure to air. The strain was identified as a Chlorella by morphological and molecular biological methods, and named Chlorella sp. BWY-1, The anaerobic digestion effluent used in this study was from a biogas plant with the raw materials of swine wastewater after solid-liquid separation. The Chlorella regularis (FACHB-729) was used as the control strain. The comparative study showed that Chlorella sp, BWY-Ihad relatively higher growth rate, biomass accumulation capacity and pollutants removal rate in BG11. and different concentrations of anaerobic digestion effluent. Chlorella sp. BWY-1 had the highest growth rate and biomass productivity (324.40 mg.L-1) in BG11, but its lipid productivity and lipid content increased with the increase of anaerobic digestion effluent concentration, In undiluted anaerobic digestion effluent, the lipid productivity and lipid content of Chlorella sp. BWY-1 were up to 44. 43% and 108. 70 mg.L-1, respectively. Those results showed that the isolated algal strain bad some potential applications in livestock wastewater treatment and bioenergy production, it could be combined with a solid-liquid separation, anaerobic fermentation and other techniques for processing livestock wastewater and producing biodiesel.

Dynamics of chloroplast genomes in green plants.

Chloroplasts are essential organelles, in which genes have widely been used in the phylogenetic analysis of green plants. Here, we took advantage of the breadth of plastid genomes (cpDNAs) sequenced species to investigate their dynamic changes. Our study showed that gene rearrangements occurred more frequently in the cpDNAs of green algae than in land plants. Phylogenetic trees were generated using 55 conserved protein-coding genes including 33 genes for photosynthesis, 16 ribosomal protein genes and 6 other genes, which supported the monophyletic evolution of vascular plants, land plants, seed plants, and angiosperms. Moreover, we could show that seed plants were more closely related to bryophytes rather than pteridophytes. Furthermore, the substitution rate for cpDNA genes was calculated to be 3.3×10(-10), which was almost 10 times lower than genes of nuclear genomes, probably because of the plastid homologous recombination machinery.

The combined and second exposure effect of copper (II) and chlortetracycline on fresh water algae, Chlorella pyrenoidosa and Microcystis aeruginosa.

In the experiment, Chlorella pyrenoidosa and Microcystis aeruginosa were chosen to test the individual, combined and second exposure effect of Cu(2+) and chlortetracycline (CTC). The 96 h EC50s of each test were calculated, with the ranges of 0.972-15.6 µmol/L (Cu(2+)), 29.5-102.5 µmol/L (CTC), 14.4-78.9 µmol/L (mixture). The combined toxicities were evaluated with toxicity units (TU) method. The toxicity of complex of Cu(2+) and chlortetracycline was analyzed using concentration addition (CA) model. In the initial test, the combined effect of the two substances was partly additive to C. pyrenoidosa and antagonistic to M. aeruginosa, while in the second exposure test, the combined effect was synergistic to both algae. The biochemical indicators measured in the experiment included chlorophyll fluorescence (Fv/Fm), MDA content, SOD activity and content of soluble proteins. When under combined stress, the biochemical features of both algae were significantly different between the initial test and the second exposure test.

Three stage cultivation process of facultative strain of Chlorella sorokiniana for treating dairy farm effluent and lipid enhancement.

Reserve lipids of microalgae are promising for biodiesel production. However, economically feasible and sustainable energy production from microalgae requires optimization of cultivation conditions for both biomass yield and lipid production of microalgae. Biomass yield and lipid production in microalgae are a contradictory problem because required conditions for both targets are different. Simultaneously, the mass cultivation of microalgae for biofuel production also depends extremely on the performance of the microalgae strains used. In this study a green unicellular microalgae Chlorella sorokiniana (DS6) isolated from the holding tanks of farm wastewater treatment plant using multi-step screening and acclimation procedures was found high-lipid producing facultative heterotrophic microalgae strain capable of growing on dairy farm effluent (DFE) for biodiesel feedstock and wastewater treatment. Morphological features and the phylogenetic analysis for the 18S rRNA identified the isolated strains. A novel three stage cultivation process of facultative strain of C. sorokiniana was examined for lipid production.

Effect of different organic matters on flocculation of Chlorella sorokiniana and optimization of flocculation conditions in swine manure wastewater.

In this study, flocculation of Chlorella sorokiniana cultivated in swine manure wastewater, BG-11 medium and BG-11 medium supplemented with different organic matters (glucose, urea and tryptone) was investigated. The results demonstrated that the minimum amount of Al(3+) required for complete flocculation in wastewater would increase substantially, and flocculation efficiency became highly sensitive to pH. Tryptone could cause similar extent of inhibition on flocculation as in wastewater. Meanwhile, glucose could increase concentrations of Algogenic Organic Matter (AOM), inhibiting flocculation strongly at higher pH, including flocculation induced by Al(3+) and autoflocculation. However, urea had little effect on flocculation of C. sorokiniana. Moreover, the major factors: dilution times, pH and flocculants dosage, which had significant impact on flocculation efficiency of C. sorokiniana in piggery wastewater, were optimized using response surface methodology (RSM). The optimal flocculation efficiency (100%) was achieved at pH 8.5, 7-folds of dilution and 52.14 mg L(-1) of Al(3+).

Regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime.

Proteomics in conjunction with biochemical strategy was employed to unravel regulation of lipid metabolism in the green microalga Chlorella protothecoides by heterotrophy-photoinduction cultivation regime (HPC). Interestingly, HPC triggered transiently synthesis of starch followed by substantial lipid accumulation. And a marked decrease in intracellular protein and chlorophyll contents was also observed after 12h of photo-induction. The highest lipid content of 50.5% was achieved upon the photo-induction stage, which represented 69.3% higher than that of the end of heterotrophic cultivation. Results suggested that turnover of carbon-nitrogen-rich compounds such as starch, protein, and chlorophyll might provide carbon or energy for lipid accumulation. The proteomics analysis indicated that several pathways including glycolysis, TCA cycle, ß-oxidation of fatty acids, Calvin cycle, photosynthesis, energy and transport, protein biosynthesis, regulate and defense were involved in the lipid biosynthesis. Malate dehydrogenase and acyl-CoA dehydrogenase were suggested as key regulatory factors in enhancing lipid accumulation.