Spirodela Polyrhiza Research Articles

The Developmental Cycle of Spirodela polyrhiza Turions: A Model for Turion-Based Duckweed Overwintering?

Duckweeds are widely distributed small, simply constructed aquatic higher plants (the Lemnaceae) found on quiet freshwater surfaces. Species inhabiting temperate climates may have to cope with long periods of severe cold during the winter season. Several duckweeds form compact resting structures from the assimilatory fronds of the growing season that can bridge inhospitable conditions in a quiescent state. Of these, turions separate from the mother fronds and overwinter on the water body bottom in a dormant state. They can surface, germinate, and sprout to resume active growth upon warming in the spring. The turions of the largest duckweed, Spirodela polyrhiza, have been intensively examined as to ultrastructure, the factors governing their formation and release from dormancy, and the signals driving their germination and sprouting and the accompanying starch degradation. Comparative transcriptomics of assimilatory fronds and dormant turions are revealing the molecular features of this developmental cycle. The results illustrate an elegant sequence of reactions that ensures aquatic survival of even severe winters by frost avoidance in a vegetative mode. Since little is known about other duckweed resting fronds, the S. polyrhiza turion developmental cycle cannot be considered to be representative of duckweed resting fronds in general but can serve as a reference for corresponding investigations.

Salinity-responsive hyperaccumulation of flavonoids in Spirodela polyrrhiza, resultant maneuvering in the structure and antimicrobial as well as azo dye decontamination profile of biofabricated zinc oxide nanoentities.

Duckweeds (Spirodela polyrrhiza) are free-floating macrophytes that grow profusely in nutrient-rich waters. Under ideal conditions, they exhibit a rapid growth rate and can absorb a substantial amount of nutrients, macromolecules, and pollutants from bodies of water. Zinc oxide nanoparticles (ZnO NPs) synthesized from plant extracts, particularly under stress conditions, have opened new research avenues in the field of nanotechnology. Under salinity stress, the accumulation of flavonoids in duckweeds can affect the structure of ZnO NPs, helping researchers ascertain their antimicrobial role. In our study, we exposed mid-log phase duckweed monocultures to 75 mM NaCl in a full-strength Murashige and Skoog medium for 7 days, followed by a 15-day recovery period. We observed significant overexpression of superoxide and hydrogen peroxide as reactive oxygen species. As a result, chlorophyll and certain metabolites were produced in lesser amounts, while flavonoid and phenol content increased by 12% and 8%, respectively. This overproduction persisted up to 10 days into the recovery treatment period but dropped by 8% and 5%, respectively, by the 15th day. The flavonoid coating transformed the NPs into rosette clusters, which exhibited reduced antimicrobial activity against Aeromonas hydrophila, a Gram-negative, fish-pathogenic bacterium. Herein, we discuss potential mechanisms for the conformational transformation of ZnO NPs into finer dimensions in response to NaCl-induced oxidative stress in duckweed. In this study, the azo dye degradation capacity of salinity-treated plants increased as the flavonoid profile became enriched. Zinc oxide nanoparticles, both prior to and after salinity treatment, were found to be efficient in scavenging azo dye and mitigating its toxicity, as evidenced by improved germination, growth, and overall plant morphometry.

Modelling and optimizing secondary metabolites production in Spirodela polyrhiza using machine learning

Spirodela. polyrhiza as one of the potential feedstocks for industrial natural dye and pharmaceutical products, efficient process to produce from it were scarce. Here, Artificial Neural Network (ANN) and Genetic Algorithm (GA) were employed for modelling and optimization respectively. Current study focus on establishing useable models and anticipating the optimal concentration of nitrate (NO3−), phosphate (PO43−) and ammonium (NH4+) to give highest relative growth rate (RGR) and produce maximum amount of anthocyanins, chlorophylls, phenolic compounds and antioxidants. In short, 12 and 11 neurons in hidden layer were found to be most suitable neuron numbers used for the modelling of growth and secondary metabolites respectively. All the models exhibited a high degree of robustness and performance with a high r2 value (higher than 0.75) and a low mean squared error (lower than 0.05). GA also anticipated the best concentration of macronutrients to achieve the maximum growth and metabolites production with significant low errors (below 7 %). The employment of machine learning was useful for such applications.

Utility of dried and fermented meal from the duckweed species <i>Spirodela polyrhiza</i> as fishmeal-protein replacer in diets for common carp fry

Utility of dried and fermented meal from the duckweed species <i>Spirodela polyrhiza</i> as fishmeal-protein replacer in diets for common carp fry

Tolerance and phytoremediation capacity of atrazine and S-metolachlor by two duckweeds.

The phytotoxicity and removal of atrazine and S-metolachlor in sterile duckweed systems were estimated in this study. Herbicides were added at environmentally relevant ranges: 0-400µg/L for atrazine or 0-200µg/L for S-metolachlor in systems with Spirodela polyrhiza or Lemna minor. Toxicity biomarkers, i.e., changes in plant biomass, surface area, chlorophyll fluorescence parameters, pigments, lipid peroxidation, protein concentration, and antioxidative enzyme activities in plants were estimated after 7days. S. polyrhiza (RGRbiomass-EC50 = 164.8µg/L) was more tolerant to atrazine than L. minor (RGRbiomass-EC50 = 101.0µg/L). Atrazine caused damage to photosystem II (ΦM), a reduction in electron transport between PSII and PSI (Φ'M), as well as disruption in energy distribution pathways (decrease in qPrel and increase in UQFrel), most prominently in L. minor. However, L. minor (RGRbiomass-EC50 = 128.9µg/L) was more tolerant to S-metolachlor than S. polyrhiza (RGRbiomass-EC50 = 15.5μg/L). The highest sensitivity of S. polyrhiza to S-metolachlor was attributed to a decrease in absorbed energy used in photochemistry (qPrel) and an increase in lipid peroxidation, indicating that S. polyrhiza plants were experiencing oxidative stress. Residual pesticide analysis in the water after seven days allowed us to conclude that plants were responsible for reducing up to 16.5% of atrazine and 28.7% of S-metolachlor in the duckweed system. S. polyrhiza showed higher atrazine phytoremediation capacity than L. minor. S. polyrhiza was more efficient at an environmentally relevant concentration of S-metolachlor (25μg/L) and L. minor at higher concentrations (200μg/L).

Biodegradation of microcystins by microbiota of duckweed Spirodelapolyrhiza

Cyanobacteria-produced allelochemicals, including hepatotoxic microcystins (MCs), exert an inhibitory effect on macrophyte growth. However, the role of macrophyte-associated bacteria and algae (macrophyte microbiota) in mitigating these immediate negative effects of cyanotoxins remains poorly understood. In this paper, we analyzed the biodegradation of microcystin-RR, MC-LR, and MC-LF by microbiota of the macrophyte Spirodela polyrhiza. The biodegradation of two MC variants was observed and LC-MS/MS analysis allowed identifying the degradation products of MC-RR (m/z 1011, 984, 969, 877, 862, 820, and 615) and MC-LR (m/z 968 and 953), including eight previously unreported products. No degradation products of MC-LF were detected, suggesting its stability and resistance under experimental conditions. NGS-based profiling of microbial consortia revealed no major differences in bacterial community composition across experimental treatments. Taxa previously reported as capable of MC degradation have been found in S. polyrhiza microbiota. Furthermore, the presence of genes encoding putative microcystinase homologues and the formation of new linear intermediates suggest a biochemical pathway that is similar, but not identical to previously reported. The ability of aquatic plant microbiota to biodegrade MCs holds environmental significance, and further studies in this field are required.

CALLUS INDUCTION FROM THE FRONDS OF GREATER DUCKWEED (<i>Spirodela polyrhiza </i>(L.) Schleid) AND ASSESSMENT OF ITS ANTIMICROBIAL EFFICACY

Greater duckweed (Spirodela polyrrhiza (L.) Schleid. are known to propagate excessively in stagnant eutrophic water bodies resulting in the contamination of daughter fronds which can affect their secondary metabolite profile. The present study was aimed in establish organogenic callus from the fronds of greater duckweeds; to optimize a protocol by utilizing different plant growth regulators for future use and to screen the antimicrobially potent metabolites present in callus. The induction of callus was successful using sterile fronds of Spirodela polyrrhiza (L.) Schleid as explants in MS, B5 and SH media fortified with plant growth regulators namely IBA and BAP. SH basal medium fortified with IBA and BA in 2:1 ratio was most effective in callus induction. B5 medium fortified with 2.0 µM IBA + 4.0 µM BAP showed highest callus induction in S. polyrrhiza with lower days to callus induction as reflective in their overall biomass. The callus extract showed efficient antimicrobial effect on Pseudomonas aeroginosa (MTCC accession No. 3451) with ampicillin and chloramphenicol as reference antibiotics. Presence of flavonoids like orientin, genistin and apigetrin were indicated in the LC-MS data which might be responsible for antimicrobial activity in a synergistic manner.

Enzymatic Characterization of SpPAL Genes in S. polyrhiza and Overexpression of the SpPAL3.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the deamination of phenylalanine, which is the initial step in the biosynthesis of phenylpropanoids. It serves as a crucial enzyme that facilitates the transfer of carbon from primary to secondary metabolism in plants. Duckweed is regarded as a promising chassis plant in synthetic biology research and application, due to its being rich in secondary metabolites and other advantages. The genes encoding PAL in Spirodela polyrhiza (L.) Schleid, the giant duckweed, were investigated in this study. Three SpPAL genes (SpPAL1-SpPAL3) were identified and cloned. All of them were successfully expressed in E. coli, and their recombinant proteins all showed PAL activity. In addition, SpPAL1 and SpPAL2 proteins could also utilize tyrosine as substrate, although the activity was low. A qRT-PCR analysis demonstrated that the expression of SpPAL3 was most pronounced in young fronds. It was found that the expression of SpPAL1 and SpPAL3 was significantly induced by MeJA treatment. Overexpression of SpPAL3 in Lemna turionifera inhibited the growth of fronds and adventitious roots in the transgenic plants, indicating the importance of SpPAL3 in duckweed besides its involvement in the secondary metabolism.

Increasing Fertilization Efficiency of Biomass Ash by the Synergistically Acting Digestate and Extract from Water Plants Sequestering CO2 in Sorghum Crops.

The utilization of biomass ash in sustainable agriculture and increasing its fertilizing efficiency by biological agents, potentially sequestering CO2, have become important issues for the global economy. The aim of this paper was to investigate the effects of ash from sorghum (Sorghum bicolor L. Moench) and Jerusalem artichoke (Helianthus tuberosus L.) biomass, a biogas plant digestate, and a Spirodela polyrhiza extract, acting alone or synergistically, on soil fertility and the development, health and physiological properties of sorghum plants. The results show novel information concerning differences in the composition and impact of ash, depending on its origin, soil properties and sorghum plant development. Sorghum ash was more effective than that from Jerusalem artichoke. Ash used alone and preferably acting synergistically with the digestate and Spirodela polyrhiza extract greatly increased soil fertility and the growth, biomass yield and health of sorghum plants. These improvements were associated with an increased chlorophyll content in leaves, better gas exchange (photosynthesis, transpiration, stomatal conductance), greater enzyme activity (acid and alkaline phosphatase, RNase, and total dehydrogenase), and a higher biomass energy value. The developed treatments improved environmental conditions by replacing synthetic fertilizers, increasing the sequestration of CO2, solving the ash storage problem, reducing the need for pesticides, and enabling a closed circulation of nutrients between plant and soil, maintaining high soil fertility.

Effects of SpGSH1 and SpPCS1 overexpression or co-overexpression on cadmium accumulation in yeast and Spirodela polyrhiza

Cadmium (Cd) is one of the most toxic elements to all organisms. Glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is considered an extremely important mechanism in Cd detoxification in plants. However, few studies have focused on the roles of glutamate-cysteine ligase (GSH1) and phytochelatin synthase (PCS1) in Cd accumulation and detoxification in plants. In this study, SpGSH1 and SpPCS1 were identified and cloned from Spirodela polyrhiza and analyzed their functions in yeast and S. polyrhiza via single- or dual-gene (SpGP1) overexpression. The findings of this study showed that SpGSH1, SpPCS1, and SpGP1 could dramatically rescue the growth of the yeast mutant Δycf1. In S. polyrhiza, SpGSH1 was located in the cytoplasm and could promote Mn and Ca accumulation. SpPCS1 was located in the cytoplasm and nucleus, mainly expressed in meristem regions, and promoted Cd, Fe, Mn, and Ca accumulation. SpGSH1 and SpPCS1 co-overexpression increased the Cd, Mn, and Ca contents. Based on the growth data of S. polyrhiza, it was recommended that biomass as the preferable indicator for assessing plant tolerance to Cd stress compared to frond number in duckweeds. Collectively, this study for the first time systematically elaborated the function of SpGSH1 and SpPCS1 for Cd detoxification in S. polyrhiza.

(Invited) The “Duckweed Dip”: DNA-Wrapped Carbon Nanotubes for Transient Gene Expression

This study reports on usage of functionalized carbon nanotubes as a transgene delivery cargo into higher plants. We show that aquatic Spirodela polyrhiza (Duckweed) plant, when incubated in a solution containing carbon nanotubes wrapped with a plasmid containing the gene for reporter protein β-glucuronidase (GUS), can directly uptake the DNA-CNTs with high efficiency. The mechanism of the DNA-CNT delivery, directly from growth media, and plasmid release is discussed. We show that transgenes encoded within the plasmids are expressed by the plants. This method allows to avoid using large doses of nanomaterials or direct infiltration of agrobacterium into plant tissue. We expect this new approach for transgene delivery will allow for more efficient plant engineering and can serve as a useful tool in synthetic biology.ACKNOWLEDGMENTThe work was supported by the National Institutes of Health/National Institute of General Medical Sciences [1R35GM133483] and Department of Defense [Contract #W911QY2220006]. This work was performed in part at the Joint School of Nanoscience and Nanoengineering, a member of the Southeastern Nanotechnology Infrastructure Corridor (SENIC) and National Nanotechnology Coordinated Infrastructure (NNCI), which is supported by the National Science Foundation [ECCS-1542174].

The immediate metabolomic effects of whole-genome duplication in the greater duckweed, Spirodela polyrhiza.

In plants, whole-genome duplication (WGD) is a common mutation with profound evolutionary potential. Given the costs associated with a superfluous genome copy, polyploid establishment is enigmatic. However, in the right environment, immediate phenotypic changes following WGD can facilitate establishment. Metabolite abundances are the direct output of the cell's regulatory network and determine much of the impact of environmental and genetic change on the phenotype. While it is well known that an increase in the bulk amount of genetic material can increase cell size, the impact of gene dosage multiplication on the metabolome remains largely unknown. We used untargeted metabolomics on four genetically distinct diploid-neoautotetraploid pairs of the greater duckweed, Spirodela polyrhiza, to investigate how WGD affects metabolite abundances per cell and per biomass. Autopolyploidy increased metabolite levels per cell, but the response of individual metabolites varied considerably. However, the impact on metabolite level per biomass was restricted because the increased cell size reduced the metabolite concentration per cell. Nevertheless, we detected both quantitative and qualitative effects of WGD on the metabolome. Many effects were strain-specific, but some were shared by all four strains. The nature and impact of metabolic changes after WGD depended strongly on the genotype. Dosage effects have the potential to alter the plant metabolome qualitatively and quantitatively, but were largely balanced out by the reduction in metabolite concentration due to an increase in cell size in this species.

Lower concentration polyethylene microplastics can influence free-floating macrophyte interactions by combined effects of many weak interactions: A nonnegligible ecological impact

Microplastics (MPs) are ubiquitous in freshwater ecosystems and their accumulation has been considered an emerging threat. Early research on the effects of MPs on macrophytes primarily focused on the toxicological impacts on individual macrophytes, with several studies suggesting that lower concentrations of MPs have little impact on macrophytes. However, the ecological implications of lower MP concentrations on macrophyte communities remain largely unexplored. Here, we experimented to assess the effects of lower concentrations including 25 mg/L, 50 mg/L, 75 mg/L, and 100 mg/L of polyethylene (PE) microplastics on Spirodela polyrhiza and Lemna minor, and their community. Our results also indicated that PE concentrations below 100 mg/L had no significant effect on relative growth rate, specific leaf area, Chlorophyll a, Chlorophyll b, Chlorophyll a + b, carotenoid, malondialdehyde (MDA), catalase, and soluble sugar of monocultural S. polyrhiza. However, a lower concentration of PE significantly decreased the MDA of monocultural L. minor and significantly affected the comprehensive index of S. polyrhiza. These findings suggested that lower concentrations of PE can influence interactions between macrophytes maybe due to the cumulative effects of many weak interactions. Additionally, our study showed that 75 mg/L and 100 mg/L PE additions decreased the competitive balance index value of two macrophytes under mixed-culture condition. This result implied that the ecological influence of lower concentration MPs on macrophytes may manifest at the community level rather than at the population level, due to species-specific responses and varying degrees of sensitivity of macrophytes to PE concentrations. Thus, our study emphasizes the need to closely monitor the ecological consequences of emerging contaminants such as MPs accumulation on macrophyte communities, rather than focusing solely on the morphology and physiology of individual macrophytes.

Influence of humic acid on the p-tert-Butylphenol removal efficiency by Spirodela polyrhiza-Tas13 association

Plant-microbe remediation technique is considered as a promising technology in removal of organic pollutants and its remediation efficiency is largely affected by a variety of surrounding environmental factors. Humic acid (HA) is the complex organic substance ubiquitous in environment, which characterized by its surfactant-like micelle microstructure and various reaction activity. In our study, a plant-microbe association with high p-tert-Butylphenol (PTBP) degradation potential constructed by Spirodela polyrhiza and Sphingobium phenoxybenzoativorans Tas13 has been used, and the influence of HA on the PTBP degradation efficiency of S. polyrhiza-Tas13 association was investigated. The result showed that the presence of HA greatly improved PTBP removal efficiency of S. polyrhiza-Tas13. The reason accounted for this may be due to the presence of HA promoted bacterial cell propagation, altered bacterial cell wall permeability, increased catechol 2,3-dioxygenase (C23O) enzyme activity of strain Tas13, rather than increasing the colonization ability of strain Tas13 on to the root surface. This study will greatly facilitate the application of aquatic plant-microbe association in environmental remediation.

Aroma and metabolite profiling in duckweeds: Exploring species and ecotypic variation to enable wider adoption as a food crop

Duckweeds (water lentils) are a nutritious human food source, with Wolffia species consumed traditionally in Eastern Asia. Duckweed contain up to 45 % protein by dry weight, high macronutrients, minerals and carotenoids. However, duckweed are not cultivated at scale and there are circa 35 other species to consider for food potential in other global regions. Here, we measured the suitability of four Lemna species and Spirodela polyrhiza for nutritional assessment, by scaling up growth of 25 ecotypes from the United Kingdom in a glasshouse. Here we showed intra- and inter-species variation of aromatic and metabolic profiles, together with biomass obtained from production. The dominant volatile organic compounds (VOCs) in duckweed are hexanal, 1-penten-3-one, 1-penten-3-ol, cis-2-pentanol and pentadecanal, with variations in amounts of 22 other compounds between species. In comparison with other leafy herbs, duckweed aroma profiles were most similar to spinach and dandelion with high ‘green’ and ‘fresh’ aroma compounds. Spirodela polyrhiza contained high flavonoids including apigenin and luteolin, offering potential benefits for health. Our results demonstrate that Lemna and Spirodela species have suitable flavonoid and amino acid profiles for nutrition. VOCs found here had positive aroma descriptors and can be used as biomarkers of freshness during storage of duckweed foodstuffs.

Sulfamethoxazole removal and fuel-feedstock biomass production from wastewater in a phyto-Fenton process using duckweed culture

The phyto-Fenton process, which generates hydroxyl radicals through Fenton and Fenton-like reactions using plant-derived hydrogen peroxide (H2O2) and ferrous iron (Fe (II)) can degrade organic pollutants. Duckweed, an aquatic plant, is promising for a co-beneficial phytoremediation process that combines wastewater treatment and biomass production for biofuel feedstock. However, the phyto-Fenton process using duckweed has not been extensively studied. Because sulfamethoxazole (SMX), a major antibiotic, is distributed widely and is an emerging contaminant, its effective removal from contaminated water is necessary. The present study investigated the possibility of the simultaneous efficient removal of SMX from polluted water and biomass production for fuel feedstock by the phyto-Fenton process using duckweed. This is the first attempt to demonstrate the co-benefits of SMX removal and biomass production using duckweed. Intracellular H2O2 was produced using four duckweeds, Lemna aequinoctialis, L. minor, Landolina punctata, and Spirodela polyrhiza, in the range of 16.7–24.6 μ mol g−1 fresh weight, and extracellular H2O2 was released into the water phase. Consequently, duckweed could be used as an H2O2 supply source for the phyto-Fenton process. Specifically, 0.5 g fresh duckweed almost completely eliminated 1 mg L−1 SMX after 5 d in 50 mL sterile modified Hoagland solution containing 10 mM Fe (II). Fe (II)-dependent elimination of SMX indicated the occurrence of phyto-Fenton reaction. The phyto-Fenton process using duckweed effectively removed SMX. S. polyrhiza duckweed similarly removed 1 mg L−1 SMX even in sewage effluent containing other organic contaminants. During this treatment, duckweed biomass was generated at 7.95 g dry weight m−2 d−1, which was converted into methane at 353 normal liters CH4 kg−1 volatile solids by anaerobic digestion. For the first time, this study clearly demonstrates the potential for simultaneous SMX removal and biomass production from SMX-contaminated wastewater using duckweed.

Comparative genomics and evidence for an unusual polyamine oxidation pathway in aquatic duckweed (Spirodela polyrhiza L.)

Polyamines (PA) cellular levels are maintained through a balance between synthesis and catabolism, achieved by two classes of enzymes polyamine oxidases (PAOs) and copper amine oxidases (CuAO). Here we investigated the occurrence, molecular evolution and role(s) of PAOs and CuAO gene families in aquatic duckweed and their comparison with other aquatic plants -sea eelgrass, bladderwort, and Lotus. We identified eight bona fide PAO genes (SpPAO1–SpPAO8) and one SpCuAO1 in the greater duckweed genome from three genome assemblies. Interestingly, duckweed PAO genes increased their number through a tandem duplication event, while contrary to this CuAO genes were significantly lost to a single gene SpCuAO1. Phylogenetic analysis revealed that tandemly duplicated SpPAO2–7 share close similarity to well-known terminal catabolism (TC) pathway PAO genes while SpPAO1 and SpPAO8 seem to segregate along with back conversion (BC) participating known PAO genes, suggesting that all tandem duplicated PAOs are involved in TC pathway which is contrary to known trend in land plants where CuAOs are mainly involved in TC pathway. Comparative transcript abundance studies indicated that all eight PAOs and one CuAO gene respond to multiple stresses and principal component analysis identifies SpPAO4 as a highly active gene in response to multiple stresses. Results showed that oxidation of higher polyamines (SPD/SPM) through the TC pathway is diversified in duckweeds. Taken together this study reveals unique insights into the genomic losses and gains of polyamine metabolism possibly involved in achieving the structural and physiological adaptations required for aquatic lifestyle of duckweeds.

Population genomics and epigenomics of Spirodela polyrhiza provide insights into the evolution of facultative asexuality

Many plants are facultatively asexual, balancing short-term benefits with long-term costs of asexuality. During range expansion, natural selection likely influences the genetic controls of asexuality in these organisms. However, evidence of natural selection driving asexuality is limited, and the evolutionary consequences of asexuality on the genomic and epigenomic diversity remain controversial. We analyzed population genomes and epigenomes of Spirodela polyrhiza, (L.) Schleid., a facultatively asexual plant that flowers rarely, revealing remarkably low genomic diversity and DNA methylation levels. Within species, demographic history and the frequency of asexual reproduction jointly determined intra-specific variations of genomic diversity and DNA methylation levels. Genome-wide scans revealed that genes associated with stress adaptations, flowering and embryogenesis were under positive selection. These data are consistent with the hypothesize that natural selection can shape the evolution of asexuality during habitat expansions, which alters genomic and epigenomic diversity levels.

Comparison of mercury (Hg) bioaccumulation with mono- and mixed Lemna minor and Spirodela polyrhiza cultures.

Mercury (Hg) is a prevalent and harmful contaminant that persists in the environment. For phytoremediation, it is important to discover which plants can bioaccumulate meaningful amounts of Hg while also tolerating its toxicity. Additionally, increasing biodiversity could create a more resilient and self-sustaining system for remediation. This study explores whether mixed populations of Lemna minor and Spirodela polyrhiza can better bioaccumulate and tolerate Hg than monocultures. Mono- and mixed cultures of L. minor and S. polyrhiza were grown in mesocosms of 0.5µg/L or 100µg/L Hg (HgCl2) spiked water for 96h. Change in weight of duckweed was used to assess Hg tolerance. Diffusive gradients in thin-films (DGTs) were used as surrogate monitoring devices for bioavailable levels of Hg. For biomass growth, the mixed culture of the L. minor was greater than the monoculture at the high dose. The L. minor accumulated more Hg in the mixed culture at the low dose while the S. polyrhiza was higher in the mixed at the high dose. Hg speciation in water was modeled using Windermere Humic Aqueous Model 7 (WHAM7) to compare the bioavailable species indicated by the DGTs. Potentially due to the controlled conditions, the WHAM7 output of bioavailable Hg was almost 1:1 to that estimated by the DGTs, indicating good predictive capability of geochemical modeling and passive sampler DGT on metal bioavailability. Overall, the mixed cultures statistically performed as well as or better than the monocultures when tolerating and bioaccumulating Hg. However, there needs to be further work to see if the significant differences translate into practical differences worth the extra resources to maintain multiple species.

An Ionic Liquid-Based Biorefinery Approach for Duckweed Utilization.

This study establishes a foundation for the ionic liquid (IL) pretreatment of duckweed biomass. An optimized IL-based process was designed to exploit the unique properties of duckweed including efficient metal removal, potential starch accumulation, and protein accumulation. Two ILs, namely, dimethylethanolammonium formate ([DMEtA][HCOO]) and N,N-dimethylbutylammonium hydrogen sulfate ([DMBA][HSO4]), were investigated for the pretreatment of two duckweed species (Spirodela polyrhiza and Lemna minor). The evaluation focused on starch recovery, sugar release, protein recovery, and metal extraction capabilities. [DMEtA][HCOO] demonstrated near-quantitative starch recoveries at 120 °C, while [DMBA][HSO4] showed similar performance at 90 °C within a reaction time of 2 h. Saccharification yields for most pulps exceeded 90% after 8 h of hydrolysis, outperforming "traditional" lignocellulosic biomasses such as miscanthus or sugarcane bagasse. Approximately 50 and 80 wt % of the protein were solubilized in [DMEtA][HCOO] and [DMBA][HSO4], respectively, while the remaining protein distributed between the pulp and lignin. However, the solubilized protein in the IL could not be recovered due to its low molecular weight. Regarding metal extraction, [DMEtA][HCOO] demonstrated higher efficiency, achieving 81% removal of Ni from Lemna minor's pulps, whereas [DMBA][HSO4] extracted only 28% of Ni with slightly higher pulp concentrations. These findings indicate the need for further optimization in concurrent metal extraction using ILs.