Fundamentals and application in phytoremediation of an efficient arsenate reducing bacterium Pseudomonas putida ARS1.

Arsenic is a ubiquitous environmental pollutant. Microbe-mediated arsenic bio-transformations significantly influence arsenic mobility and toxicity. Arsenic transformations by soil and aquatic organisms have been well documented, while little is known regarding effects due to endophytic bacteria. An endophyte Pseudomonas putida ARS1 was isolated from rice grown in arsenic contaminated soil. P. putida ARS1 shows high tolerance to arsenite (As(III)) and arsenate (As(V)), and exhibits efficient As(V) reduction and As(III) efflux activities. When exposed to 0.6 mg/L As(V), As(V) in the medium was completely converted to As(III) by P. putida ARS1 within 4 hr. Genome sequencing showed that P. putida ARS1 has two chromosomal arsenic resistance gene clusters (arsRCBH) that contribute to efficient As(V) reduction and As(III) efflux, and result in high resistance to arsenicals. Wolffia globosa is a strong arsenic accumulator with high potential for arsenic phytoremediation, which takes up As(III) more efficiently than As(V). Co-culture of P. putida ARS1 and W. globosa enhanced arsenic accumulation in W. globosa by 69%, and resulted in 91% removal of arsenic (at initial concentration of 0.6 mg/L As(V)) from water within 3 days. This study provides a promising strategy for in situ arsenic phytoremediation through the cooperation of plant and endophytic bacterium.

Frond architecture of the rootless duckweed Wolffia globosa.

BACKGROUND: The plant body in duckweed species has undergone reduction and simplification from the ancient Spirodela species towards more derived Wolffia species. Among the five duckweed genera, Wolffia members are rootless and represent the smallest and most reduced species. A better understanding of Wolffia frond architecture is necessary to fully explore duckweed evolution. RESULTS: We conducted a comprehensive study of the morphology and anatomy of Wolffia globosa, the only Wolffia species in China. We first used X-ray microtomography imaging to reveal the three-dimensional and internal structure of the W. globosa frond. This showed that new fronds rapidly budded from the hollow reproductive pocket of the mother fronds and that several generations at various developmental stages could coexist in a single W. globosa frond. Using light microscopy, we observed that the meristem area of the W. globosa frond was located at the base of the reproductive pocket and composed of undifferentiated cells that continued to produce new buds. A single epidermal layer surrounded the W. globosa frond, and the mesophyll cells varied from small and dense palisade-like parenchyma cells to large, vacuolated cells from the ventral to the dorsal part. Furthermore, W. globosa fronds contained all the same organelles as other angiosperms; the most prominent organelles were chloroplasts with abundant starch grains. CONCLUSIONS: Our study revealed that the reproductive strategy of W. globosa plants enables the rapid accumulation of biomass and the wide distribution of this species in various habitats. The reduced body plan and size of Wolffia are consistent with our observation that relatively few cell types are present in these plants. We also propose that W. globosa plants are not only suitable for the study of structural reduction in higher plants, but also an ideal system to explore fundamental developmental processes of higher plants that cannot be addressed using other model plants.

Extraction, Enzymatic Modification, and Anti-Cancer Potential of an Alternative Plant-Based Protein from Wolffia globosa.

The global plant-based protein demand is rapidly expanding in line with the increase in the world's population. In this study, ultrasonic-assisted extraction (UAE) was applied to extract protein from Wolffia globosa as an alternative source. Enzymatic hydrolysis was used to modify the protein properties for extended use as a functional ingredient. The successful optimal conditions for protein extraction included a liquid to solid ratio of 30 mL/g, 25 min of extraction time, and a 78% sonication amplitude, providing a higher protein extraction yield than alkaline extraction by about 2.17-fold. The derived protein was rich in essential amino acids, including leucine, valine, and phenylalanine. Protamex and Alcalase were used to prepare protein hydrolysates with different degrees of hydrolysis, producing protein fragments with molecular weights ranging between <10 and 61.5 kDa. Enzymatic hydrolysis caused the secondary structural transformations of proteins from ß-sheets and random coils to α-helix and ß-turn structures. Moreover, it influenced the protein functional properties, particularly enhancing the protein solubility and emulsifying activity. Partial hydrolysis (DH3%) improved the foaming properties of proteins; meanwhile, an excess hydrolysis degree reduced the emulsifying stability and oil-binding capacity. The produced protein hydrolysates showed potential as anti-cancer peptides on human ovarian cancer cell lines.

Wolffia globosa-Based Nutritious Snack Formulation with High Protein and Dietary Fiber Contents.

Wolffia globosa (W. globosa) or duckweed is a small aquatic plant with high protein, dietary fiber, and lipid contents that can be combined with food products to develop nutritious snacks as one strategy to mitigate malnutrition. Here, response surface methodology (RSM) with mixture design was used to develop snacks from W. globosa freeze-dried powder (WP). The physical properties, proximate analysis, amino acid profiles, sensory evaluation, phytochemical analysis, antioxidant properties, and genotoxicity (Ames test) of the snacks were evaluated. The optimal W. globosa snack formula was 64% glutinous rice flour, 10% tapioca flour, and 26% WP, giving a highly desirable liking score of 1.00. Addition of WP increased crude protein, essential amino acids, and dietary fiber compared with the control snack by 51%, 147%, and 83%, respectively. According to the Thai recommended daily intakes, the developed W. globosa snack had high protein and dietary fiber. Phytochemical contents and antioxidant activities of the W. globosa snack such as total phenolic contents (TPCs), total flavonoid contents (TFCs), ferric ion reducing antioxidant power (FRAP) activity, and oxygen radical absorbance capacity (ORAC) activity were significantly higher than the control snack. The novel combination of WP with snack product ingredients greatly enhanced nutritional value.

Antimicrobial and Functional Properties of Duckweed (Wolffia globosa) Protein and Peptide Extracts Prepared by Ultrasound-Assisted Extraction.

Wolffia globosa is an interesting alternative plant-based protein source containing up to 40% protein dry weight. Dried duckweed protein extract (PE) was obtained using ultrasound-assisted extraction (UAE) before isoelectric precipitation (pH 3.5) to yield protein concentrate (PC) and protein solution (PS). The PC was hydrolyzed using Alcalase enzyme to obtain protein concentrate hydrolysate (PCH). Among all fractions, PCH exhibited antimicrobial properties by decreasing populations of Vibrio parahaemolyticus and Candida albicans at 0.43 ± 1.31 log reduction (66.21%) and 3.70 ± 0.11 log reduction (99.98%), respectively. The PE and PS also showed high solubilities at pH 8 of 90.49% and 86.84%, respectively. The PE demonstrated the highest emulsifying capacity (EC) (71.29%) at pH 4, while the highest emulsifying stability (ES) (~98%) was obtained from the PE and PS at pH 6 and pH 2, respectively. The major molecular weights (Mw) of the PE, PC, PCH and PS were observed at 25, 45, 63 and 100 kDa, with a decrease in the Mw of the PCH (<5 kDa). The PCH contained the highest total amino acids, with aspartic acid and glutamic acid being the major components. The results revealed the antimicrobial and functional properties of duckweed protein and hydrolysate for the first time and showed their potential for further development as functional food ingredients.

The Metabolomic-Gut-Clinical Axis of Mankai Plant-Derived Dietary Polyphenols.

BACKGROUND: Polyphenols are secondary metabolites produced by plants to defend themselves from environmental stressors. We explored the effect of Wolffia globosa 'Mankai', a novel cultivated strain of a polyphenol-rich aquatic plant, on the metabolomic-gut clinical axis in vitro, in-vivo and in a clinical trial. METHODS: We used mass-spectrometry-based metabolomics methods from three laboratories to detect Mankai phenolic metabolites and examined predicted functional pathways in a Mankai artificial-gut bioreactor. Plasma and urine polyphenols were assessed among the 294 DIRECT-PLUS 18-month trial participants, comparing the effect of a polyphenol-rich green-Mediterranean diet (+1240 mg/polyphenols/day, provided by Mankai, green tea and walnuts) to a walnuts-enriched (+440 mg/polyphenols/day) Mediterranean diet and a healthy controlled diet. RESULTS: Approximately 200 different phenolic compounds were specifically detected in the Mankai plant. The Mankai-supplemented bioreactor artificial gut displayed a significantly higher relative-abundance of 16S-rRNA bacterial gene sequences encoding for enzymes involved in phenolic compound degradation. In humans, several Mankai-related plasma and urine polyphenols were differentially elevated in the green Mediterranean group compared with the other groups (p < 0.05) after six and 18 months of intervention (e.g., urine hydroxy-phenyl-acetic-acid and urolithin-A; plasma Naringenin and 2,5-diOH-benzoic-acid). Specific polyphenols, such as urolithin-A and 4-ethylphenol, were directly involved with clinical weight-related changes. CONCLUSIONS: The Mankai new plant is rich in various unique potent polyphenols, potentially affecting the metabolomic-gut-clinical axis.

Safety of Wolffia globosa powder as a Novel food pursuant to Regulation (EU) 2015/2283.

Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on Wolffia globosa powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. Wolffia globosa is an aquatic plant, one out of the 38 species of the water lentil family which is composed by five genera (i.e. Spirodela, Landoltia, Lemna, Wolffiella and Wolffia). The NF is produced by cultivation of Wolffia globosa plants under controlled conditions, washing with hot water and drying. The main constituents of the NF are protein, fibre and fat. The Panel notes that the concentration of trace elements and contaminants in the NF is highly dependent on the conditions of cultivation of the plant and the fertiliser composition. The NF is intended to be used as food ingredient in a variety of food categories and as food supplement. The target population is the general population except for food supplements which are intended to be consumed exclusively by adults. The Panel considers that with the exception of concerns related to the manganese intake, taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous. The subchronic toxicity study provided with the NF revealed a number of significant findings and the Panel considers the middle dose (6.5 g/kg body weight (bw) per day) as the no observed adverse effect level (NOAEL). Based on the protein concentration, the Panel considers that the consumption of the NF may trigger allergic reactions. The Panel concluded that an increase in manganese intake from the NF used as food ingredient or food supplements is of safety concern and the safety of the NF cannot be established.

Expression of LamB Vaccine Antigen in Wolffia globosa (Duck Weed) Against Fish Vibriosis.

Vibriosis is a commonly found bacterial disease identified among fish and shellfish cultured in saline waters. A multitude of Vibrio species have been identified as the causative agents. LamB, a member of outer membrane protein (OMPs) family of these bacteria is conserved among all Vibrio species and has been identified as an efficient vaccine candidate against vibriosis. Rootless duckweed (Wolffia) is a tiny, edible aquatic plant possessing characteristics suitable for the utilization as a bioreactor. Thus, we attempted to express a protective edible vaccine antigen against fish vibriosis in nuclear-transformed Wolffia. We amplified LamB gene from virulent Vibrio alginolyticus and it was modified to maximize the protein expression level and translocate the protein to the endoplasmic reticulum (ER) in plants. It was cloned into binary vector pMYC under the control of CaMV 35S promoter and introduced into Wolffia globosa by Agrobacterium-mediated transformation. Integration and expression of the LamB gene was confirmed by genomic PCR and RT-PCR. Western blot analysis revealed accumulation of the LamB protein in 8 transgenic lines. The cross-protective property of transgenic Wolffia was evaluated by orally vaccinating zebrafish through feeding fresh transgenic Wolffia and subsequently challenging with virulent V. alginolyticus. High relative percent survival (RPS) of the vaccinated fish (63.3%) confirmed that fish immunized with transgenic Wolffia were well-protected from Vibrio infection. These findings suggest that Wolffia expressed LamB could serve as an edible plant-based candidate vaccine model for fish vibriosis and feasibility of utilizing Wolffia as bioreactor to produce edible vaccines.

Wolffia globosa-Mankai Plant-Based Protein Contains Bioactive Vitamin B12 and Is Well Absorbed in Humans.

BACKGROUND: Rare plants that contain corrinoid compounds mostly comprise cobalamin analogues, which may compete with cobalamin (vitamin B12 (B12)) metabolism. We examined the presence of B12 in a cultivated strain of an aquatic plant: Wolffia globosa (Mankai), and predicted functional pathways using gut-bioreactor, and the effects of long-term Mankai consumption as a partial meat substitute, on serum B12 concentrations. METHODS: We used microbiological assay, liquid-chromatography/electrospray-ionization-tandem-mass-spectrometry (LC-MS/MS), and anoxic bioreactors for the B12 experiments. We explored the effect of a green Mediterranean/low-meat diet, containing 100 g of frozen Mankai shake/day, on serum B12 levels during the 18-month DIRECT-PLUS (ID:NCT03020186) weight-loss trial, compared with control and Mediterranean diet groups. RESULTS: The B12 content of Mankai was consistent at different seasons (p = 0.76). Several cobalamin congeners (Hydroxocobalamin(OH-B12); 5-deoxyadenosylcobalamin(Ado-B12); methylcobalamin(Me-B12); cyanocobalamin(CN-B12)) were identified in Mankai extracts, whereas no pseudo B12 was detected. A higher abundance of 16S-rRNA gene amplicon sequences associated with a genome containing a KEGG ortholog involved in microbial B12 metabolism were observed, compared with control bioreactors that lacked Mankai. Following the DIRECT-PLUS intervention (n = 294 participants; retention-rate = 89%; baseline B12 = 420.5 ± 187.8 pg/mL), serum B12 increased by 5.2% in control, 9.9% in Mediterranean, and 15.4% in Mankai-containing green Mediterranean/low-meat diets (p = 0.025 between extreme groups). CONCLUSIONS: Mankai plant contains bioactive B12 compounds and could serve as a B12 plant-based food source.

Protein bioavailability of Wolffia globosa duckweed, a novel aquatic plant - A randomized controlled trial.

BACKGROUND & AIMS: While the world is extensively looking for alternatives to animal protein sources, it is not clear which plant sources can provide the requisite full complement of essential amino acids (EAAs). Wolffia globosa is an aquatic, edible duckweed, the smallest plant on earth, and it offers all nine EAAs, dietary fibers, polyphenols, iron, zinc and B12 vitamin. This work was designed to evaluate Mankai (a newly developed high-protein strain of W. globosa) as an optional bioavailable source of EAAs for humans (primary outcome), and of further nutrients such as vitamin B12, in comparison to well-established animal and plant protein sources; cheese and peas, respectively. METHODS: 36 men, subjected for 3 days to a stable diet and subsequent overnight (12 h) fast, were randomized to consume one of three iso-protein (30 g) based test-meals (soft cheese, green peas, Mankai). Blood samples were collected at 0, 30, 90 and 180 min. RESULTS: The 3 h blood concentrations of the EAAs: histidine, phenylalanine, threonine, lysine, and tryptophan, triggered by intake of Mankai, was essentially significant as compared to baseline (p < 0.05) and similar to that of soft cheese and pea changes (p > 0.05 between groups). Although branched-chain-amino-acids (leucine/isoleucine, valine) increased significantly by Mankai within 3 h (p < 0.05 vs. baseline), the change was relatively higher for cheese as compared to Mankai or peas (p < 0.05 between groups). The increase in vitamin B12 by Mankai was higher as compared to changes induced by either cheese (p=0.007) or peas (p=0.047, between groups). CONCLUSIONS: Mankai may provide a high-quality substitute source for animal protein, and a potential bioavailable source of vitamin B12.

Development of Efficient Protocols for Stable and Transient Gene Transformation for Wolffia Globosa Using Agrobacterium.

Members of the Wolffia genus are fascinating plants for many biologists as they are the smallest flowering plants on Earth and exhibit a reduced body plan that is of great interest to developmental biologists. There has also been recent interest in the use of these species for bioenergy or biorefining. Molecular and developmental studies have been limited in Wolffia species due to the high genome complexity and uncertainties regarding the stable genetic transformation. In this manuscript we present new protocols for both stable and transient genetic transformation for Wolffia globosa using Agrobacterium tumefaciens. For the transient transformation, we used Wolffia fronds whereas we used clusters for the stable transformation. As proof of concept we transformed two synthetic promoter constructs driving expression of the GUS marker gene, that have previously been used to monitor auxin and cytokinin output in a variety of species. Using these approaches we obtained a Transformation Efficiency (TE) of 0.14% for the stable transformation and 21.8% for the transient transformation. The efficiency of these two methods of transformation are sufficient to allow future studies to investigate gene function. This is the first report for successful stable transformation of W. globosa.

The different response mechanisms of Wolffia globosa: Light-induced silver nanoparticle toxicity.

Silver nanoparticles (AgNPs) have emerged as a promising bactericide. Plants are a major point of entry of contaminants into trophic chains. Here, the physiological responses of Wolffia globosa to AgNPs have been probed using different light schemes, and these data may reveal new insights into the toxic mechanism of AgNPs. W. globosa was grown in culture medium and treated with different concentrations of AgNPs for 24h under pre- and post-illuminated conditions. However, fluorescence quenching, the accumulation of sugar and the reduction of Hill reaction activity were found in response to the AgNP-stresses. In the pre-illuminated condition, oxidative damage was obvious, as indicated by the higher malondialdehyde (MDA) content and an up-regulation of superoxide dismutase (SOD) activity. The maximum increases of MDA content and SOD activity were 1.14 and 2.52 times the respective controls when exposed to 10mg/L AgNPs. In contrast, in the post-illuminated condition, the alterations in photosynthetic pigment and soluble proteins content were more significant than the alterations in oxidative stress. The contents of chlorophyll a, carotenoids and soluble protein decreased to 77.7%, 66.2% and 72.9% of the controls after treatment with the highest concentration of AgNPs (10mg/L). Based on the different physiological responses, we speculated that in the pre-illuminated condition, oxidative stress was responsible for the decline in the oxygen evolution rate, while in the post-illuminated condition, the decrease in the Hill reaction activity could be attributed to the blocking of electron transfer and an insufficient proton supply. Our findings demonstrate that environmental factors regulate the physiological responses of plants to AgNPs through distinct mechanisms.

Evaluation of food chain transfer of the antibiotic oxytetracycline and human risk assessment.

There has been recent concern regarding the possibility of antibiotics entering the aquatic food chain and impacting human consumers. This work reports experimental results of the bioconcentration of the antibiotic oxytetracycline (OTC) by the Asian watermeal plant (Wolffia globosa Hartog & Plas) and bioaccumulation of OTC in watermeal and water by the seven-striped carp (Probarbus jullieni). They show, for the first time, the extent to which OTC is able to transfer from water to plant to fish and enter the food chain. The mean bioconcentration factor (dry weight basis) with watermeal was 1.28 × 10(3) L kg(-1). Separate experiments were undertaken to characterize accumulation of OTC by carp from water and watermeal. These showed the latter pathway to be dominant under the conditions employed. The bioconcentration and biomagnification factors for these processes were 1.75 L kg(-1) and 2 × 10(-4) kg g(-1) respectively. Using an aqueous concentration range of 0.34-3.0 µg L(-1), hazard quotients for human consumption of contaminated fish of 1.3 × 10(-2) to 1.15 × 10(-1) were derived.