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At least 19 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2022

Genome sequence, phylogenetic analysis, and structure-based annotation reveal metabolic potential of Chlorella sp. SLA-04

Algae are a broad class of photosynthetic eukaryotes that are phylogenetically and physiologically diverse. Most of the phylogenetic diversity has been inferred from 18S rDNA sequencing since there are only a few complete genomes available in public databases. Here we use ultra-long-read Nanopore sequencing to determine a gapless, telomere-to-telomere complete genome sequence of Chlorella sp. SLA-04, previously described as Chlorella sorokiniana SLA-04. Chlorella sp. SLA-04 is a green alga that grows to high cell density in a wide variety of environments - high and neutral pH, high and low alkalinity, and high and low salinity. SLA-04's ability to grow in high pH and high alkalinity media without external CO 2 supply is favorable for large-scale algal biomass production. Phylogenetic analysis performed using ribosomal DNA and conserved protein sequences consistently reveal that Chlorella sp. SLA-04 forms a distinct lineage from other strains of Chlorella sorokiniana. We complement traditional genome annotation methods with high throughput structural predictions and demonstrate that this approach expands functional prediction of the SLA-04 proteome. Genomic analysis of the SLA-04 genome identifies the genes capable of utilizing TCA cycle intermediates to replenish cytosolic acetyl-CoA pools for lipid production. We also identify a complete metabolic pathway for sphingolipid anabolism that may allow SLA-04 to readily adapt to changing environmental conditions and facilitate robust cultivation in mass production systems. Altogether, this work clarifies the phylogeny of Chlorella sp. SLA-04 within Trebouxiophyceae and demonstrates how structural predictions can be used to improve annotation beyond sequencebased methods.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

An efficient Chlorella sp.-Cupriavidus necator microcosm for phenol degradation and its cooperation mechanism.

In this work, a Chlorella sp.-Cupriavidus necator (C. necator) microcosm was artificially established for phenol degradation. Additionally, the cooperation relationship between Chlorella sp. and C. necator was initially demonstrated, and then the effects of Chlorella sp./C. necator inoculation ratio, light intensity, temperature and pH on the performance of this microcosm were systematically evaluated and optimized. The optimal conditions for phenol degradation were as follows: a Chlorella sp./C. necator inoculation ratio of 1:1, a light intensity of 110 μmol m -2 s -1 , a temperature in the range of 25-32 °C and a pH in the range of 5.5-7.5. Under optimal conditions, this microcosm could degrade phenol with a maximum concentration of 1200 mg L -1 within 60 h. It was found that only when the phenol concentration was reduced to the tolerance concentration of microalgae, that is, the last stage of phenol degradation, the cooperation effect could be generated, indicating that the tolerance of microalgae to phenol may be more important than its degradation performance. Comparative transcriptomic analysis was conducted to discuss the cooperation mechanism of this microcosm subject to high phenol concentrations. The up-regulation of genes involved in photosynthesis and carbon fixation of Chlorella sp. demonstrated the CO 2 and O 2 exchange between Chlorella sp. and C. necator and their cooperation relationship. This study suggests that this microcosm has great potential for the bioremediation of phenol contaminants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Comparison of Auxenochlorella protothecoides and Chlorella spp. Chloroplast Genomes: Evidence for Endosymbiosis and Horizontal Virus-like Gene Transfer

Resequencing of the chloroplast genome (cpDNA) of Auxenochlorella protothecoides UTEX 25 was completed (GenBank Accession no. KC631634.1), revealing a genome size of 84,576 base pairs and 30.8% GC content, consistent with features reported for the previously sequenced A. protothecoides 0710, (GenBank Accession no. KC843975). The A. protothecoides UTEX 25 cpDNA encoded 78 predicted open reading frames, 32 tRNAs, and 4 rRNAs, making it smaller and more compact than the cpDNA genome of C. variabilis (124,579 bp) and C. vulgaris (150,613 bp). By comparison, the compact genome size of A. protothecoides was attributable primarily to a lower intergenic sequence content. The cpDNA coding regions of all known Chlorella species were found to be organized in conserved colinear blocks, with some rearrangements. The Auxenochlorella and Chlorella species genome structure and composition were similar, and of particular interest were genes influencing photosynthetic efficiency, i.e., chlorophyll synthesis and photosystem subunit I and II genes, consistent with other biofuel species of interest. Phylogenetic analysis revealed that Prototheca cutis is the closest known A. protothecoides relative, followed by members of the genus Chlorella. The cpDNA of A. protothecoides encodes 37 genes that are highly homologous to representative cyanobacteria species, including rrn16, rrn23, and psbA, corroborating a well-recognized symbiosis. Several putative coding regions were identified that shared high nucleotide sequence identity with virus-like sequences, suggestive of horizontal gene transfer. Despite these predictions, no corresponding transcripts were obtained by RT-PCR amplification, indicating they are unlikely to be expressed in the extant lineage.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Phylogenetic analyses and reclassification of the oleaginous marine species Nannochloris sp. “desiccata" (Trebouxiophyceae, Chlorophyta), formerly Chlorella desiccata , supported by a high-quality genome assembly

Microalgae are diverse, with many gaps remaining in phylogenetic and physiological understanding. Thus, studying new microalgae species increases our broader comprehension of biological diversity, and evaluation of new candidates as algal production platforms can lead to improved productivity under a variety of cultivation conditions. Chlorella is a genus of fast-growing species often isolated from freshwater habitats and cultivated as a source of nutritional supplements. However, the use of freshwater increases competition with other freshwater needs. We identified Chlorella desiccata to be worthy of further investigation as a potential algae production strain, due to its isolation from a marine environment and its promising growth and biochemical composition properties. Long-read genomic sequencing was conducted for C. desiccata UTEX 2526, resulting in a high-quality, near chromosome level, diploid genome with an assembly length of 21.55 Mbp in only 18 contigs. We also report complete circular mitochondrial and chloroplast genomes. Phylogenomic and phylogenetic analyses using nuclear, chloroplast, 18S rRNA, and actin sequences revealed that this species clades within strains currently identified as Nannochloris (Trebouxiophyceae, Chlorophyta), leading to its reclassification as Nannochloris sp. “desiccata” UTEX 2526. The mode of cell division for this species is autosporulation, differing from the type species N. bacillaris. As has occurred across multiple microalgae genera, there are repeated examples of Nannochloris species reclassification in the literature. This high-quality genome assembly and phylogenetic analysis of the potential algal production strain Nannochloris sp. “desiccata” UTEX 2526 provides an important reference and useful tool for further studying this region of the phylogenetic tree.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Improving biomass and lipid yields of Desmodesmus armatus and Chlorella vulgaris through mutagenesis and high-throughput screening

Advancement of microalgae for use as biofuel feedstock depends on bioprospecting novel strains and then applying classical strain improvement techniques to enhance desirable traits. Here we successfully demonstrate this approach using two novel strains of green algae, identified as Desmodesmus armatus and Chlorella vulgaris, to improve biomass and lipid yields. UV irradiation was utilized to generate random mutations and the resulting populations were analyzed via flow cytometry to quantify lipid fluorescence while enriching for high lipid variants using fluorescence activated cell sorting. Secondary screening was performed via in situ fluorescence staining to identify the single highest lipid producing mutants from each strain. This approach produced a Desmodesmus armatus mutant that exhibited both increased biomass productivity and higher lipid content, which doubled lipid yield from the culture. The resulting Chlorella vulgaris mutant exhibited an increase in growth only, resulting in a 33% increase in lipid yield.

09 BIOMASS FUELS↗
DOE OSTI2022

Contrary effects of phytoplankton Chlorella vulgaris and its exudates on mercury methylation by iron- and sulfate-reducing bacteria

Mercury (Hg) is a pervasive environmental pollutant and poses serious health concerns as inorganic Hg(II) can be converted to the neurotoxin methylmercury (MeHg), which bioaccumulates and biomagnifies in food webs. Phytoplankton, representing the base of aquatic food webs, can take up Hg(II) and influence MeHg production, but currently little is known about how and to what extent phytoplankton may impact Hg(II) methylation by itself or by methylating bacteria it harbors. This study investigated whether some species of phytoplankton could produce MeHg and how the live or dead phytoplankton cells and excreted algal organic matter (AOM) impact Hg(II) methylation by several known methylators, including iron-reducing bacteria (FeRB), Geobacter anodireducens SD-1 and Geobacter sulfurreducens PCA, and the sulfate-reducing bacterium (SRB) Desulfovibrio desulfuricans ND132 (or Pseudodesulfovibrio mercurii). Here our results indicate that, among the 4 phytoplankton species studied, none were capable of methylating Hg(II). However, the presence of phytoplankton cells (either live or dead) from Chlorella vulgaris (CV) generally inhibited Hg(II) methylation by FeRB but substantially enhanced methylation by SRB D. desulfuricans ND132. Enhanced methylation was attributed in part to CV-excreted AOM, which increased Hg(II) complexation and methylation by ND132 cells. In contrast, inhibition of methylation by FeRB was attributed to these bacteria incapable of competing with phytoplankton for Hg(II) binding and uptake. These observations suggest that phytoplankton could play different roles in affecting Hg(II) methylation by the two groups of anaerobic bacteria, FeRB and SRB, and thus shed additional light on how phytoplankton blooms may modulate MeHg production and bioaccumulation in the aquatic environment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Re-Programing Glucose Catabolism in the Microalga Chlorella sorokiniana under Light Condition

The microalga Chlorella sorokiniana has attracted much attention for lipid production and wastewater treatment. It can perform photosynthesis and organic carbon utilization concurrently. To understand its phototrophic metabolism, a biomass compositional analysis, a 13 C metabolic flux analysis, and metabolite pool size analyses were performed. Under dark condition, the oxidative pentose phosphate pathway (OPP) was the major route for glucose catabolism (88% carbon flux) and a cyclic OPP–glycolytic route for glucose catabolism was formed. Under light condition, fluxes in the glucose catabolism, tricarboxylic acid (TCA) cycle, and anaplerotic reaction (CO 2 fixation via phosphoenolpyruvate carboxylase) were all suppressed. Meanwhile, the RuBisCO reaction became active and the ratio of its carbon fixation to glucose carbon utilization was determined as 7:100. Moreover, light condition significantly reduced the pool sizes of sugar phosphate metabolites (such as E4P, F6P, and S7P) and promoted biomass synthesis (which reached 0.155 h –1 ). In addition, light condition increased glucose consumption rates, leading to higher ATP and NADPH production and a higher protein content (43% vs. 30%) in the biomass during the exponential growth phase.

13C-MFA↗
DOE OSTI2022

Mercury Reduction, Uptake, and Species Transformation by Freshwater Alga Chlorella vulgaris under Sunlit and Dark Conditions

As a major entry point of mercury (Hg) to aquatic food webs, algae play an important role in taking up and transforming Hg species in aquatic ecosystems. However, little is known how and to what extent Hg reduction, uptake, and species transformations are mediated by algal cells and their exudates, algal organic matter (AOM), under either sunlit or dark conditions. In this study, using Chlorella vulgaris (CV) as one of the most prevalent freshwater model algal species, we show that solar irradiation could enhance the reduction of mercuric Hg(II) to elemental Hg(0) by both CV cells and AOM. AOM reduced more Hg(II) than algal cells themselves due to cell surface adsorption and uptake of Hg(II) inside the cells under solar irradiation. Synchrotron radiation X-ray absorption near-edge spectroscopy (SR-XANES) analyses indicate that sunlight facilitated the transformation of Hg to less bioavailable species, such as β-HgS and Hg-phytochelatins, compared to Hg(Cysteine) 2 -like species formed in algal cells in the dark. These findings highlight important functional roles and potential mechanisms of algae in Hg reduction and immobilization under varying lighting conditions and how these processes may modulate Hg cycling and bioavailability in the aquatic environment.

54 ENVIRONMENTAL SCIENCES↗
DOE OSTI2020

Association between algal productivity and phycosphere composition in an outdoor Chlorella sorokiniana reactor based on multiple longitudinal analyses

Summary Microalgae as a biofuel source are of great interest. Bacterial phycosphere inhabitants of algal cultures are hypothesized to contribute to productivity. In this study, the bacterial composition of the Chlorella sorokiniana phycosphere was determined over several production cycles in different growing seasons by 16S rRNA gene sequencing and identification. The diversity of the phycosphere increased with time during each individual reactor run, based on Faith’s phylogenetic diversity metric versus days post‐inoculation ( R = 0.66, P < 0.001). During summer months, Vampirovibrio chlorellavorus , an obligate predatory bacterium, was prevalent. Bacterial sequences assigned to the Rhizobiales, Betaproteobacteriales and Chitinophagales were positively associated with algal biomass productivity. Applications of the general biocide, benzalkonium chloride, to a subset of experiments intended to abate V. chlorellavorus appeared to temporarily suppress phycosphere bacterial growth, however, there was no relationship between those bacterial taxa suppressed by benzalkonium chloride and their association with algal productivity, based on multinomial model correlations. Algal health was approximated using a model‐based metric, or the ‘Health Index’ that indicated a robust, positive relationship between C. sorokiniana fitness and presence of members belonging to the Burholderiaceae and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium clade. Bacterial community composition was linked to the efficiency of microalgal biomass production and algal health.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Carbon-Storing Structural Material Based on Wastewater-Cultivated Chlorella Sorokiniana

Microalgae cultivation is a promising technology for carbon sequestration and wastewater remediation. It is economically and logistically critical to find viable applications for the large quantities of produced biomass. In current research, a structural material is developed by using nearly 100% Chlorella sorokiniana. The otherwise non-cohesive microalgae cells are first surface-activated and then compacted under a relatively high pressure. The resulting material is a dense solid, stronger than typical steel-reinforced concrete in flexural tests. The processing procedure is simple and fast, and the setup is scalable. This technique may be useful for next-generation green construction and provide a use-case for sustainable microalgae biomass.

Yi, Haozhe↗
DOE OSTI2020

Similarities and Differences in the Effects of Toxic Concentrations of Cadmium and Chromium on the Structure and Functions of Thylakoid Membranes in Chlorella variabilis

Trace metal contaminations in natural waters, wetlands, and wastewaters pose serious threats to aquatic ecosystems—mainly via targeting microalgae. In this work, we investigated the effects of toxic amounts of chromium and cadmium ions on the structure and function of the photosynthetic machinery of Chlorella variabilis cells. To halt the propagation of cells, we used high concentrations of Cd and Cr, 50–50 mg L –1 , in the forms of CdCl 2 x 2.5 H 2 O and K 2 Cr 2 O 7 , respectively. Both treatments led to similar, about 50% gradual diminishment of the chlorophyll contents of the cells in 48 h, which was, however, accompanied by a small (~10%) but statistically significant enrichment (Cd) and loss (Cr) of ß-carotene. Both Cd and Cr inhibited the activity of photosystem II (PSII)—but with more severe inhibitions with Cr. On the contrary, the PsbA (D1) protein of PSII and the PsbO protein of the oxygen-evolving complex were retained more in Cr-treated cells than in the presence of Cd. These data and the higher susceptibility of P700 redox transients in Cr-treated cells suggest that, unlike with Cd, PSII is not the main target in the photochemical apparatus. These differences at the level of photochemistry also brought about dissimilarities at higher levels of the structural complexity of the photosynthetic apparatus. Circular dichroism (CD) spectroscopy measurements revealed moderate perturbations in the macro-organization of the protein complexes—with more pronounced decline in Cd-treated cells than in the cells with Cr. Also, as reflected by transmission electron microscopy and small-angle neutron scattering, the thylakoid membranes suffered shrinking and were largely fragmented in Cd-treated cells, whereas no changes could be discerned with Cr. The preservation of integrity of membranes in Cr-treated cells was most probably aided by high proportion of the de-epoxidized xanthophylls, which were absent with Cd. It can thus be concluded that beside strong similarities of the toxic effects of Cr and Cd, the response of the photosynthetic machinery of C. variabilis to these two trace metal ions substantially differ from each other—strongly suggesting different inhibitory and protective mechanisms following the primary toxic events.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Protein Extraction, Precipitation, and Recovery from Chlorella sorokiniana Using Mechanochemical Methods

Protein extraction, precipitation, and recovery methods were evaluated by this study using a green alga—Chlorella sorokiniana. A mechanochemical cell disruption process was applied to facilitate protein extraction from microalgal biomass. Optimization of the mechanochemical process resulted in milling conditions that achieved a protein extraction of 52.7 ± 6.45%. The consequent acid precipitation method was optimized to recover 98.7% of proteins from the microalgal slurry. The measured protein content of the protein isolate was 41.4% w/w. These results indicate that the precipitation method is successful at recovering the extracted proteins in the algal slurry; however, the removal of non-protein solids during centrifugation and pH adjustment is not complete. The energy balance analysis elucidated that the energy demand of the protein extraction and recovery operation, at 0.83 MJ/kg dry algal biomass, is much lower than previous studies using high-pressure homogenization and membrane filtration. This study concludes that mechanochemical protein extraction and recovery is an effective, low-energy processing method, which could be used by algal biorefineries to prepare algal proteins for value-added chemical production as well as to make algal carbohydrates and lipids in the residual biomass more accessible for biofuel production.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

DISCOVR strain pipeline screening – Part II: Winter and summer season areal productivities and biomass compositional shifts in climate-simulation photobioreactor cultures

Assessing the seasonal biomass productivity and compositional shift dynamics under simulated outdoor culture conditions of the top 22 algae strains selected during Tier I flask screening is an important step in the further prioritization of strains with regard to outdoor pond cultivation. These top 22 strains were subjected to Tier II testing in the PNNL Laboratory Environmental Algae Pond Simulator (LEAPS) photobioreactors, simulating light and temperature conditions of 20 cm deep outdoor ponds during the Arizona winter and summer season. All strains were grown in two consecutive nutrient-replete batch culture experiments at their particular optimal medium salinity to quantify their respective seasonal linear-phase areal biomass productivities. To determine biomass compositional shifts in response to nutrient-depletion, the LEAPS cultures were allowed to enter a 9-day nutrient depletion phase at the end of the second batch run. The following strains were evaluated in winter-season climate-simulated cultures and are listed in the order from highest (7.9 g m -2 day -1 ) to lowest (2.3 g m -2 day -1 ) areal N-replete biomass productivity: Monoraphidium minutum 26B-AM, Tetraselmis striata LANL1001, Chlorella vulgaris LRB AZ-1201, Micractinium reisseri NREL14-F2, Monoraphidium sp. MONOR1, Chlorella vulgaris NREL4-C12, Scenedesmus obliquus UTEX393, Scenedesmus acutus LRB-AP-0401, Nannochloropsis oceanica CCAP849/10, and Stichococcus minutus CCALA727. The following strains were evaluated in summer-season climate-simulated cultures and are listed in the order from highest (31.8 g m -2 day -1 ) to lowest (14.2 g m -2 day -1 ) areal N-replete biomass productivity: Picochlorum renovo NREL39-A8, Scenedesmus obliquus UTEX393, Porphyridium cruentum CCMP675, Picochlorum celeri TG2-WT-CSM/EMRE, Chlorella sorokiniana DOE1116, Stichococcus minor CCMP819, Picochlorum oklahomensis CCMP2329, Chlorella sorokiniana DOE1412 (UTEXB3016), Scenedesmus rubescens NREL46B-D3, Picochlorum soloecismus DOE101, Tetraselmis striata LANL1001, Scenedesmus obliquus DOE 0152.z, and Agmenellum quadruplicatum UTEX2268. All LEAPS cultures experienced a significant reduction in areal biomass productivity in response to nutrient-depletion, from 7-16% in the winter season simulation and up to 1-60% in the summer season simulation. Finally, for 10 of the strains tested, the carbohydrate content more than doubled upon nutrient depletion, and for 9 strains, the lipid content increased by over 50% of the initial content.

09 BIOMASS FUELS↗
DOE OSTI2023

DISCOVR strain pipeline screening – Part I: Maximum specific growth rate as a function of temperature and salinity for 38 candidate microalgae for biofuels production

Here, to identify high productivity strains for microalgal biofuels generation, the maximum specific growth rate of 38 strains was measured as a function of salinity (i.e., 5, 15, and 35 PSU) and temperature (i.e., at 8 temperatures along a linear gradient from ca. 5 to 45°C) to determine the most suitable growth medium salinity and best growing season, respectively, for outdoor raceway pond cultivation. The following strains were evaluated: Agmenellum quadruplicatum UTEX 2268, Anabaena sp. ATCC 33081, Arthrospira fusiformis UTEX 2721, Arthrospira platensis UTEX 3086, Chlorella vulgaris NREL 4-C12, Chlorella autotrophica CCMP 243, Chlorella sorokiniana DOE1044, Chlorella sorokiniana DOE 1116, Chlorella sorokiniana DOE 1412 (UTEXB3016), Chlorella vulgaris LRB AZ-1201, Chlorococcum littorale UTEX 117, Chlorococcum sp. UTEX-B P7, Chloromonas reticulata CCALA 870, Coelastrella sp. DOE 0202, Cyanobacterium sp. AB1, Micractinium reisseri NREL 14-F2, Microchloropsis gaditana CCMP1894, Microchloropsis salina CCMP 1776, Monoraphidium sp. MONOR1, Monoraphidium minutum 26B-AM, Nannochloropsis oceanica CCAP 849/10, Oscillatoria cf. priestleyi CCMEE 5020.1-1, Picochlorum celeri TG2-WT-CSM/EMRE, Picochlorum oklahomensis CCMP 2329, Picochlorum renovo NREL 39-A8, Picochlorum soloecismus DOE 101, Porphyridium cruentum CCMP 675, Scenedesmus acutus LRB-AP-0401, Scenedesmus obliquus DOE 0152.z, Scenedesmus obliquus UTEX393,Scenedesmus rubescens NREL 46B-D3, Scenedesmus sp. IITRIND2, Stichococcus minor CCMP 819, Stichococcus minutus CCALA 727, Synechococcus elongatus UTEX2973.1, Tetraselmis striata LANL 1001, Tisochrysis lutea CCMP 1324, and Tribonema minus UTEXB3156. For each strain, the identity and the presence of bacterial cohorts was determined using 18S and 16S rDNA sequencing, respectively. The maximum specific growth rate versus temperature data were also used to determine the activation energies (Arrhenius equation) for most strains. For all strains, the measured salinity and temperature tolerance data were compared to those reported in the literature. The fastest growing strains were down-selected for subsequent biomass productivity measurements in climate-simulation photobioreactors, as reported in the next paper in the issue.

18S and 16S rDNA sequencing↗