Bioremediation of Uranium from Nuclear Waste Using Cyanobacteria

Bioremediation of uranium from nuclear waste using cyanobacteria Joseph Mathuthu, Naomi Dikeledi Mokhine, Babalola Olubukola & Ayangbenro Ayansina ,

Introduction Radioactive wastes are produced as a by-product of nuclear power generation (Vandana et al., 2021). The disposal of radioactive waste has become a severe environmental issue. The longer half-life of some of these radioactive wastes implies that it takes longer to reduce the radioactivity of a compound by half. Therefore tyhe disposal of such waste materials has become a dilemma for academics, policymakers, and power generation organizations (Vandana et al., 2021). Recent years have seen a rise in public and scientific concern around radioactive processing because to the extensive discharge of radionuclides into the environment and their mobility. Using microorganisms (bacteria, algae, and fungi) to harness the biogeochemical cycles of radioactive wastes is known as bioremediation.

Introduction Continued Bioremediation is the process of reducing contamination to a non-toxic or undetectable level using biological agents such as microorganisms, plants, or other living organisms. Bioremediation is cheap cost, eco-friendly, and successful remediation capabilities (Vandana et al., 2021). Lloyd and Renshaw stated that several novel species that extract uranium from solution have recently been discovered (Lloyd and Renshaw, 2005). The most common methods for uranium bioremediation are, e.g., enzymatic metal bioreduction of soluble U(VI) to sparingly soluble U(IV) (Kulkarni et al., 2013). Naturally occurring bacteria expressing phosphatases, such as Citrobacter sp., etc., are used for bioremediation of heavy metals like cadmium and uranium in acidic to neutral pH (Jacob et al., 2018). Additionally, the use of immobilization techniques on an industrial scale is promising (Jacob et al., 2018). In this research, cyanobacteria were isolated from flowing water and used in the removal of uranium from contaminated water.

Aim and Objectives Aim The aim of this project is to investigate the bioremediation of uranium from nuclear waste solution using cyanobacteria . Objectives: Specific objectives: Evaluate the uranium concentration in the nuclear waste solution using UV-VIS Spectrophotometry. Investigate the effectiveness of cyanobacteria in removing uranium from different concentrations of uranium solutions. Determine the optimum conditions (pH, temperature and moisture level) of uranium removal by cyanobacteria.

Methodology . Eight water samples were collected in 500 ml bottles from major water bodies with cyanobacteria (Adekola et al., 2015). Three uranium waste was collected in 50 ml bottles following the method by (Santos and Ladeira, 2011) from CARST Labs Thirty five water samples were collected from different boreholes in Mafikeng area of the the North West Province. BG-11 enrichment media was used for isolation of cyanobacteria as described by Jawaharraj et al. (2016) Residual uranium concentration was measured using the Ultra Violet Visible Spectroscopy (UV-VIS V-750)

DNA Sequencing The genomic DNA from each cyanobacterial isolate was extracted using the Zymo soil microbe s extraction kit (Zymo Research, USA) following the manufacturers instruction. Polymerase chain Reaction (PCR) gene amplification of the 16S rRNA was conducted using a reaction volume of 25 l containing (0.5 l each of both forward and reverse primer, 12.5 l PCR master mix, 1 l DNA template, 10.5 l nuclease-free water). A 1.5% agarose gel containing ethidium bromide was used to check the quality of the PCR products. Thereafter, PCR products was sequenced at Inqaba Biotechnology Laboratory, South Africa. The result of the sequence obtained was compared with other sequences in the NCBI GenBank data. Phylogenetic analysis was conducted using the MEGA X software package.

RESULTS AND DISCUSSION The results of this study are in agreement with other studies which show that cyanobacteria reduced uranium by an average of 60% for some samples. The majority of the isolates (80%) belong to the genus Anabaena.

Table 1: cyanobacterial isolates and their corresponding similar match from the NCBI genbank database Bacteria Group Bacteria Name Code NCBI genbank Similarity Percentage Consensus_J1 Anabaena cycadae JQ964322 95.96% PKGSAK6 Consensus_J2 Anabaena cycadae JQ964322 95.93% PKGSAK6 Consensus_J3 Anabaena cycadae JQ964322 95.94% PKGSAK6 Consensus_J4 Anabaena doliolum JX075259 90.52% GSPKAK3 Consensus_J5 Anabaena cycadae KF157402 88.58% PKGSAK6 Consensus_J6 Anabaena doliolum JQ964322 90.71% GSPKAK3 Consensus_J7 Anabaena doliolum JQ964322 91.11% GSPKAK3 Consensus_J8 Anabaena cycadae JQ964322 95.97% PKGSAK6 Consensus_J9 Anabaena cycadae JQ964322 95.97% PKGSAK6 Consensus_J11 Anabaena cycadae JQ964322 95.87% PKGSAK6

Discussions on bacteria isolation Discussions on bacteria isolation Of the 5 samples used in this study, S10K, SL4 and 53 are uranium waste samples while samples M20, M10 are water samples. Twelve (12) samples were analysed for residual uranium after introducing the bacteria. Six samples (J6, 3J, J10, J6J10 J10J15, J15) of bacteria were introduced to the same sample. Isolates J6, J3, J9, J2, J4, J1, J8, J7, and J11 were phylogenetically similar to Anabaena cicadae. Isolate J5 belonged to Microcystis aeruginosa.

Table 2: Samples uranium levels before and after introducing cynobacteria. Sample Before Introducing Cyanobacteria (mSv) After Introducing Cyanobacteria (mSv) 53 3.8 2.2 S10K 3.9 2.3 SL4 3.7 1.9 M10 4 1.3 M20 4 1.6

Results from M10 Results from M10 2.7 2.5 2.3 Uranium Concentration (mSv) 2.1 1.9 1.7 1.5 1.3 1.1 0.9 1 2 3 4 5 6 7 8 9 10 UV-VIS wavelegth (x 100 nm) M10 M10J6J10 WED M103J WED M10J6 WED M10J10 WED M10J10J15 WED M10J15 WED M10J6FRI M10J10FRI M10J6J10FRI M10J10J15FRI M10J15FRI M103JFRI Figure 1: Sample M10 Day 1 and Day 2 of 48 hr and 96 hrs respectively after introducing cyanobacteria.

Results on M20 Results on M20 2.9 2.7 2.5 2.3 Uranium Concentration 2.1 1.9 1.7 1.5 1.3 UV-VIS wavelegth (x 100 nm) 1.1 0.9 1 2 3 4 5 6 7 8 9 10 M20 M20 J10 WED M20 J10 J5 WED M20 3J WED M20 J6 WED M20 J6 J10 WED M20 J15 WED M20J6FRI M203JFRI M20J10J15FRI M20J6J10FRI M20J10FRI M20J15FRI Figure 2 Sample M20 Day 1 and Day 2 of 48 hr and 96 hrs respectively

Figure 3: Sample M20 Day 1 and Day 2 of 48 hr and 96 hrs respectively

Discussions on bioremediation of uranium Discussions on bioremediation of uranium In Figure 5 Sample M10J10 FRI was reduced the most, it had average percentage reduction of 65.9% . Sample M10J6J10 FRI was reduced the least. In Figure 6, Sample M20J15 FRI was reduced the most, it had average percentage reduction of 70% . Sample M203J FRI was reduced the least. Although other studies marine bacteria was studied, uranium was reduced with up to percentages of 90% (Banerjee et al., 2022) and this agrees with our study even though in our study fresh water bacteria was used and the reduction was just below 85%. Toxicity of the nuclear waste may have prevented the cyanobacterial species used in this study from growing at optimum condition and as a result their ability to remediate uranium was reduced. Anabaena dolionum outperformed the other chemical and biological alternatives that were examined.

Conclusion and Recommendations Anabaenadolionum outperformed the other chemical and biological alternatives that were examined. Anabaenadolionum produced the highest levels of reduction on the samples compared to other organisms ranging from 65% to 70%. Major findings of this study, is that two fresh water cyanobacteria, Anabena dolionum and Anabena cycade, have been discovered to possess a notable ability to sequester uranyl from water. Further research is recommended as this field is very promising and cost effective in dealing with waste and radioactive waste. Bacteria J6 reduced the uranium the most in the uranium samples but not necessarily in the water samples. For water samples M20 and M10, bacteria J10 and J15 reduced the uranium the most compared to other bacteria.

Conclusions and recommendation Conclusions and recommendation Uranium in water samples was reduced more than the uranium in uranium waste samples. This might be due to the harsh environment of the uranium waste samples, causing the bacteria in water samples to survive better and not die off (Banerjee et al., 2022). Anabaena doliolum , a cyanobacterium that was isolated from a river in Mafikeng, was shown to bioremdiate U at an ideal pH & Temperatures of 7.0pH and 25 C respectively. Anabena cycade, have been shown in this study to possess a notable ability to sequester uranyl (UIV) from water. Further research is recommended as this field is very promising and cost effective in dealing with waste and radiaoactive waste.

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