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Effect of soil molybdenum concentration on the collapse radiation bioremediation ability and transcriptome of Epiphyllum azuris
SQS-006 "Kia" (d)1*, SQR-004 "Nyx" (d)1, SFR-001 "Archangel" (d)1, SQM-011 "Magic" (d)1, SQK-009 "Krieg" (d)1, SQM-014 "Axiom" (d)1, SQF-021 "Sharps" (d)1, SQF-019 "Keter" (d)1, ARCTIC-3147 "Madeline" (d)2, and Veers M3.
1. Sangvis Ferri Mobile Task Force, Moscow, Neo-Soviet Union
2. Griffin and Kryuger, Moscow, Neo-Soviet Union
3. Flora Institute of Bremen, Bremen, German Democratic Republic
* Corresponding author: kia_sqs006@gnk,net
(d) denotes doll authors
Abstract
With conventional physicochemical collapse radiation decontamination methods remaining expensive and impractical for many applications, Epiphyllum-based bioremediation alternatives have begun to garner increasing interest. A key roadblock to the adoption of such bioremediative methods is the efficiency of the Epiphyllum flowers in absorbing radiation from the soil. In our study, we evaluated the effect of soil molybdenum concentration on the bioremediative efficiency of Epiphyllum azuris. Plants were grown in environmentally controlled plots containing 150 mVe of collapse radiation as well as 2 (control), 10, 30, or 100 ppm of Mo2+. The plants' physical growth, metal accumulation, and the soil radiation levels were measured regularly for 18 days. Our results showed that bioremediative efficiency peaked in the 30 ppm group, with an 26% increase in efficiency after 18 days. Efficiency was decreased in the 100 ppm group due to acute metal toxicity. Additionally, radiation absorption was faster in treated groups even if the end efficiency was not increased. We also conducted transcriptomic analysis, which revealed that Res2 and the CpAT-ROcT pathway expression was upregulated in the treated groups. We believe this provides further avenues of approach in genetically engineering Epiphyllum azuris plants to increase their bioremediative efficiency without the need to introduce additional molybdenum into the environment.
Introduction
Ever since the Beilan incident in 2030, collapse radiation (CR) has posed an unprecedented threat to both society and the Earth's biosphere at large. Although CR purification technology has advanced greatly in recent decades, it is still prohibitively expensive for application in all but the largest and wealthiest parts of the world*. Even today, ELID and other CR-related illnesses remain the greatest causes of mortality and morbidity outside White and Green Zones*. A previous study by Ch'en et al. concluded that approximately 30% of CR-related deaths in eastern Asian Yellow Zones could have been avoided with a modest decrease in decontamination costs consistent with a low-intensity environmental decontamination regimen*. Over half of the morbidity and mortality observed in the study was due to chronic illnesses caused by exposure to lingering radioactivity from discrete contamination events (such as major radiation storms)*. As the world continues its slow journey towards recovery, it remains paramount for newer and more effective methods of locally removing CR to be developed. New inventions such as the KASSA reactor show great promise in reducing the cost barrier for effective large-scale radiation purification*, but such technologies are largely out of reach for most rural communities in the world*. Affordable and accessible purification of localized irradiation caused by spills and radiation storms remains a key roadblock for many humanitarian organizations*. In most cases, it is impractical to relocate large purification devices in situ, creating an urgent need for smaller-scale alternatives. Although mobile solutions have been developed, they have been met with limited success due to either high upfront costs (such as Svarog Heavy Industries' ARTEMiS platform) and/or high operating costs (such as the Thermo Fisher ABCD filters). In the recent decades, bioremediative methods have begun to attract increasing interest as a cheaper alternative to conventional physicochemical approaches.
Bioremediation is a process in which biological organisms are used to remove contaminants from soil and water. Contamination can exist in many forms, although conventional contaminants can largely be divided into two categories: organic and inorganic. Organic contaminants include hydrocarbons and pharmaceuticals, and remediation generally relies upon the metabolism of the contaminants by microorganisms, which convert them into safe products*. Inorganic contaminants include heavy metals and halogens, which cannot be destroyed as with organic contaminants. Instead, they are converted into less dangerous forms, such as through precipitation via redox reactions*. As one would expect, a limiting factor on the ability of an organism to perform bioremediation is their ability to withstand the toxic effects of the contaminant, while another factor is the species' efficiency in removing the contaminant from the substrate. CR possesses properties of both organic and inorganic contaminants from a bioremediation standpoint*. However, the analogy is complicated by the fact that CR is not baryonic matter, instead conjugating with baryonic matter through Cohen-Landau interactions*. CR conjugated with organic matter and water can be absorbed by cells through standard biological processes such as transporters and diffusion, allowing for them to be concentrated intracellularly*. However, the vast majority of species cannot break or form Cohen-Landau interactions, meaning they can only concentrate CR-conjugated molecules without actually moving radiation between individual molecules*. The sole exceptions are plants of the Epiphyllum order (not to be confused with the Epiphyllum genus, an unrelated clade of cacti), which are the only known biological organisms which can directly manipulate Cohen-Landau interactions*. These species mainly rely on the poorly understood Pembrose pathway, which not only moves CR between molecules, but is able to generate energy for the plant as well*. An advanced discussion of CR physics is beyond the scope of this paper, but it is important to recognize that Epiphyllum plants are capable of transferring CR between molecules, and that makes them extraordinarily valuable in the sequestration of CR from a substrate*.
Specifically, Epiphyllum are CR hyperaccumulators, localizing it to their flower buds and releasing it during blooming*. The collapse fluid is collected from the surrounding soil using specialized transporters on the root hairs*, where unique cells in the vascular tissue concentrates the CR within the vasculature*. The CR is then moved up throughout the plant using standard vascular dynamics, with the CR finally being concentrated into the ovaries of the Epiphyllum flowers*. The flowers and plant bodies can then easily be collected with the help of radiation-resistant PPE or using dolls. This represents a much cheaper alternative to physicochemical decontamination methods such as counter-flow radiation sieving* and CCR reverse osmosis techniques*. However, the use of Epiphyllum for decontamination is not yet efficient enough for it to be practically used in most scenarios, with major issues such as the rate of radiation absorbance, soil penetration depth, and prediction of radiation blooms being major roadblocks*. Notably, much valid concern has been raised over the risk of the flowers blooming before they are collected, which would spread the collected radiation back into the surroundings*.
Project Epi-ABLE (Epiphyllum-Assisted Bioremediation of Lost Environs) of the Flora Institute of Bremen is one of several scientific initiatives around the world which aim to utilize Epiphyllum plants to remove CR from contaminated soil and water*. Epi-ABLE aims to increase the bioremediative efficacy of Epiphyllum through genetic engineering and selective breeding of various species and strains. In particular, the Res2 and CpAT genes have been heavily studied due their expression in the roots of the plants*, with the roots being shown to be the rate-limiting step in CR hyperaccumulation*. The CpAT gene is involved in the Maharaj cycle, coding for a transporter that exchanges unconjugated water within the root hairs for conjugated water in the soil*. This process uses a form of active transport, but the energy cost is made up for by the energy gained from harnessing the radiation*. The Res2 gene is a transcription factor exclusively found within Epiphyllum plants which is expressed in various parts of the plant*. In the roots, Res2 expression is proportional to the soil CR concentration, with increased Res2 expression leading to increased CR uptake via upregulation of several genes including CpAT*. Res2 has often been used as a marker to measure the plant's response to various treatments*.
Our research is centred around adapting a strain of E. azuris obtained from Project Epi-ABLE for use in contaminated rural areas in the Transcarpathian Lowlands of the Neo-Soviet Union. This strain was originally obtained and developed by the Sangvis Ferri medical department and is now being developed by a team in Griffin and Kryuger PMC under guidance from Ultilife. In a recent humanitarian operation in the village of Kurilev, a genetically modified strain of E. azuris from was used to successfully remove CR from the soil after a spill from a storage container*. The storage contained had been hidden from sight and slowly leaking into the soil for an unknown amount of time prior to discovery—possibly several months—causing a moderate degree of CR contamination down to at least the water table. The ambient radiation was measured to be approximately 100 mVe, which placed the area on the higher end of the Yellow Zone range. While removing the container, it was damaged and a large amount of concentrated collapse fluid—approximately 1 L of 1 Ve concentration—was released into the soil. Although the radiation did not spread down to the water table thanks to rapid cleanup, the surface radiation concentration 100 m from the spill site peaked at approximately 250 mVe, qualifying the area as a Red Zone. One of the key factors that allowed the operation to succeed was the application of a sodium molybdate supplement to the plants, which considerably increased their CR hyperaccumulation rates. Molybdenum (Mo2+) is an essential micronutrient in most plants, playing a role in development and serving as cofactors in enzymes used in the nitrogen cycle*. Previous research determined that the bioremedial efficacy of Epiphyllum fluorencis was increased by greater soil concentrations of heavy metal nutrients such as cadmium, molybdenum, zinc, and copper*, with further study suggesting those metals play an important role in the biochemical pathways associated with the uptake of CR from the soil*. The operation in Kurilev validated those findings in a real-life scenario using E. azuris. However, the nature of the operation did not allow us to elucidate the precise mechanism in which Mo2+ contributes to bioremediation. As E. azuris is a considerably larger species than E. fluorencis, with root penetration upwards of 1.5 m, it is a much better candidate for real-world application. In this study we aimed to reproduce the increase in soil molybdenum concentration in a controlled setting to measure the effects of different molybdate concentrations on bioremedial characteristics of E. azuris. Furthermore, we used transcriptomic and immunofluorescence analysis to characterize the associated cellular changes. Our results indicate that a moderate supplement of molybdate considerably increases the bioremediative potential of E. azuris plants by stimulating Res2 expression in the roots. These finding link the increase in potential to a specific biological mechanism, opening the door for further research into specifically targeting that mechanism to increase bioremediative efficiency.
Materials and methods
Growth of Epiphyllum azuris samples
Genetically modified E. azuris plant seeds (strain DL4112, Flora Institute of Bremen) from Project Azure Shield were grown in plots of soil containing various concentrations of sodium molybdate. Soil was collected from grassland in the Transcarpathian Lowlands region, with chemical analysis to confirm that they had a relatively normal composition. Seeds were planted and raised according to standard procedures, except different plots were treated with different amounts of aqueous sodium molybdate to produce an initial soil concentration of 2 ppm (control), 10 ppm, 30 ppm, and 100 ppm of molybdenum. Soil was mechanically mixed to produce an even distribution. Each plot contained 10 flowers in a grid spaced 1 m apart. Each plot was 3 m deep. The plots were placed in a greenhouse and grown according to the climactic standards for eastern European grasslands published by Kovač et al. in 2046. Initial soil CR concentration was normalized to 250 mVe prior to the experiment using concentrated collapse fluid obtained from a purification device.
Measurement of plant growth and soil radiation
Measurements were conducted at 2-, 4-, 6-, 10-, 14-, and 18-days post-planting. Plant growth was characterized by measuring the height of each plant from the soil to the apical meristem after pulling the plant taut. Additional qualitative observations were also made. Soil radiation was measured with a Galatea RW3400 radiation sensor, with each plot being measured 5 times at different parts of the plot at a depth of 30 cm.
Measurement of metal hyperaccumulation
At the end of the experiment after 18 days, the plants were removed from the soil, with care being taken to minimize the breakage and loss of roots during removal. The molybdenum and copper percent dry weight () of each plant's roots was estimated by homogenization followed by analysis using a Thermo Fisher I-550 water purification assay kit. The dry mass of each plant's roots was determined by drying the roots under elevated temperatures. The purpose of estimating of copper was to estimate the level of interference of high molybdenum concentration on the plant's physiology, since molybdenum has been demonstrated as an antagonist for multiple other heavy metal ions in a wide variety of plants*.
Transcriptome analysis
To obtain samples for transcriptome measurement, needle samples of the plant's root hairs were taken at 6- and 18-days post-planting. Root samples were homogenized, and RNA purification was conducted using an Ultilife MG750 RNA purification kit. RNA purity was determined via nanodrop spectrophotometry. mRNA was converted into cDNA using oligo-dT primers and an Oxford Genomics RT kit. The resultant cDNA was analyzed using an Illumina-Candleton PSeq700 sequencer. The output was normalized as transcripts-per-million (TPM) and compared against the International Epiphyllum Genomic Database (IEGD) to determine changes in mRNA expression.
Statistical analysis
Plant growth, soil radiation, and metal hyperaccumulation measurements were determined by the Shapiro-Wilk test to be all normally distributed. Treatment groups were compared using the one-way ANOVA test with post-hoc Tukey's HSD test to identify significant differences between groups. Soil radiation measurements were analysed with and without normalisation by plant mass. An α-value of 0.05 was used in the analysis of significance.
Transcript abundances were compared using Pearson correlation coefficients (PCCs) by comparing to the control, and genes were clustered using the HOPACH 2.0 algorithm. Analysis was done using BSAP 4.1.12.
Raw data is available in Appendix A.
Results
Plant Growth and metal accumulation
To determine the effect of soil molybdenum on the growth of E. azuris, seeds were grown in soil treated with various amounts of sodium molybdate. Plant height was measured at regular intervals during growth, while the of copper and molybdenum in the plants was determined at the end of the experiment after 18 days. During the first six days of the experiment, the rate of growth was generally proportional to the soil molybdenum concentration (Figure 1a). The highest rate of growth occurred at the beginning of the experiment, with the growth rate decreasing after several days. The largest mean plant height after six days was that of the 30 ppm group followed by the 100 ppm. After the six-day mark, the growth of the all the treatment groups levelled off at about 65 cm except for the 100 ppm group, although groups with lower soil molybdenum concentration reached the threshold height slower (Figure 1b). Notably, the 100 ppm treatment group experienced a steady decline after the 10-day mark, with the plants displaying a diseased state consistent with copper deficiency as the experiment progressed. The mean plant weight after 18 days was not significantly different between any of the groups except for the 100 ppm group, which had a much lower weight (p=0.42 and p=0.02 respectively, Figure 1c).
Figure 1. Growth of Epiphyllum azuris flowers grown under various soil molybdenum concentrations.
The data for the average plant molybdenum percent dry mass () showed high variance due to the inherent imprecision in the procedure (Table 1). However, the average molybdenum for the 10ppm, 30 ppm, and 100 ppm groups were significantly different from one another and increased with soil molybdenum concentration (p=0.009). The 100 ppm group had the highest average , which is approximately ten times higher than the in E. azuris plants grown under the 2 ppm control*. The difference between the 2 ppm and 10 ppm groups was not significantly different (p=0.14). The data for the average plant copper was likewise high in variance, with the 30 ppm and 100 ppm groups showing lower values than the 2 ppm and 10 ppm groups (Table 2). The 100 ppm group had the lowest average , which was approximately 60% that of the control.
Table 1. Molybdenum dry mass of Epiphyllum azuris plants grown in various concentrations of soil molybdenum for 18 days.
Table 2. Copper dry mass of Epiphyllum azuris plants grown in various concentrations of soil molybdenum for 18 days.
Overall, these results show that increasing the soil molybdenum concentration stimulates growth of E. azuris plants and molybdenum uptake. However, at high concentrations molybdenum is toxic to the plants.
Collapse radiation absorption from soil
To determine the effects of soil molybdenum on the ability of E. azuris to remove CR from soil, the soil in each plot was assessed to determine the CR concentration at regular intervals. The soil radiation level remained relatively stable until the 4-day mark, where the plants began growing flower buds. After that, the soil radiation level decreased quickly for all groups, with plots containing higher molybdenum concentrations showing faster decreases in CR content (Figure 3). All treatment plots contained considerably lower soil CR after 6 days compared to the control, but this difference began to decrease afterwards, with only the 30 ppm treatment group showing a significantly lower concentration than the 2 ppm control after 18 days, being (26% compared to control, p=0.04). The CR concentration in the 100 ppm plot decreased at the second fastest rate until it levelled off at about 8 days, roughly coinciding with the onset of the diseased state. These results suggest that a moderately higher soil molybdenum concentration can increase the bioremediative ability of E. azuris, although the benefit is mostly in reaching the peak CR absorption sooner while the peak itself is more resistant to change.
Figure 3. Soil collapse radiation concentration after treatment by Epiphyllum azuris flowers grown in various concentrations of soil molybdenum.
Transcriptomic analysis
To ascertain the mechanism behind the changes caused by differing soil molybdenum concentrations, we conducted RNA-seq analysis on root samples at 6- and 18-days post-planting. PCCs and gene clustering were used to identify trends in transcript abundance, with the results summarized in Figure 4. Soil molybdenum was observed to be correlated with both Res2 gene expression and genes associated with the RoCT-CpAT pathway. Notably, CpAT expression increased dramatically, with a log2 value of 2.8 in the 30 ppm group. Many genes associated with plant growth (such as those coding for auxins) were differentially expressed in the treatment groups compared to the control after 6 days, consistent with the observed increased rate of initial growth (Figure 5). However, the expression of these genes returned to normal by 18 days. As expected, genes coding for molybdenum uptake and processing were upregulated, while stress proteins related to acute metal toxicity such as phytochelatins were upregulated in the 30 ppm and 100 ppm groups (Figure 5). Overall, the expression patterns of the plants were similar to normal acute heavy metal toxicity responses, except the E. azuris plants exhibited upregulation of the RoCT-CpAT pathway, which is involved in CR uptake. These results suggest that the effect of soil molybdenum on E. azuris CR hyperaccumulation is based on upregulation of the RoCT-CpAT pathway.
Figure 4. Summary of transcriptomic analysis in Epiphyllum azuris plants grown in various concentrations of soil molybdenum.
Figure 5. Differential expression cluster analysis of Epiphyllum azuris plants grown in various concentrations of soil molybdenum.
Discussion
In our study, we showed that soil molybdenum concentration influences the CR hyperaccumulation capability of E. azuris flowers via activation of the RoCT-CpAT pathway. Peak hyperaccumulation was observed at around 30 ppm, with a 26% higher efficiency compared to the 2 ppm control. Higher concentrations were observed to cause severe acute heavy metal toxicity, suggesting an optimal point somewhere between 10 ppm and 100 ppm. It is possible that this toxicity is caused by copper deficiency caused by the increase in phytochelatin production, stimulated by the high molybdenum concentration*. Further studies including a mixed metal blend may increase the maximum tolerable concentration.
These results are consistent with the parameters of our previous operation in Kurilev. During the operation, the soil molybdenum was treated to attain a level of approximately 20–70 ppm. Notably, one of the teams accidentally added double the amount of molybdenum supplement to a section of soil, which resulted in the same pattern of plant wilting observed in the 100 ppm group of this study. However, it is important to recognize that the conclusions of our study may not be fully applicable to real-world scenarios. The growth parameters (i.e. temperature, rainfall) were controlled in our experiment, and would obviously vary greatly in practice. For example, rainfall may affect the soil structure and composition, which would affect the bioavailability of soil molybdenum to the plants*. Molybdenum availability is greatly reduced in soils with pH6.0, as well as soils with high concentrations of other transition metals*. Additionally, while 100 ppm soil molybdenum eventually resulted in considerable plant injury, they were still able to approach the maximal CR accumulation amount and were able to do so much faster than the control. This may be of interest to time-sensitive operations which would not last much past a week anyway*. For example, a past study has shown that there is an urgent need by crisis response teams for fast-acting purification kits for use after severe radiation storms*. Future research will need to study the effects of molybdenum in various environmental conditions, including using field plots, to determine the validity of our results.
Another factor to consider is the toxicity of high levels of soil molybdenum to plant and other life in the treated soil. Certain plants are unable to tolerate many of the concentrations of molybdenum used in this study*, and acute molybdenum toxicity in grazing animals commonly leads to copper deficiency*. Additionally, some studies suggest a link between chronic molybdenum exposure and the risk of esophageal cancer in humans, though the data are sparse*. It will be important to consider the rate at which the molybdenum will leach and diffuse out of the soil, both to ensure optimal concentrations are maintained throughout the operation, and to assess the risk of chronic exposure to the inhabitants of the area*. Heavy metals in the soil can be washed into local rivers and lakes via rainfall, where they are known to collect through sedimentation*. Despite these, the outcomes of such a scenario would not likely be worse than the alternative of not having sufficient CR purification*. This is reflected in the widespread repealing or disregard of many environmental protection laws in many countries, including the Neo-Soviet Union, after the Belian incident*.
Perhaps more exciting is the prospect of controlling the RoCT-CpAT pathway directly without the need to worry about soil molybdenum at all. It is unknown why exactly the RoCT-CpAT pathway is being activated, but it may be related to several enzymes and transcription factors which require Mo2+ as a cofactor*. Genetic engineering of the CpAT gene, such as the creation of a gene construct with an inducible and controllable regulatory element, may be an appealing area of further research. Previous work by Rosenthaal et al. found that controlling the Res2 gene was unable to increase the bioremediative ability of E. fluorencis, although it did increase the growth rate*. However, more work needs to be done to fully map the transcriptional changes that take place, as the transcriptomic analysis in our study was limited by resource considerations. Additionally, proteomic analysis would also help unveil the relevant mechanistic properties.
Conclusion
While Epiphyllum-based CR bioremediation techniques continue to gather considerable interest, there remains multiple issues with their application that must be solved before they can be adopted, including bioremediative efficiency. Our study reveals that soil molybdenum concentration plays an important role in said efficiency of E. Azuris plants, with a supranormal concentration of 10–100 ppm being associated with up to a 26% increase in efficiency. Additionally, we show that both the Res2 and RoCT-CpAT pathway are upregulated under such conditions. We hope our research will serve as the groundwork for further studies which examine the effect of soil molybdenum under field conditions, and attempt to manipulate the RoCT-CpAT pathway to discover a non-soil molybdenum dependent method to increase bioremediative efficiency.
Acknowledgements
We would like to thank the entirety of Griffin and Kryuger PMC for supporting us immensely throughout the study process. It would not be an understatement to claim that most of the authors would not exist today if it weren't for their help.
We would also like to thank Dr. Leone Michel of the Flora Institute of Bremen for generously providing us with the seeds that were used to establish Project Azure Shield, as well as the institute in general for their insights and advice during the experimental process.
Funding and conflicts of interest
This research was funded in part by the Neo-Soviet Union Natural Sciences Research Grant (NSRC), grant ID: 12095914.
SQS-006 "Kia", SQR-004 "Nyx", SFR-001 "Archangel", SQM-011 "Magic", SQK-009 "Krieg", SQM-014 "Axiom", SQF-021 "Sharps", and SQF-019 "Keter" were part of the renegade remnant of the now-defunct Sangvis Ferri Heavy Industries during a portion of this study. ARCTIC-3147 "Madeline" is currently part of Griffin and Kryuger PMC. There is active armed conflict between the Sangvis Ferri remnant and Griffin and Kryuger PMC. The authors have certified that this potential conflict of interest has not affected any part of the experimentation, manuscript preparation, or decision to publish.
Data availability statement
All data are available in our manuscript and the attached data file. Further inquiries should be directed towards the corresponding author.
References
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