The NanDeSyn database for Nannochloropsis systems and synthetic biology.

Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community-based annotation and mining of the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi-omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single-cell.cn). Via the Tripal toolkit, it features user-friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.

Heterogeneity of intron presence/absence in Olifantiella sp. (Bacillariophyta) contributes to the understanding of intron loss.

Although hypotheses have been proposed and developed to interpret the origins and functions of introns, substantial controversies remain about the mechanism of intron evolution. The availability of introns in the intermediate state is quite helpful for resolving this debate. In this study, a new strain of diatom (denominated as DB21-1) was isolated and identified as Olifantiella sp., which possesses multiple types of 18S rDNAs (obtained from genomic DNA; lengths ranged from 2,056 bp to 2,988 bp). Based on alignments between 18S rDNAs and 18S rRNA (obtained from cDNA; 1,783 bp), seven intron insertion sites (IISs) located in the 18S rDNA were identified, each of which displayed the polymorphism of intron presence/absence. Specific primers around each IIS were designed to amplify the introns and the results indicated that introns in the same IIS varied in lengths, while terminal sequences were conserved. Our study showed that the process of intron loss happens via a series of successive steps, and each step could derive corresponding introns under intermediate states. Moreover, these results indicate that the mechanism of genomic deletion that occurs at DNA level can also lead to exact intron loss.

UDP-glucose pyrophosphorylase as a target for regulating carbon flux distribution and antioxidant capacity in Phaeodactylum tricornutum.

UDP-glucose pyrophosphorylase (UGPase) is a key enzyme for polysaccharide synthesis, and its role in plants and bacteria is well established; however, its functions in unicellular microalgae remain ill-defined. Here, we perform bioinformatics, subcellular localization as well as in vitro and in vivo analyses to elucidate the functions of two UGPs (UGP1 and UGP2) in the model microalga Phaeodactylum tricornutum. Despite differences in amino acid sequence, substrate specificity, and subcellular localization between UGP1 and UGP2, both enzymes can efficiently increase the production of chrysolaminarin (Chrl) or lipids by regulating carbon flux distribution without impairing growth and photosynthesis in transgenic strains. Productivity evaluation indicate that UGP1 play a bigger role in regulating Chrl and lipid production than UGP2. In addition, UGP1 enhance antioxidant capacity, whereas UGP2 is involved in sulfoquinovosyldiacylglycerol (SQDG) synthesis in P. tricornutum. Taken together, the present results suggest that ideal microalgal strains can be developed for the industrial production of Chrl or lipids and lay the foundation for the development of methods to improve oxidative stress tolerance in diatoms.

An attempt to simultaneously quantify the polysaccharide, total lipid, protein and pigment in single Cyclotella cryptica cell by Raman spectroscopy.

BACKGROUND: At present, the conventional methods for determining photosynthetic products of microalgae are usually based on a large number of cell mass to reach the measurement baseline, and the result can only reveal the average state at the population level, which is not feasible for large-scale and rapid screening of specific phenotypes from a large number of potential microalgae mutants. In recent years, single-cell Raman spectra (SCRS) has been proved to be able to rapidly and simultaneously quantify the biochemical components of microalgae. However, this method has not been reported to analyze the biochemical components of Cyclotella cryptica (C. cryptica). Thus, SCRS was first attempt to determine these four biochemical components in this diatom. RESULTS: The method based on SCRS was established to simultaneously quantify the contents of polysaccharide, total lipids, protein and Chl-a in C. cryptica, with thirteen Raman bands were found to be the main marker bands for the diatom components. Moreover, Partial Least Square Regression (PLSR) models based on full spectrum can reliably predict these four cellular components, with Pearson correlation coefficient for these components reached 0.949, 0.904, 0.801 and 0.917, respectively. Finally, based on SCRS data of one isogenic sample, the pairwise correlation and dynamic transformation process of these components can be analyzed by Intra-ramanome Correlation Analysis (IRCA), and the results showed silicon starvation could promote the carbon in C. cryptica cells to flow from protein and pigment metabolism to polysaccharide and lipid metabolism. CONCLUSIONS: First, method for the simultaneous quantification of the polysaccharide, total lipid, protein and pigment in single C. cryptica cell are established. Second, the instant interconversion of intracellular components was constructed through IRCA, which is based on data set of one isogenic population and more precision and timeliness. Finally, total results indicated that silicon deficiency could promote the carbon in C. cryptica cells to flow from protein and pigment metabolism to polysaccharide and lipid metabolism.

Enhancement of hemostatic properties of Cyclotella cryptica frustule through genetic manipulation.

BACKGROUND: The silicified cell wall of diatoms, also known as frustule, shows huge potential as an outstanding bio-nanomaterial for hemostatic applications due to its high hemostatic efficiency, good biocompatibility, and ready availability. As the architectural features of the frustule determine its hemostatic performance, it is of great interest to develop an effective method to modify the frustule morphology into desired patterns to further improve hemostatic efficiency. RESULTS: In this study, the gene encoding Silicalemma Associated Protein 2 (a silicalemma-spanning protein) of Cyclotella cryptica (CcSAP2) was identified as a key gene in frustule morphogenesis. Thus, it was overexpressed and knocked down, respectively. The frustule of the overexpress lines showed no obvious alteration in morphology compared to the wild type (WT), while the size, specific surface area (BET), pore volume, and pore diameter of the knockdown strains changed greatly. Particularly, the knockdown frustules achieved a more pronounced coagulation effect and in vivo hemostatic performance than the WT strains. Such observations suggested that silicalemma proteins are ideal genetic encoding targets for manipulating frustule morphology associated hemostatic properties. Furthermore, the Mantel test was adopted to identify the key morphologies associated with C. cryptica bleeding control. Finally, based on our results and recent advances, the mechanism of frustule morphogenesis was discussed. CONCLUSION: This study explores a new strategy for enhancing the hemostatic efficiency of the frustule based on genetic morphology modification and may provide insights into a better understanding of the frustule morphogenesis mechanism.

Screening and application of Chlorella strains on biosequestration of the power plant exhaust gas evolutions of biomass growth and accumulation of toxic agents.

To use microalgae for the biosequestration of carbon dioxide (CO2) emitted from the coal-fired power plants, the screening of high CO2 tolerant microalgae and their accumulation of toxic agents have attracted significant research attention. This study evaluated 10 Chlorella strains for high CO2 tolerance using combined growth rates and growth periods subjected to logistic parameters. We selected LAMB 31 with high r (0.89 ± 0.10 day-1), high k (6.51 ± 0.19), and medium Tp (5.17 ± 0.15 day) as a candidate for CO2 biosequestration. Correspondingly, six genes involving carbon fixation and metabolism processes were upregulated in LAMB 31 under high CO2 conditions, verifying its high CO2 tolerant ability. LAMB 31 cultures exposed to exhaust gas of power plant under different flow rates grew well, but the high flow rate (0.6 L/h) showed inhibition effects compared with low flow rates (0.2 and 0.3 L/h) at the end of the culturing period. The toxic agents in the exhaust gas including sulfur, arsenic, and mercury accumulated in LAMB 31 biomass but were deemed safe for use in the production of both human food and animal feed based on the National Food Safety Standard in China. This study showed a complete process involving high CO2 tolerant microalgae screening, high CO2 tolerant verification, and in situ application in a power plant. Data results provide valuable information as the basis for future research studies in microalgae application on CO2 mitigation at emission sources.

Energy metabolism and intracellular pH regulation reveal different physiological acclimation mechanisms of Chlorella strains to high concentrations of CO2.

The intolerance of high CO2 in the exhaust gas is the "bottleneck" limiting the wide application of microalgae for CO2 biosequestration. Around this topic, we selected high-CO2-tolerant (LAMB 33 and 31) and nontolerant (LAMB 122) Chlorella strains to study their different energy metabolisms and cytoplasmic pH regulations in response to high CO2. Under 40 % CO2, LAMB 33 and 31 both showed elevated ATP synthesis, accelerated ATP consumption and fast cytoplasmic pH regulation while exhibiting different acclimating strategies therein: chloroplast acclimations were reflected by high chlorophyll contents in 33 but photosystem transitions in 31; faster mitochondrial acclimations occurred in 33 than in 31; cellular organic carbon mainly flowed to monosaccharide synthesis for 33 but to monosaccharide and protein synthesis for 31; and cytoplasmic pH regulation was attributed to V-ATPase in 31 but not in 33. All the above metabolic processes gradually collapsed in 122, leading to growth inhibition. Our study identified different metabolic acclimation strategies among Chlorella strains to high CO2 and provided new traits for breeding microalgae for CO2 biosequestration.

How do microalgae in response to biological pollution treat in cultivation? A case study investigating microalgal defense against ciliate predator Euplotes vannus.

Microalgae have significant amounts of proteins, lipids, carotenoids, vitamins, minerals, and unique pigments. However, with the gradual expansion of microalgae cultivation, hostile biological pollution seriously restricted the large-scale microalgae cultivation and limited the exploitation of its biological resources. Moreover, protozoan poses the greatest threat to microalgae cultivation. Here, the relationship between six marine economic microalgae populations and their ciliate predator Euplotes vannus was examined. And four concentrations were designed for each type of microalgae to carry out the experiment. It was revealed that four species of microalgae inhibit the ciliate population growth at high density. Furthermore, the experiment which was the influence of microalgae at three different growth stages on the growth of the ciliates for these four kinds of high-density inhibitory microalgae was designed. The microalgae inhibitory effects were already exhibited at the end of the exponential growth phase, and it was significantly inhibited during the stationary growth phase. As the microalgae concentration increased, the inhibitory effect became more pronounced. This study provides fundamental data for screening protozoan-inhibiting microalgae and shows potential to be used in algae cultivation.

Effects of fluctuating temperature in open raceway ponds on the biomass accumulation and harvest efficiency of Spirulina in large-scale cultivation.

It is of great significance to select strains with wide adaptability to temperature range for large-scale commercial cultivation of Spirulina. The aim of this study was to comprehend how the strain H-208 grew and whether this strain had any advantages in temperature adaptation compared with local production strain during the large-scale cultivation in Inner Mongolia. The results showed that the strain H-208 could adapt to the new environmental condition quickly, and the daily average biomass dry weight of strain H-208 was 49% and 52% more than that of production strain M-1 in first cycle (20.24 g/m2/day) and second cycle (16.90 g/m2/day) of acclimation experiment, respectively. The growth rate of strain H-208 was 0.055 and 0.066 g/L/day from July 22 to July 25 and from July 26 to July 29, respectively, while the growth rate of strain M-1 was only 0.036 and 0.032 g/L/day, respectively, during the same cultured days in 605-m2 raceway ponds before high temperature. The harvesting efficiency of H-208 and M-1 was 95.1% and 72.1% before high temperature, and that was 95.3% and 52.5% after being stressed by high temperature, respectively. Meanwhile, it was also observed that the filaments of the two strains contracted and their pitches were smaller than that before high temperature stress, especially the strain M-1. In 20-m2 raceway ponds of recovery experiment after high temperature, the percentage of daily average biomass dry weight of strain H-208 was 68% more than that of strain M-1, which demonstrated that strain H-208 could recover and grow rapidly, and its self-regulation ability was superior to that of strain M-1.

Tentative identification of key factors determining the hemostatic efficiency of diatom frustule.

It is increasingly essential to develop excellent materials for rapid hemorrhage control. Our previous study showed that centric diatoms such as frustules were superior to QuikClot® in hemostasis, however, related studies in pennate diatoms are still scarce. The morphological and physicochemical properties of pennate diatoms are quite different from those of centric diatoms, meaning that significant differences may also be observed from their hemostatic effects. Thus, the hemostasis effects of four pennate diatom frustules (Cocconeiopsis orthoneoides, Navicula avium, Navicula sp., and Pleurosigma indicum) were investigated in this study. Herein, all diatom frustules demonstrated outstanding hemostasis performance. For example, the in vitro coagulation time of C. orthoneoides (100.33 ± 9.5 s) was 32.4% lower than that of QuikClot®. Meanwhile, the hemostatic times of C. orthoneoides in the rat tail amputation and femoral artery models were 82 s and 180 s, respectively, only around one-half and one-third of the QuikClot® values. Moreover, the blood loss amounts of C. orthoneoides in the rat tail amputation and femoral artery model were 73.4% and 61% less than that of QuikClot®. Besides that, diatom frustules also exhibited favorable biocompatibility (hemolysis ratio <5%, MEFs cell viabilities >80%, and no inflammation). To find out the key factors underlying the hemostatic effect of frustules, Pearson correlation analysis was further performed in this study. The results demonstrated that the coagulation reaction time (R) was negatively correlated with the specific surface area and liquid absorbability but positively with the diatom pore diameter. The angle α, indicating the clot formation rate, was negative to the diatom size and pore diameter. Additionally, MA also showed a negative correlation with the BET value. This study can enrich our knowledge about the application potential of diatoms in the field of bleeding control and is helpful in deepening our understanding about the hemostatic mechanism of frustules.

The molecular mechanisms of Chlorella sp. responding to high CO2: A study based on comparative transcriptome analysis between strains with high- and low-CO2 tolerance.

High CO2 acclimation for microalgae has attracted large research attention owing to the usefulness of microalgae in bio-sequestration of CO2 from the emission source. In this study, one high CO2 tolerant (LAMB 31) and non-tolerant (LAMB 122) Chlorella sp. strains were transferred from air to 40% CO2, during which four time points were chosen for comparative transcriptome analysis. Gene changes started in the lag phase (T1) of population growth with more genes (7889) upregulated in LAMB 31 than in LAMB 122 (1092). Further function enrichments indicated: In LAMB 31, up-regulation of genes in cyclic electron transportation, F-type ATPase and Calvin cycle were associated with the enhancement of carbon fixation abilities; upregulation of genes in phosphorylation together with V-ATPase, which contributed to cytoplasmatic pH stability; Lastly, enhancement of carbon metabolisms including TCA cycle and glycolysis accelerated the consumption of cellular organic carbon. Most of the genes in these pathways and processes showed downregulation in LAMB 122. This study disclosed the most complete transcriptional molecular mechanisms of Chlorella sp. responding to high CO2 by combining CO2 fixation, transportation, and metabolic processes. The results provided valuable genetic information for future screening and breeding of microalgae with high-CO2 tolerance for more efficient CO2 bio-sequestration.

A review on the progress, challenges and prospects in commercializing microalgal fucoxanthin.

Fucoxanthin, the most abundant but nearly untapped carotenoid resource, is in the spotlight in the last decade from various perspectives due to a wide range of bioactivities and healthy benefits. The exploitation of fucoxanthin for nutraceutical and pharmaceutical purposes encompasses enormous scientific and economic potentials. Traditional production of fucoxanthin from brown algae (macroalgae) is constrained by limited yield and prohibitively high cost. Microalgae, as the most diverse photoautotrophs, hold the promises as sustainable sources and ideal cell factories for commercial fucoxanthin production, owing to their rich fucoxanthin content and excellent biomass productivity. In this work, the recent progress in upstream (microalgae selection, optimization of culture conditions, trophic modes, cultivation strategies and biosynthesis pathway) as well as downstream processes (extraction) of fucoxanthin production has been comprehensively and critically reviewed. The major bottlenecks, such as screening of fucoxanthin-producers, conflict between biomass and fucoxanthin accumulation under high light condition, unclear steps in biosynthesis pathway and limited evaluation of outdoor scale-up cultivation and extraction, have been pinpointed. Most importantly, the applications of emerging and conventional techniques facilitating commercialization of microalgal fucoxanthin are highlighted. The reviewed and evaluated include breeding and high-throughput screening methods of elite strains; flashing light effect inducing concurrent biomass and fucoxanthin accumulation; fucoxanthin biosynthesis and the regulatory mechanisms associating with its accumulation elucidated with the development of genetic engineering and omics techniques; and photobioreactors, harvesting and extraction techniques suitable for scaling up fucoxanthin production. In conclusion, the prospects of microalgal fucoxanthin commercialization can be expected with the joint development of fundamental phycology and biotechnology.

Potential to resist biological contamination in marine microalgae culture: Effect of extracellular substances of Nannochloropsis oceanica on population growth of Euplotes vannus and other protozoa.

The commercially important marine microalgae Nannochloropsis oceanica is easily ingested by protozoan predators during large-scale cultivation. However, investigations into the effect of microalgae on the growth of protozoa are scant. A feeding experiment was conducted with Euplotes vannus grazing on different concentrations of N. oceanica. The ciliate population was significantly lower in the high concentration of algae than that in the low or medium algal concentration treatments. The density of ciliates cultured in algae filtrate media was significantly lower than that in lysate media and the blank control. Furthermore, the algal cell filtrate was added to three other protozoan populations, and they all gradually lost their ability to move and their body shape changed. This study investigated the interactions between N. oceanica and protozoan predators and provides insight on using microalgal extracellular substances to control biological contamination in the future.

The regulating mechanisms of CO2 fixation and carbon allocations of two Chlorella sp. strains in response to high CO2 levels.

The extreme high CO2 in industrial exhaust gas cannot be tolerated by microalgae is the key challenge for the application of microalgae in CO2 bio-sequestration. To provide better insights for this challenge, we chose one high CO2 tolerant (Chlorella sp. LAMB 31) and non-tolerant (Chlorella sp. LAMB 122) Chlorella sp. to examine their different CO2 fixation and carbon allocation responses to 40% CO2. The results indicated LAMB 31 had a 24-h "lag phase" of biomass increase, during which the transition from PSII-PSI and the increase of lipid synthesis happened to acclimate high CO2 conditions, followed by the increase of pigments synthesis, carbon fixation rates and polysaccharide productions. However, no acclimating mechanism was observed in LAMB 122, whose biomass, photosynthesis and material synthesis were all gradually collapsed under 40% CO2. Finally, four parameters including Chl a, polysaccharides, carbon fixation rates and MDA were selected to be good physiological biomarkers for high CO2 tolerant strains screenings in the future.

Treating wastewater by indigenous microalgae strain in pilot platform located inside a municipal wastewater treatment plant.

Various resources from a municipal wastewater treatment plant (MWTP) are available for microalgae cultivation plants, suggesting that a combination of these technologies can be used to produce microalgae biomass and remove contaminants at a low cost. In this study, the growth performance and nutrient removal efficiency of an indigenous Scenedesmus sp. in various wastewater media with different exchange patterns were investigated firstly, then transferred to a pilot-scale photobioreactor (located inside a MWTP) for bioremediation use. The temperature and pH of the platform were maintained at 15-30°C and 7.6, respectively. The N H 4 + -   N , N O 3 - -   N , and P O 4 3 - -   P of the wastewater could be reduced to below 0.05, 0.40, and 0.175 mg L-1, respectively. Our results indicate that microalgae cultivation using the resources of a MWTP can achieve high algal biomass productivity and nutrient removal rate. Our study also suggests that efficient technology for controlling zooplankton needs to be developed.

Growth-promoting bacteria double eicosapentaenoic acid yield in microalgae.

High-yielding microalgae present an important commodity to sustainably satisfy burgeoning food, feed and biofuel demands. Because algae-associated bacteria can significantly enhance or reduce yields, we isolated, identified and selected highly-effective "probiotic" bacterial strains associated with Nannochloropsis oceanica, a high-yielding microalga rich in eicosapentaenoic acid (EPA). Xenic algae growth was significantly enhanced by co-cultivation with ten isolated bacteria that improved culture density and biomass by 2.2- and 1.56-fold, respectively (1.39 × 108 cells mL-1; 0.82 g L-1). EPA contents increased up to 2.25-fold (to 39.68% of total fatty acids). Added probiotic bacteria possessed multiple growth-stimulating characteristics, including atmospheric nitrogen fixation, growth hormone production and phosphorous solubilization. Core N. oceanica-dominant bacterial microbiomes at different cultivation scales included Sphingobacteria, Flavobacteria (Bacteroidetes), and α, γ-Proteobacteria, and added probiotic bacteria could be maintained. We conclude that the supplementation with probiotic algae-associated bacteria can significantly enhance biomass and EPA production of N. oceanica.

Genome assembly of Nannochloropsis oceanica provides evidence of host nucleus overthrow by the symbiont nucleus during speciation.

The species of the genus Nannochloropsis are unique in their maintenance of a nucleus-plastid continuum throughout their cell cycle, non-motility and asexual reproduction. These characteristics should have been endorsed in their gene assemblages (genomes). Here we show that N. oceanica has a genome of 29.3 Mb consisting of 32 pseudochromosomes and containing 7,330 protein-coding genes; and the host nucleus may have been overthrown by an ancient red alga symbiont nucleus during speciation through secondary endosymbiosis. In addition, N. oceanica has lost its flagella and abilities to undergo meiosis and sexual reproduction, and adopted a genome reduction strategy during speciation. We propose that N. oceanica emerged through the active fusion of a host protist and a photosynthesizing ancient red alga and the symbiont nucleus became dominant over the host nucleus while the chloroplast was wrapped by two layers of endoplasmic reticulum. Our findings evidenced an alternative speciation pathway of eukaryotes.

Large-Scale Cultivation of Spirulina for Biological CO2 Mitigation in Open Raceway Ponds Using Purified CO2 From a Coal Chemical Flue Gas.

In order to select excellent strains with high CO2 fixation capability on a large scale, nine Spirulina species were cultivated in columnar photobioreactors with the addition of 10% CO2. The two species selected (208 and 220) were optimized for pH value, total dissolved inorganic carbon (DIC), and phosphorus content with intermittent CO2 addition in 4 m2 indoor raceway ponds. On the basis of biomass accumulation and CO2 fixation rate in the present study, the optimum pH, DIC, and phosphate concentration were 9.5, 0.1 mol L-1, and 200 mg L-1 for both strains, respectively. Lastly, the two strains selected were semi-continuously cultivated successfully for CO2 mitigation in 605 m2 raceway ponds aerated with food-grade CO2 purified from a coal chemical flue gas on a large scale. The daily average biomass dry weight of the two stains reached up to 18.7 and 13.2 g m-2 d-1, respectively, suggesting the two Spirulina strains can be utilized for mass production.

Influence of the physicochemical characteristics of diatom frustules on hemorrhage control.

Uncontrollable hemorrhage is the main cause of death in military and civilian accidents. It is therefore necessary and an urgent requirement to develop a safe and efficient hemostatic material. In this study, the hemostatic performance of frustules of three centric diatom species (Thalassiosira weissflogii, Thalassiosira sp., and Cyclotella cryptica) with similar shapes and different sizes was investigated. The complicated structure of T. weissflogii, leading to its highest BET surface area (169.5 m2 g-1) and liquid absorption (51.4 ± 1.6 times the weight of liquid), exhibited the shortest hemostasis time (158 ± 8.19 s) in in vitro blood coagulation. Thalassiosira sp. had a shorter hemostasis time (167.33 ± 14.74 s) than that of QuikClot® and C. cryptica, indicating that diatom size also played an important role in hemostasis due to the interface reaction between the material and plasma protein. The in vivo hemostasis results further confirmed this conclusion. Diatom frustules also exhibited favorable blood compatibility (<5%), and no significant cell toxicity could be observed from the three frustules. Our results suggest that the coagulation effect of frustules is strengthened upon a decrease in the size and increase in the liquid absorbability. This report provides valuable information for the medical application of diatom frustules in the field of hemorrhage control.

Two-step process: Enhanced strategy for wastewater treatment using microalgae.

Microalgae possess many advantages, but the lack of a suitable strategy to simultaneously facilitate their low cost cultivation and high value productions limits their commercial applications. In this study, two microalgae strains (RT_C and RT_F) isolated from a municipal wastewater treatment plant were used to establish a two-step wastewater treatment process. During step-1, RT_C was cultivated in composite wastewater due to its high tolerance of sludge centrate; followed by step-2, in which the supernatant generated from RT_C culture was used to cultivate RT_F. The NH4+-N, PO43--P, and COD in the wastewater were removed almost completely using this strategy. Moreover, the majority of the metal ions in the wastewater were absorbed by RT_C during step-1, and thus the powdered RT_F only contained low levels of toxic metals. Our results demonstrate that this two-step process is effective for removing pollutants and while generating a powder sufficiently clean for extracting valuable compounds.