Mitigation of Arsenic Accumulation in Crop Plants Using Biofertilizer

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Hoang, Kyoung-Woong Kim This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2164650/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Mar, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted You are reading this latest preprint version Abstract Elevated levels of arsenic in crop plants have been found in various regions worldwide, especially where agricultural soils have been affected by arsenic-enriched aquifer and human activities including mining, smelting, pesticide application, and so forth. Given the highly toxic nature of arsenic, remediation should be carried out immediately to reduce this potentially toxic element transport from soil to crop plants. This study focused on the utilization of biofertilizer which is a combination of arsenic-accumulating microorganisms and adsorbent (carrier) in order to achieve high efficiency of arsenic immobilization and ability to apply in the field. Thirty-two bacterial strains were isolated from 9 soil samples collected from Dongjin and Duckum mining areas in Korea using nutrient medium amended with 2 mM sodium arsenite. Among isolates, strain DE12 identified as Bacillus megaterium exhibited the greatest arsenic accumulation capacity (0.236 mg/g dry biomass) and ability to resist up to 18 mM arsenite. Among three agricultural waste adsorbents studied, rice straw was proved to have higher adsorption capacity (0.104 mg/g) than rice husk and corn husk. Therefore, rice straw was chosen to be the carrier to form biofertilizer together with strain DE12. Inoculation of biofertilizer in soil showed reduction of arsenic content in edible part of lettuce, water spinach, and sweet basil by 17.5%, 34.1%, and 34,1%, respectively compared to control group. The use of biofertilizer may open up the potential application in the field for other food plants. arsenic accumulation bacteria biofertilizer Bacillus megaterium soil remediation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Grain and vegetables have long been recognized as important sources of food for the world’s population. Therefore, cultivating such plants on contaminated land can potentially lead to accumulation of toxicants including arsenic in edible parts of plants, subsequently leading to accumlation in human body. Unforturnately, numerous studies have reported elevated concentrations of total arsenic in crop plants worldwide due to high levels of arsenic in soil and irrigation water (Bhattacharya et al. 2010 ; Das et al. 2004 ; Hoang et al. 2021 ; Li et al. 2017 ; Nguyen et al. 2019 ). While the Chinese National limit for As is 0.5 mg/kg in vegetables (Clever and Jie 2014 ) with maximum level of total arsenic at 0.3 mg/kg in rice (Codex Alimentarius Commission 2014 ), arsenic contents from rice and vegetables samples from contaminated sites have exceeded these thresholds. Given the highly toxic nature of As, it is necessary to perform proper treatments immediately to reduce arsenic transport from soil and irrigation water to food chains. For soil remediation, biological methods are considered greener compared with chemical or physical technologies. However, they are less stable as they depend on microbial adaptability to the local environment. Recently, biofertilizer has received considarable attention because of its potential to provide better survival rate for inoculant microorganisms. Biofertilizer is defined as a fertilizer consisting of microorganisms with specific functions and organic materials (or carrier). Therefore, it has effects of both microorganisms and organic carrier (Sun et al. 2020 ). Biofertilizer has been long investigated for many purposes, mostly for enhancing nutrient supplement and plant biomass (Mukhtar et al. 2017 ; Naher et al. 2021 ; Sohaib et al. 2020 ). Several studies have also reported the use of biofertilizer for immobilization of heavy metals in soil to increase biomass and decrease toxicant such as Cd, Hg, Pb, Cr, Co and Ni concentrations in edible parts of lettuce and wheat compared with the control (Hassan et al. 2017 ; T. Wang et al. 2017 ). However, few such studies have been conducted about arsenic. There are several mechanims behind the way microorganisms interact with arsenic compounds. However, these bioactivities will eventually lead to either decrement or enhancement of As bioavailability in the environment. Various studies have been conducted to examine the role of specific microorganisms in protecting crops by making As less bioavailable in cultivating soil. For instance, inoculation of Trichoderman asperellum , an As-resistant fungal strain, can increase As availability in Chenzhou soils and As content in water spinach (Su et al. 2017 ). Meanwhile, Brevundimonas diminuta , an As-accumulating bacterial strain, can help reduce arsenic uptake in edible parts of rice plants and effectively sequester As in root systems (N. Singh et al. 2016 ). As-tolerant bacteria including Ralstonia eutropha, Rhizobium tropici , and Exiguobacterium aurantiacum can alleviate As and Cd contents in edible parts of Chinese cabbage and radish (X. Wang et al. 2017 ). However, microbial inoculants generally have a short shelf life as the population of microoganisms will soon decline after innoculation due to unpredictable abiotic stresses (such as pH, moisture, temperature, salinity, and elevated level of toxicants) and biotic stresses as they have to compete with indigenous micro-flora and protozoans for energy, carbon and nutrients (Siddiq et al. 2018 ). Therefore, the presence of carrier material plays very important role in microbial survival. In this study, agricultural wastes including rice husk, rice straw, and coconut husk were examined as carriers for biofertilizer. The main reason for that is because lignocellulosic fibres are porous and can swell in contact with water (Budd and Herrington 1989 ), meaning that they can provide habitats for inoculant bacteria and possess good moisture adsorption capacity. Besides, they are low-cost, renewable materials and available in adequate amounts. Furthermore, lignocellulosic materials have shown to be promising biosorbents of arsenic via main interactions including complexation, electrostatic attraction, ion exchange, and precipitation (Maia et al. 2021 ), which could help fortify the purpose of biofertilizer in this study. In addition, open burning is one of the most common waste straw mangement practices performed by farmers all over the world, leading to emission of lethal greenhouse gases (G. Singh et al. 2021 ). Therefore, utilization of agricultural waste can help reduce the environment burden. Objectives of this study were to screen for arsenite-resistant bacteria and suitable carrier for biofertilizer and to examine the potential applicability of biofertilizer in contaminated cultivating soil. The application of biofertilizer could be a useful strategy for immobilizing arsenic in soils and preventing accumulation of this element in crop plants. Materials And Methods Site description and sample collection Sampling sites for this study were arsenic contaminated mining areas in South Korea (Fig. 1 ). Dongjin Au-Ag-Cu mine is situated in Jinan-gun, Jeollabuk-do with such ore minerals as chalcopyrite, galena, sphalerite, arsenopyrite, and pyrite. Small-scale mining activities had been carried out in this area until 1965 (Lee et al. 1996 ). However; several studies have thus far proclaimed significant levels of heavy metal and metalloids in the vicinity due to mine waste (Na et al. 1997 ; Yoo et al. 2014 ). Duckum Au-Ag mine is located in Naju-si, Jeollanam-do. After it was abandoned, this mining area was left behind with 645160 m 3 of tailing and 225000 m 3 of waste rock piles, threatening the surrounding environment (Kim et al. 2002 ). Soil samples were collected and sealed in sterilized plastic bags and kept at 4 \(℃\) for until further analysis. For arsenic content analysis, soil samples were dried, sieved through 200-µm-mesh, and digested in aqua regia. The mixture was then centrifuged at 3000 rpm for 10 min and filtered through a 0.45-µm syringe filter before it was analyzed by Inductively coupled plasma - optical emission spectrometry (ICP-OES) (Ko et al. 2020 ). Arsenic levels in collected soil samples are shown in Table 1 . Table 1 Total arsenic concentrations in soil samples Sampling sites ID Coordinates Arsenic concentration (mg/kg) Dongjin DJ1 35.6812; 127.3581 18.8 DJ2 35.7078; 127.3569 38.6 DJ3 35.7090; 127.3568 52.8 DJ4 35.7102; 127.3558 269.0 Duckum DE1 34.9797; 126.5920 5.5 DE2,3 34.9794; 126.5942 8.6 DE4 34.9782; 126.5909 39.4 DE5 34.9785; 126.5910 45.08 Isolation of arsenite-resistant bacteria After 0.1 g of fresh soil sample was added to 100 mL saline solution, serial dilution was performed. Then 100 uL aliquot of each diluted sample was spread onto nutrient agar (peptone 5 g/L, NaCl 5 g/L, yeast extract 2 g/L, agar 15 g/L) amended with 2 mM of sodium arsenite. After incubating at 30 \(℃\) for 3 days, distinct colonies were selected based on morphology and subcultured on fresh media (Dey et al. 2016 ). Cellular morphology of each isolate was observed using a bright field microscope. In addition, screening of all isolates on blood agar plates (KisanBio) was performed to examine hemolytic activity. Evaluation of arsenite resistance and accumulation In order to evaluate arsenite resistivity, minimum inhibition concentrations (MIC) of all isolates were determined. MIC was determined as the lowest arsenite concentration that inhibited visible microbial growth. 1% of 24-hour grown culture of each isolated strain was inoculated into nutrient broth (peptone 5 g/L, NaCl 5 g/L, yeast extract 2 g/L) amended with different concentrations of sodium arsenite (2–20 mM) in a 96-well-plate. Bacterial growth was then tested through the presence of colony on a nutrient agar plate (Banerjee et al. 2011 ). To determine arsenite accumulation by bacterial cells, 1% of 24-hour grown culture of each isolate was inoculated into 5 mL nutrient broth in the presence of 2 mM sodium arsenite. Bacterial biomass was harvested by centrifugation (3000 rpm, 10 min) at room temperature and washed twice with saline solution. After the supernatant was carefully removed, cell pellets were then air dried for 2 days and dissolved in 200 µL of concentrated nitric acid for 2 days at room temperature (Kostal et al. 2004 ). Arsenic content in biomass was then determined by Inductively coupled plasma mass spectrometry (ICP-MS). Identification of selected bacterial strain Those strains capable of tolerating and accumulating arsenite were selected for identification by analyzing their 16 rRNA genes. PCR amplification of 16s rRNA gene fragments was done using 27F (5’-AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-TACCGTTACCTTGTTACGACTT-3’) primers. DNA fragments were sequenced with primers 785F 5’ (GGATTAGATACCCTGGTA-3’) and 907R (5’-CCGTCAATTAMTTTRAGTTT-3’). Nucleotide sequences were compared to sequence databases in the NCBI GenBank using nucleotide BLAST. Bacterial 16S rRNA gene sequences obtained from this study were aligned with other sequences from the database and phylogenetical analysis was made by neighbour-joining method with 1000 bootstrap replicates using MEGA 11 software. Physiological characteristics of bacterial colonies were examined using biochemical test kits (KisanBio). Different biochemical properties of bacterial isolates such as catalase activity (Cat), Voges-Proskauer test (VP), utilization of different carbon sources including arabinose (Ara), lactose (Lac), mannitol (Man), glucose (Glu) and decarboxylation of amino acid including ornithine (Orn), lysine (Lys), and arginine (Arg) were tested. Adsoprtion capacity of carrier Adsorbents (rice straw, rice husk, coconut husk) were washed thrice with DI water, dried, and passed through a 200 \(\mu\) M-mesh sieve. Then 10% adsorbents (w/v) were added to solution of 10 mg/L sodium arsenite (pH 6). These solutions were mixed at room temperature on a rotary shaker for 24 h. At the end of experiment, residual concentration in the supernatant liquid was analyzed by ICP-OES (Podder and Majumder 2015 ). Adsorption capacity of each carrier was calculated with the following equation: $${q}_{e}=\frac{\left({C}_{0}-{C}_{t}\right)V}{m}$$ where, \({q}_{e}\) : adsorption capacity of adsorbent (mg metal/g dry weight) \({C}_{0}\) : the initial concentration of arsenic in the solution (mg /L) \({C}_{t}\) : the final concentration of arsenic in the solution (mg /L) \(V\) : the volume of the metal solution (L) \(m\) : the dry weight of the adsorbent (g) Biofertilizer formation Selected adsorbent and bacteria strain were used to formulate biofertilizer. To become a carrier, adsorbent was washed thrice with deionized water, dried, ground, and sieved through 200 \(\mu\) m mesh before autoclaving twice. Then 40 mL of overnight grown culture of selected strain was added to 40 g of sterilized carrier material stored in an aseptic container and incubated at 30°C for 2 days. After incubation, biofertilizer portions in all containers were mixed altogether and kept in a sterilized plastic bag. Measurement of colony forming units (CFU) in formulated biofertilizer were made after 7 days of packaging. Observation of viability was carried out using the spread plate method and the number of colonies was calculated using the total plate count (CFU/ml) method (Hassan and Bano 2015 ). Plant cultivation and analysis Preparation of cultivating soil Contaminated soil was collected in Duckum mining area (34.978376; 126.590974). Cultivating soil was attained by mixing contaminated soil and purchased fertile soil at a ratio of 1:1. Arsenic concentration of mixed soil analyzed by ICP-OES was 64.09 mg/kg. Mixed soil was then homogenized with coconut peat at a ratio of 3:1 to improve water retention capacity and increase available nutrient content. The mixture was then air-dried at room temperature for 7 days for cultivation. Biofertilizer was then inoculated in cultivating soil with particular ratios to obtain same bacterial concentration in soil. Mixtures were then left in shade to settle for 2 days. Treatment comprised fertile soil (FS) (non-contaminated soil), contaminated soil (CS), and biofertilizer + contaminated soil (BF-CS). For the BF-CS group, 10% (w/w) biofertilizer was added into contaminated soil before cultivation. Seed preparation and germination Seeds of lettuce ( Lactuca sativa ), water spinach ( Ipomoea aquatica ), and sweet basil ( Ocimum basilicum ) were surface-sterilized with 95% ethanol for 2 min followed by shaking with 10% chlorox for 2–3 min (Hassan and Bano 2015 ). Pot experiment was carried out in a greenhouse of School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, South Korea. Disinfected seeds were germinated in cultivating soil for one week. After germination, uniform seedlings were thinned to four plants per pot with triplicates for each treatment. All plants were cultivated under ambient temperature and irrigated without leachate with a light and darkness regime. Pots were placed randomly in the greenhouse and moved every day to ensure that they received the same light. As water spinach is a semi-aquatic vegetable, submerged condition was applied only to this plant type. Lettuce and water spinach plants were harvest after 40 days while sweet basil plants were collected after 60 days of cultivation. Arsenic analysis Collected crop samples were rinsed thrice with deionized water to remove soil particles or dust on the surface and separated into roots and shoots (edible parts). All samples were then air-dried at room temperature for 4 days and homogenized using a mortar and pestle. Then 0.05 g of each sample was digested with 3 mL of concentrated nitric acid (65%) at 220 \(℃\) for 20 min in a microwave digestion system. After digestion, the extract was filled up with deionized water to 10 mL and the solution was filtered through a 0.45-µm syringe filter. Total arsenic and heavy metal concentrations were then detected by ICP-MS (Park and Choi 2013 ). Data quality control and statistical analysis All samples were digested in triplicate. Accuracy of the digestion method was evaluated by the analysis of Standard Reference Materials (SRM 2711a, SRM 1573a) in the same way as treated sample. Recovery rates for SRM 2711a and SRM 1573a were 104% and 91%, respectively. Calculation and statistical data analyses were performed using Microsoft Excel 2016. The sampling map was built with QGIS (version 3.24) software. Figures were created with OriginPro 2021. Results And Discussion Isolation of arsenite-resistant bacteria Thirty-two bacterial strains were isolated from one surface soil sample (0–20 cm depth) (DE2) and eight subsurface soil (below 20 cm depth) samples collected from Dongjin and Duckum mining areas. Most colonies had a white to milky color. The rest had a yellow to orange color. Glistening, water-like colonies were observed for half of isolates. This is a character of a bacterial colony that produces slime or a capsule. These microbial surfaces can function as a protection layer for cells against dewatering or toxic substances (Sheng et al. 2010 ). Gram-staining results varied among isolates. Spore formation was found for DJ24, DJ25, DJ33, DE12, DE13, and DE14. Cellular shapes of all strains were mostly bacilli except for DJ31, DE16, DE42, and DE52 which were cocci (as shown in Fig. 2 ). Of all bacterial isolates, DJ36, DJ44, DE17, DE21, DE41, and DE51 were found to have hemolysis activities. Thus, they were not used for further experiments. Evaluation of arsenite resistance and accumulation by bacterial cells To examining potential arsenite resistivity of isolates, minimum inhibition concentrations (MICs) were determined and recorded in Table 2 . Overall, bacterial strains isolated from Dongjin mining site exhibited greater abilities to tolerate arsenite over those from contaminated areas in Duckum, which might be due to higher concentrations of total arsenic in general in Dongjin soil samples. Among these strains, DJ22 and DJ41 thrived the best in the presence of arsenite with ability to sustain their growth in the presence of over 20 mM of sodium arsenite. Interestingly, these two strains had several similar characteristics: smooth, yellow colonies; Gram-positive, and cellular size of \(0.5\times 0.6 \mu m\) . Strains DJ25 and DE12 also exhibited excellent resistance ability, with MIC values of 20 mM and 18 mM, respectively. Gram-staining of these two strains results showed the presence of spore formation, an attribute of several bacteria to resist extreme external conditions. Microscopic observation of several strains revealed capsular material surrounding these bacteria, which could be one of the reasons for their high resistance to arsenic. All strains that were able to tolerate above 4 mM of arsenite were used for further experiments. Table 2 Minimum inhibition concentrations (MICs) of arsenite for bacterial isolates Isolates DJ11 DJ21 DJ22 DJ23 DJ24 DJ25 DJ26 DJ28 DJ31 MIC (mM) 10 4 > 20 4 6 20 16 16 4 Isolates DJ33 DJ35 DJ37 DJ41 DJ42 DE11 DE12 DE13 DE14 MIC (mM) 4 12 10 > 20 8 12 18 4 4 Isolates DE16 DE24 DE31 DE33 DE34 DE42 DE52 DE53 MIC (mM) 14 6 6 6 6 4 16 6 Arsenite accumulating abilities of isolated strains are illustrated in Fig. 3 . Isolate DJ24 showed the greatest arsenic accumulation ability (0.247 mg/g dry biomass). Results of MICs for arsenite and arsenic accumulation suggested that bacterial response toward arsenite varied among isolates. Particularly, strains DJ22 and DJ41 with the highest resistant abilities were among the bacteria that accumulated the lowest amount of arsenic, which indicated that these strains had mechanisms other than accumulation to survive in the presence of arsenite. Isolate DE12, which possessed high arsenic resistant and accumulating abilities (0.236 mg/g dry biomass) was then selected for species identification. Identification of selected bacterial strains Based on results of 16S rRNA gene sequence and phylogenetic analysis, DE12 was designated as a species beling to Bacillus genera and shared a high similarity with Bacillus megaterium as shown in Fig. 4 . Detailed characterization of DE12 was also performed to help assure its identification and to explore its bioctechnologically important traits. Table 3 displays results for particular biochemical tests of strain DE12 and other bacteria identified as Bacillus megaterium from previous studies. It also describes biochemical characteristics of Bacillus megaterium in Bergey’s Manual (Whitman 2009 ). Physiological characteristics of strain DE12 were found to be similar to those of other bacteria listed. In addition, DE12 was found to be a Gram-positive bacterium with an average size of \(1.5 \times 2.3 \mu m\) . It was able to form spores, a dormant form of bacteria to resist physical and chemical influences. There has been no evidence of pathogenic traits of this species. Moreover, Bacillus megaterium has been claimed to be of economic importance due to its ability to produce several crucial enzymes (Sura and Hiremath 2019 ), Therefore, strain DE12 was chosen to be the bacterium for biofertilizer formation. Table 3 Physiological characteristics of strain DE12 and Bacillus megaterium in other studies. Reference Size (µm) Spore Gram Cat Ara Lac Man Glu VP Orn Lys Arg This study (DE12) 1.5 \(\times\) 2.3 + + + + + + + - - - - Sura and Hiremath 2019 \(\ge\) 0.9 \(\times\) (2–4) + + + + + + - Andriani et al. 2017 + + + + + - Bergey’s Manual (1.2–1.5) \(\times\) (2–5) + + + + + + + - - - - +: positive reaction; -: negative reaction Adsorption capacity of carrier Although agricultural wastes, which are carbonaceous materials, are usually used for biochar formation to enhance the adsorption capacity of pollutants, production cost of biochar could make it harder for biofertilizer to be applied in a large scale in paddy field. In this study, experiment was conducted only on raw materials. Rice straw was chosen for its high adsorption capacity to be the carrier to form biofertilizer together with strain DE12. Coconut husk exhibited the lowest adsorption capacity at 0.016 mg/kg among studied materials. Adsorption capacity of coconut husk in this study was lower than that of coconut fiber in the study of Nashine and Tembhurkar ( 2016 ) at over 0.03 mg As (III)/ g adsorbent with the same adsorbent dose (10 g/L) but higher initial arsenite concentration (0.380 mg/L) (Nashine and Tembhurkar 2016 ). Rice straw was found to have higher adsorption capacity (0.104 ± 0.008 mg/g) than rice husk (0.066 ± 0.009 mg/g). This could be explained by differences in chemical compositions between rice straw and rice husk. Lignin and silica are two components presented in significant amounts in these parts of rice plant. Both of these components can reduce the binding between accessible functional groups on rice husks and rice straw surfaces and adsorbate ions/molecules (Chakraborty et al. 2011 ). According to a previous study, lignin content in rice husk amounted up to 22.5%, which was significantly higher than the lignin content in rice straw (13.5%) (Rosado et al. 2021 ). Silica was also found to be more abundant in rice husk (93%) than in rice straw (82%) (Damanhuri et al. 2020 ). Therefore, the presence of high content of lignin and silica can hinder rice husk from becoming a potential adsorbent material. Biofertilizer Formation And Plant Analysis Strain DE12 and rice straw were selected as components of biofertilizer. Biofertilizer is generally recommended to be free from contaminants and should contain a microbial load of approximately 10 7 cells per gram carrier to achieve the best support for plant growth (Sethi and Adhikary 2012 ). For measurements made at 7 days after incubation, the formulation contained \(1.5 \times {10}^{9}\) CFU/g, which could be applied to the field. This result is also similar to previous studies on biofertilizer preparation containing \(13 \times {10}^{8}\) CFU/g and \(19 \times {10}^{8}\) CFU/g of Pseudomonas moraviensis and B. cereus , respectively (Hassan and Bano 2015 ). For the BF-CS group, 10 % (w/w) biofertilozer wa introduced to mixed soil and left in shade for 2 days before being used for cultivation. Plant samples were collected after particular cultivation time. The difference observed between length of plants in cultivated group was not significant. The study of Egodawatta et al. ( 2018 ) has shown a poor relationship between both length and biomass (in root and shoot) of water spinach and exposure to As concentration under 100 mg/kg in natural contaminated soils. Codling ( 2014 ) has also reported an insignificant reduction in lettuce yield when lettuce is cultivated in naturally As-contaminated soil (133–153 mg As/kg). Overall, As was accumulated the most in roots of all three cultivated plant types. High arsenic and metal concentrations are usually expected in roots because of their direct contact with these elements. Concentrations of toxic elements are generally decreased sharply from roots to shoots and stems. Kumwimba et al. ( 2013 ) have studied As adsorption in five lettuce cultivars and found that the average As concentration in roots was 12–31 times higher than that in shoots (Kumwimba et al. 2013 ), which is quite similar to the present study (9–25 times for lettuce). These results indicate a role of roots in restricting the transport of these compounds from soil to aerial parts of these plants. In addition, cultivating leafy vegetables in soil with elevated concentrations of potentially toxic elements can result in alarming levels of these compounds in shoot and root samples. Contents of arsenic in CS group were 6–18 times higher than those in FS group which was cultivated in non-contaminated soil. In addition, China has released National Food Safety Standard of Maximum Levels of Contaminants in Foods and set limit for As at 0.5 mg/kg in vegetables (Clever and Jie 2014 ). Results showed that concentrations of these elements in all samples cultivated in CS group (cultivated in contaminated soil) exceeded this standard. Therefore, these vegetables are not recommended for fresh consumption. Interestingly, As contents in edible parts of sweet basil harvested after 60 days were comparable to those in water spinach, which was cultivated in contaminated soil for 40 days. Amounts of arsenic compounds accumulated in plant biomasses of these two plants were also noticeably higher than those in lettuce. This could be due to the promoted arsenic accumulation under flooding condition of water spinach. In this study, arsenic contents in shoots and roots of CS group were 5.08 mg/kg and 84.73 mg/kg, respectively. These levels were quite similar to aresnic content in shoots of water spinach at 5.18 mg/kg dry weight when cultivated in a flooding area with soil containing 72.1 mgAs/kg in a previous study of Liao et al. (2021). The study also reported that arsenic content in water spinach cultured in aerobic condition with the same soil was 1.57 mg/kg. This result demonstrated that higher As concentration in pore water of flooding soil could increase the accumulation of As in roots and shoots compared to that in aerobic soil. In this study, arsenic contents in BF-CS were lower than those in CS, meaning that inoculation of biofertilizer could reduce arsenic accumulation in studied plants. However, the efficiency of biofertilizer in alleviating arsenic accumulation was different among cultivating plants. For lettuce, As concentrations in shoots and roots of BF-CS group (1.29 mg/kg and 27.86 mg/kg) were slightly lower than those of the CS group (1.56 mg/kg and 38.18 mg/kg). Accordingly, the application of biofertilizer appeared to bring little benefit in alleviating As accumulation lettuce. For sweet basil, inoculation of biofertilizer reduced 34.1% of As uptake in edible parts of sweet basil, from 5.16 mg/kg (CS group) to 3.40 mg/kg (BF-CS group). The presence of biofertilizer in water spinach shared the same efficiency as sweet basil, reducing arsenic content in shoots from 5.07 mg/kg (CS group) to 3.34 mg/kg (BF-CS group). Although the introduction of biofertilizer into cultivating soil did diminish up to one third of arsenic level in edible parts of these plants, it was unable to meet the standard for safe consumption. Therefore, screening for more desirable carriers for better bacterial survival support and arsenic adsorption should be carried out to raise the efficiency of biofertilizer. Conclusion Strain DE12, which was identified as Bacillus megaterium , showed great abilities to resist and accumulate arsenite among strains isolated from arsenic contaminated soil in South Korea. Results of adsorption capacitiy determination revealed that rice straw was able to adsorb the highest amount of arsenite than other studied adsorbent. The combination of this carrier and strain DE12 was able to reduce arsenic contents in edible parts of lettuce, water spinach, and sweet basil compared to the control group. Application of this biofertilizer has potential to produce biofertilizer that can help reduce arsenic accumulation in other crop plants cultivated in paddy soil with elevated arsenic levels. However, further studies are needed to determine interactions among bacteria, carrier, and As species in both submerged and aerobic soil as well as to improve the performance of carrier. In addition, investigations in cultivation especially in field trials are required to evaluate the efficiency of the biofertilizer. 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K., & Jolley, D. F. (2018). As and Sb are more labile and toxic to water spinach (Ipomoea aquatica) in recently contaminated soils than historically co-contaminated soils. Environmental Science: Processes and Impacts , 20 (5), 833–844. https://doi.org/10.1039/c8em00057c Hassan, T. U., & Bano, A. (2015). Role of carrier-based biofertilizer in reclamation of saline soil and wheat growth. Archives of Agronomy and Soil Science , 61 (12), 1719–1731. https://doi.org/10.1080/03650340.2015.1036045 Hassan, T. U., Bano, A., & Naz, I. (2017). Alleviation of heavy metals toxicity by the application of plant growth promoting rhizobacteria and effects on wheat grown in saline sodic field. International Journal of Phytoremediation , 19 (6), 522–529. https://doi.org/10.1080/15226514.2016.1267696 Hoang, A. T. P., Prinpreecha, N., & Kim, K. (2021). Influence of Mining Activities on Arsenic Concentration in Rice in Asia : A Review, 1–14. Kim, J.-Y., Kim, K.-W., Lee, J.-U., Lee, J.-S., & Cook, J. (2002). 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Assessment of two carrier materials for phosphate solubilizing biofertilizers and their effect on growth of wheat (Triticum aestivum L.). Microbiological Research , 205 (August), 107–117. https://doi.org/10.1016/j.micres.2017.08.011 Na, C.-K., Lee, M.-S., & Chung, J.-I. (1997). Pollution of Heavy Metals in Paddy Soils Around the Downstream Area of Abandoned Metal Mine and Efficiency of Reversed Soil Method as Its Remediation. Econ. Environ. Geol. , 30 (2), 123–135. Naher, U. A., Biswas, J. C., Maniruzzaman, M., Khan, F. H., Sarkar, M. I. U., Jahan, A., et al. (2021). Bio-Organic Fertilizer: A Green Technology to Reduce Synthetic N and P Fertilizer for Rice Production. Frontiers in Plant Science , 12 (March), 1–14. https://doi.org/10.3389/fpls.2021.602052 Nashine, A. L., & Tembhurkar, A. R. (2016). Equilibrium, kinetic and thermodynamic studies for adsorption of As(III) on coconut (Cocos nucifera L.) fiber. Journal of Environmental Chemical Engineering , 4 (3), 3267–3273. https://doi.org/10.1016/j.jece.2016.06.005 Nguyen, T. P. M., Nguyen, T. P. T., Bui, T. H., & Nguyen, T. H. (2019). Concentration of arsenic in groundwater, vegetables, human hair and nails in mining site in the Northern Thai Nguyen province, Vietnam: human exposure and risks assessment. Human and Ecological Risk Assessment: An International Journal , 25 (3), 602–613. https://doi.org/10.1080/10807039.2018.1483189 Park, J. H., & Choi, K. K. (2013). Risk assessment of Abandoned Jukjeon metal mine in South Korea following the Korean Guidelines. Human and Ecological Risk Assessment , 19 , 754–766. https://doi.org/10.1080/10807039.2012.708274 Podder, M. S., & Majumder, C. B. (2015). SD/MnFe2O4 composite, a biosorbent for As(III) and As(V) removal from wastewater: Optimization and isotherm study. Journal of Molecular Liquids , 212 , 382–404. https://doi.org/10.1016/j.molliq.2015.09.011 Rosado, M. J., Rencoret, J., Marques, G., Gutiérrez, A., & del Río, J. C. (2021). Structural Characteristics of the Guaiacyl-Rich Lignins From Rice (Oryza sativa L.) Husks and Straw. Frontiers in Plant Science , 12 (February), 1–17. https://doi.org/10.3389/fpls.2021.640475 Sethi, S. K., & Adhikary, S. P. (2012). Cost effective pilot scale production of biofertilizer using Rhizobium and Azotobacter. African Journal of Biotechnology , 11 (70), 13490–13493. https://doi.org/10.5897/ajbx11.012 Sheng, G. P., Yu, H. Q., & Li, X. Y. (2010). Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnology Advances , 28 (6), 882–894. https://doi.org/10.1016/j.biotechadv.2010.08.001 Siddiq, S., Saleem, U., Ahmad, K., Anayat, A., Affan, Q. M., Anwar, M. F., et al. (2018). Comparison of Conventional and Non-Conventional Carriers for Bacterial Survival and Plant Growth. International Journal of Agriculture Innovations and Research , 6 (4), 126–129. https://www.researchgate.net/publication/324829669 Singh, G., Gupta, M. K., Chaurasiya, S., Sharma, V. S., & Pimenov, D. Y. (2021). Rice straw burning: a review on its global prevalence and the sustainable alternatives for its effective mitigation. Environmental Science and Pollution Research , 28 (25), 32125–32155. https://doi.org/10.1007/s11356-021-14163-3 Singh, N., Marwa, N., Mishra, J., Verma, P. C., Rathaur, S., & Singh, N. (2016). Brevundimonas diminuta mediated alleviation of arsenic toxicity and plant growth promotion in Oryza sativa L . Ecotoxicology and Environmental Safety , 125 , 25–34. https://doi.org/10.1016/j.ecoenv.2015.11.020 Sohaib, M., Zahir, Z. A., Khan, M. Y., Ans, M., Asghar, H. N., Yasin, S., & Al-Barakah, F. N. I. (2020). Comparative evaluation of different carrier-based multi-strain bacterial formulations to mitigate the salt stress in wheat. Saudi Journal of Biological Sciences , 27 (3), 777–787. https://doi.org/10.1016/j.sjbs.2019.12.034 Su, S., Zeng, X., Bai, L., Williams, P. N., Wang, Y., Zhang, L., & Wu, C. (2017). Inoculating chlamydospores of Trichoderma asperellum SM-12F1 changes arsenic availability and enzyme activity in soils and improves water spinach growth. Chemosphere , 175 (12), 497–504. https://doi.org/10.1016/j.chemosphere.2017.02.048 Sun, B., Gu, L., Bao, L., Zhang, S., Wei, Y., Bai, Z., et al. (2020). Application of biofertilizer containing Bacillus subtilis reduced the nitrogen loss in agricultural soil. Soil Biology and Biochemistry , 148 (June), 107911. https://doi.org/10.1016/j.soilbio.2020.107911 Sura, N. K., & Hiremath, L. (2019). Isolation of Bacillus megaterium and its Commercial Importance. International Journal of ChemTech Research , 12 (04), 30–36. https://doi.org/10.20902/ijctr.2019.120405 Wang, T., Sun, H., Ren, X., Li, B., & Mao, H. (2017). Evaluation of biochars from different stock materials as carriers of bacterial strain for remediation of heavy metal-contaminated soil. Scientific Reports , 7 (1), 1–10. https://doi.org/10.1038/s41598-017-12503-3 Wang, X., Nie, Z., He, L., Wang, Q., & Sheng, X. (2017). Isolation of As-tolerant bacteria and their potentials of reducing As and Cd accumulation of edible tissues of vegetables in metal(loid)-contaminated soils. Science of the Total Environment , 579 , 179–189. https://doi.org/10.1016/j.scitotenv.2016.10.239 Whitman, W. B. (Ed.). (2009). Bergey’s Manual of Systematic Bacteriology. Volume three: The Firmicutes . Bergey’s Manual of Systematic Bacteriology (second.). New York, NY: Springer New York. https://doi.org/10.1007/978-0-387-68489-5 Yoo, E. J., Lee, J. A., Park, J. S., Lee, K., Lee, W. S., Han, J. S., & Choi, J. W. (2014). Tracing lead pollution sources in abandoned mine areas using stable Pb isotope ratios. Environmental Monitoring and Assessment , 186 (2), 781–789. https://doi.org/10.1007/s10661-013-3416-8 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 18 Mar, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2164650","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":144324687,"identity":"858abcd6-cfda-4bc7-8f43-1a9328632ac7","order_by":0,"name":"Anh T.P. Hoang","email":"","orcid":"","institution":"Gwangju Institute of Science and Technology (GIST)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Anh","middleName":"T.P.","lastName":"Hoang","suffix":""},{"id":144324688,"identity":"518ed9a8-79a8-4fd9-90b2-4cd3e699b79f","order_by":1,"name":"Kyoung-Woong Kim","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBACCQY2hgMMFQw8UH4CsVrOALWwkaKFgbENyCJai+SMtMRDN+fVysjPb2D88IMhLZ+gFmmJtAOHc7cd5zE4xsAs2cOQY9lASIucdHoDUMsxHgOgw6QZGCoMCNoC0TLnGI98GwPzb6K0SEuDHNZQw8NwjIENaEsOYS2S858lHM45dgDol8Q2yx6DNMJaJM4cM/6cU1NnL998+PCNHxXJhLVAwWEgZmxgYCBaAwNDHfFKR8EoGAWjYOQBAPe+N4fA0/hqAAAAAElFTkSuQmCC","orcid":"","institution":"Gwangju Institute of Science and Technology (GIST)","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Kyoung-Woong","middleName":"","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2022-10-14 05:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2164650/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2164650/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-32825-w","type":"published","date":"2024-03-18T08:14:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":27945405,"identity":"36c7530a-0967-47d5-97db-c74e44a464ed","added_by":"auto","created_at":"2022-10-18 16:32:40","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":589063,"visible":true,"origin":"","legend":"\u003cp\u003eLocations of soil sampling sites from Dongjin (left) and Duckum (right) mining areas.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/6ecf6d5dd3d45454804ee235.png"},{"id":27945409,"identity":"dd3e513a-d964-4b0a-ab8a-77c4d0df104b","added_by":"auto","created_at":"2022-10-18 16:32:41","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2030199,"visible":true,"origin":"","legend":"\u003cp\u003eCellular morphology of isolated strains under bright-field microscope (1000x magnification)\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/ee0ac4a23b999cefce796644.png"},{"id":27945406,"identity":"3c046e6c-0476-4c04-9e78-11a6d3e0b56f","added_by":"auto","created_at":"2022-10-18 16:32:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30123,"visible":true,"origin":"","legend":"\u003cp\u003eArsenic accumulating abilities of isolated strains.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/a18a2b16d274a3632d408357.png"},{"id":27945408,"identity":"897fdb54-6ca2-4100-8bb5-df47a5a998b4","added_by":"auto","created_at":"2022-10-18 16:32:41","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":392286,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of strain DE12 constructed based on 16s rDNA sequences\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/780b778f7cb4c0e68e6b1b46.png"},{"id":27945407,"identity":"c7b431b3-2f2c-4dcc-9e7c-1cbf6493f00d","added_by":"auto","created_at":"2022-10-18 16:32:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":32507,"visible":true,"origin":"","legend":"\u003cp\u003eTotal arsenic concentrations in shoots and roots of studied plants\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/8d6683444f67a4b906024ce8.png"},{"id":53149499,"identity":"675e3258-2f4b-405c-94fe-c83116df21c6","added_by":"auto","created_at":"2024-03-21 08:14:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3100025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2164650/v1/9173e0f4-2166-420b-bef1-9a2a9d271ede.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitigation of Arsenic Accumulation in Crop Plants Using Biofertilizer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGrain and vegetables have long been recognized as important sources of food for the world\u0026rsquo;s population. Therefore, cultivating such plants on contaminated land can potentially lead to accumulation of toxicants including arsenic in edible parts of plants, subsequently leading to accumlation in human body. Unforturnately, numerous studies have reported elevated concentrations of total arsenic in crop plants worldwide due to high levels of arsenic in soil and irrigation water (Bhattacharya et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Das et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Hoang et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Nguyen et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). While the Chinese National limit for As is 0.5 mg/kg in vegetables (Clever and Jie \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) with maximum level of total arsenic at 0.3 mg/kg in rice (Codex Alimentarius Commission \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), arsenic contents from rice and vegetables samples from contaminated sites have exceeded these thresholds. Given the highly toxic nature of As, it is necessary to perform proper treatments immediately to reduce arsenic transport from soil and irrigation water to food chains.\u003c/p\u003e \u003cp\u003eFor soil remediation, biological methods are considered greener compared with chemical or physical technologies. However, they are less stable as they depend on microbial adaptability to the local environment. Recently, biofertilizer has received considarable attention because of its potential to provide better survival rate for inoculant microorganisms. Biofertilizer is defined as a fertilizer consisting of microorganisms with specific functions and organic materials (or carrier). Therefore, it has effects of both microorganisms and organic carrier (Sun et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Biofertilizer has been long investigated for many purposes, mostly for enhancing nutrient supplement and plant biomass (Mukhtar et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Naher et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Sohaib et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Several studies have also reported the use of biofertilizer for immobilization of heavy metals in soil to increase biomass and decrease toxicant such as Cd, Hg, Pb, Cr, Co and Ni concentrations in edible parts of lettuce and wheat compared with the control (Hassan et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; T. Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, few such studies have been conducted about arsenic.\u003c/p\u003e \u003cp\u003eThere are several mechanims behind the way microorganisms interact with arsenic compounds. However, these bioactivities will eventually lead to either decrement or enhancement of As bioavailability in the environment. Various studies have been conducted to examine the role of specific microorganisms in protecting crops by making As less bioavailable in cultivating soil. For instance, inoculation of \u003cem\u003eTrichoderman asperellum\u003c/em\u003e, an As-resistant fungal strain, can increase As availability in Chenzhou soils and As content in water spinach (Su et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Meanwhile, \u003cem\u003eBrevundimonas diminuta\u003c/em\u003e, an As-accumulating bacterial strain, can help reduce arsenic uptake in edible parts of rice plants and effectively sequester As in root systems (N. Singh et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). As-tolerant bacteria including \u003cem\u003eRalstonia eutropha, Rhizobium tropici\u003c/em\u003e, and \u003cem\u003eExiguobacterium aurantiacum\u003c/em\u003e can alleviate As and Cd contents in edible parts of Chinese cabbage and radish (X. Wang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, microbial inoculants generally have a short shelf life as the population of microoganisms will soon decline after innoculation due to unpredictable abiotic stresses (such as pH, moisture, temperature, salinity, and elevated level of toxicants) and biotic stresses as they have to compete with indigenous micro-flora and protozoans for energy, carbon and nutrients (Siddiq et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Therefore, the presence of carrier material plays very important role in microbial survival. In this study, agricultural wastes including rice husk, rice straw, and coconut husk were examined as carriers for biofertilizer. The main reason for that is because lignocellulosic fibres are porous and can swell in contact with water (Budd and Herrington \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1989\u003c/span\u003e), meaning that they can provide habitats for inoculant bacteria and possess good moisture adsorption capacity. Besides, they are low-cost, renewable materials and available in adequate amounts. Furthermore, lignocellulosic materials have shown to be promising biosorbents of arsenic via main interactions including complexation, electrostatic attraction, ion exchange, and precipitation (Maia et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which could help fortify the purpose of biofertilizer in this study. In addition, open burning is one of the most common waste straw mangement practices performed by farmers all over the world, leading to emission of lethal greenhouse gases (G. Singh et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Therefore, utilization of agricultural waste can help reduce the environment burden.\u003c/p\u003e \u003cp\u003eObjectives of this study were to screen for arsenite-resistant bacteria and suitable carrier for biofertilizer and to examine the potential applicability of biofertilizer in contaminated cultivating soil. The application of biofertilizer could be a useful strategy for immobilizing arsenic in soils and preventing accumulation of this element in crop plants.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003eSite description and sample collection\u003c/h2\u003e\n \u003cp\u003eSampling sites for this study were arsenic contaminated mining areas in South Korea (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Dongjin Au-Ag-Cu mine is situated in Jinan-gun, Jeollabuk-do with such ore minerals as chalcopyrite, galena, sphalerite, arsenopyrite, and pyrite. Small-scale mining activities had been carried out in this area until 1965 (Lee et al. \u003cspan class=\"CitationRef\"\u003e1996\u003c/span\u003e). However; several studies have thus far proclaimed significant levels of heavy metal and metalloids in the vicinity due to mine waste (Na et al. \u003cspan class=\"CitationRef\"\u003e1997\u003c/span\u003e; Yoo et al. \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Duckum Au-Ag mine is located in Naju-si, Jeollanam-do. After it was abandoned, this mining area was left behind with 645160 m\u003csup\u003e3\u003c/sup\u003e of tailing and 225000 m\u003csup\u003e3\u003c/sup\u003e of waste rock piles, threatening the surrounding environment (Kim et al. \u003cspan class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eSoil samples were collected and sealed in sterilized plastic bags and kept at 4\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for until further analysis. For arsenic content analysis, soil samples were dried, sieved through 200-\u0026micro;m-mesh, and digested in aqua regia. The mixture was then centrifuged at 3000 rpm for 10 min and filtered through a 0.45-\u0026micro;m syringe filter before it was analyzed by Inductively coupled plasma - optical emission spectrometry (ICP-OES) (Ko et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Arsenic levels in collected soil samples are shown in Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\n \u003ctable border=\"1\" id=\"Tab1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eTotal arsenic concentrations in soil samples\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSampling sites\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eID\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCoordinates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eArsenic concentration (mg/kg)\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eDongjin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.6812; 127.3581\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.7078; 127.3569\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e38.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.7090; 127.3568\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e52.8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e35.7102; 127.3558\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e269.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" rowspan=\"4\"\u003e\n \u003cp\u003eDuckum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.9797; 126.5920\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE2,3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.9794; 126.5942\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.9782; 126.5909\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e39.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e34.9785; 126.5910\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e45.08\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003c/p\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003eIsolation of arsenite-resistant bacteria\u003c/h2\u003e\n \u003cp\u003eAfter 0.1 g of fresh soil sample was added to 100 mL saline solution, serial dilution was performed. Then 100 uL aliquot of each diluted sample was spread onto nutrient agar (peptone 5 g/L, NaCl 5 g/L, yeast extract 2 g/L, agar 15 g/L) amended with 2 mM of sodium arsenite. After incubating at 30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for 3 days, distinct colonies were selected based on morphology and subcultured on fresh media (Dey et al. \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Cellular morphology of each isolate was observed using a bright field microscope. In addition, screening of all isolates on blood agar plates (KisanBio) was performed to examine hemolytic activity.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003eEvaluation of arsenite resistance and accumulation\u003c/h2\u003e\n \u003cp\u003eIn order to evaluate arsenite resistivity, minimum inhibition concentrations (MIC) of all isolates were determined. MIC was determined as the lowest arsenite concentration that inhibited visible microbial growth. 1% of 24-hour grown culture of each isolated strain was inoculated into nutrient broth (peptone 5 g/L, NaCl 5 g/L, yeast extract 2 g/L) amended with different concentrations of sodium arsenite (2\u0026ndash;20 mM) in a 96-well-plate. Bacterial growth was then tested through the presence of colony on a nutrient agar plate (Banerjee et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eTo determine arsenite accumulation by bacterial cells, 1% of 24-hour grown culture of each isolate was inoculated into 5 mL nutrient broth in the presence of 2 mM sodium arsenite. Bacterial biomass was harvested by centrifugation (3000 rpm, 10 min) at room temperature and washed twice with saline solution. After the supernatant was carefully removed, cell pellets were then air dried for 2 days and dissolved in 200 \u0026micro;L of concentrated nitric acid for 2 days at room temperature (Kostal et al. \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e). Arsenic content in biomass was then determined by Inductively coupled plasma mass spectrometry (ICP-MS).\u003c/p\u003e\n \u003cdiv class=\"Section3\" id=\"Sec6\"\u003e\n \u003ch2\u003eIdentification of selected bacterial strain\u003c/h2\u003e\n \u003cp\u003eThose strains capable of tolerating and accumulating arsenite were selected for identification by analyzing their 16 rRNA genes. PCR amplification of 16s rRNA gene fragments was done using 27F (5\u0026rsquo;-AGAGTTTGATCMTGGCTCAG-3\u0026rsquo;) and 1492R (5\u0026rsquo;-TACCGTTACCTTGTTACGACTT-3\u0026rsquo;) primers. DNA fragments were sequenced with primers 785F 5\u0026rsquo; (GGATTAGATACCCTGGTA-3\u0026rsquo;) and 907R (5\u0026rsquo;-CCGTCAATTAMTTTRAGTTT-3\u0026rsquo;). Nucleotide sequences were compared to sequence databases in the NCBI GenBank using nucleotide BLAST. Bacterial 16S rRNA gene sequences obtained from this study were aligned with other sequences from the database and phylogenetical analysis was made by neighbour-joining method with 1000 bootstrap replicates using MEGA 11 software.\u003c/p\u003e\n \u003cp\u003ePhysiological characteristics of bacterial colonies were examined using biochemical test kits (KisanBio). Different biochemical properties of bacterial isolates such as catalase activity (Cat), Voges-Proskauer test (VP), utilization of different carbon sources including arabinose (Ara), lactose (Lac), mannitol (Man), glucose (Glu) and decarboxylation of amino acid including ornithine (Orn), lysine (Lys), and arginine (Arg) were tested.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec7\"\u003e\n \u003ch2\u003eAdsoprtion capacity of carrier\u003c/h2\u003e\n \u003cp\u003eAdsorbents (rice straw, rice husk, coconut husk) were washed thrice with DI water, dried, and passed through a 200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eM-mesh sieve. Then 10% adsorbents (w/v) were added to solution of 10 mg/L sodium arsenite (pH 6). These solutions were mixed at room temperature on a rotary shaker for 24 h. At the end of experiment, residual concentration in the supernatant liquid was analyzed by ICP-OES (Podder and Majumder \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Adsorption capacity of each carrier was calculated with the following equation:\u003c/p\u003e\n \u003cdiv class=\"Equation\" id=\"Equa\"\u003e\n \u003cdiv class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e$${q}_{e}=\\frac{\\left({C}_{0}-{C}_{t}\\right)V}{m}$$\u003c/div\u003e\n \u003c/div\u003e\n \u003cp\u003ewhere,\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\({q}_{e}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e : adsorption capacity of adsorbent (mg metal/g dry weight)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\({C}_{0}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e : the initial concentration of arsenic in the solution (mg /L)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\({C}_{t}\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e : the final concentration of arsenic in the solution (mg /L)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(V\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e : the volume of the metal solution (L)\u003c/p\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u0026nbsp;\u003cspan class=\"mathinline\"\u003e\\(m\\)\u003c/span\u003e\u0026nbsp;\u003c/span\u003e : the dry weight of the adsorbent (g)\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003eBiofertilizer formation\u003c/h2\u003e\n \u003cp\u003eSelected adsorbent and bacteria strain were used to formulate biofertilizer. To become a carrier, adsorbent was washed thrice with deionized water, dried, ground, and sieved through 200 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003em mesh before autoclaving twice. Then 40 mL of overnight grown culture of selected strain was added to 40 g of sterilized carrier material stored in an aseptic container and incubated at 30\u0026deg;C for 2 days. After incubation, biofertilizer portions in all containers were mixed altogether and kept in a sterilized plastic bag. Measurement of colony forming units (CFU) in formulated biofertilizer were made after 7 days of packaging. Observation of viability was carried out using the spread plate method and the number of colonies was calculated using the total plate count (CFU/ml) method (Hassan and Bano \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003ePlant cultivation and analysis\u003c/h2\u003e\n \u003cdiv class=\"Section3\" id=\"Sec10\"\u003e\n \u003ch2\u003ePreparation of cultivating soil\u003c/h2\u003e\n \u003cp\u003eContaminated soil was collected in Duckum mining area (34.978376; 126.590974). Cultivating soil was attained by mixing contaminated soil and purchased fertile soil at a ratio of 1:1. Arsenic concentration of mixed soil analyzed by ICP-OES was 64.09 mg/kg. Mixed soil was then homogenized with coconut peat at a ratio of 3:1 to improve water retention capacity and increase available nutrient content. The mixture was then air-dried at room temperature for 7 days for cultivation. Biofertilizer was then inoculated in cultivating soil with particular ratios to obtain same bacterial concentration in soil. Mixtures were then left in shade to settle for 2 days. Treatment comprised fertile soil (FS) (non-contaminated soil), contaminated soil (CS), and biofertilizer\u0026thinsp;+\u0026thinsp;contaminated soil (BF-CS). For the BF-CS group, 10% (w/w) biofertilizer was added into contaminated soil before cultivation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec11\"\u003e\n \u003ch2\u003eSeed preparation and germination\u003c/h2\u003e\n \u003cp\u003eSeeds of lettuce (\u003cem\u003eLactuca sativa\u003c/em\u003e), water spinach (\u003cem\u003eIpomoea aquatica\u003c/em\u003e), and sweet basil (\u003cem\u003eOcimum basilicum\u003c/em\u003e) were surface-sterilized with 95% ethanol for 2 min followed by shaking with 10% chlorox for 2\u0026ndash;3 min (Hassan and Bano \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Pot experiment was carried out in a greenhouse of School of Earth Sciences and Environmental Engineering, Gwangju Institute of Science and Technology, South Korea. Disinfected seeds were germinated in cultivating soil for one week. After germination, uniform seedlings were thinned to four plants per pot with triplicates for each treatment. All plants were cultivated under ambient temperature and irrigated without leachate with a light and darkness regime. Pots were placed randomly in the greenhouse and moved every day to ensure that they received the same light. As water spinach is a semi-aquatic vegetable, submerged condition was applied only to this plant type. Lettuce and water spinach plants were harvest after 40 days while sweet basil plants were collected after 60 days of cultivation.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec12\"\u003e\n \u003ch2\u003eArsenic analysis\u003c/h2\u003e\n \u003cp\u003eCollected crop samples were rinsed thrice with deionized water to remove soil particles or dust on the surface and separated into roots and shoots (edible parts). All samples were then air-dried at room temperature for 4 days and homogenized using a mortar and pestle. Then 0.05 g of each sample was digested with 3 mL of concentrated nitric acid (65%) at 220\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(℃\\)\u003c/span\u003e\u003c/span\u003e for 20 min in a microwave digestion system. After digestion, the extract was filled up with deionized water to 10 mL and the solution was filtered through a 0.45-\u0026micro;m syringe filter. Total arsenic and heavy metal concentrations were then detected by ICP-MS (Park and Choi \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec13\"\u003e\n \u003ch2\u003eData quality control and statistical analysis\u003c/h2\u003e\n \u003cp\u003eAll samples were digested in triplicate. Accuracy of the digestion method was evaluated by the analysis of Standard Reference Materials (SRM 2711a, SRM 1573a) in the same way as treated sample. Recovery rates for SRM 2711a and SRM 1573a were 104% and 91%, respectively. Calculation and statistical data analyses were performed using Microsoft Excel 2016. The sampling map was built with QGIS (version 3.24) software. Figures were created with OriginPro 2021.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"Results And Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003eIsolation of arsenite-resistant bacteria\u003c/h2\u003e\n \u003cp\u003eThirty-two bacterial strains were isolated from one surface soil sample (0\u0026ndash;20 cm depth) (DE2) and eight subsurface soil (below 20 cm depth) samples collected from Dongjin and Duckum mining areas. Most colonies had a white to milky color. The rest had a yellow to orange color. Glistening, water-like colonies were observed for half of isolates. This is a character of a bacterial colony that produces slime or a capsule. These microbial surfaces can function as a protection layer for cells against dewatering or toxic substances (Sheng et al. \u003cspan class=\"CitationRef\"\u003e2010\u003c/span\u003e). Gram-staining results varied among isolates. Spore formation was found for DJ24, DJ25, DJ33, DE12, DE13, and DE14. Cellular shapes of all strains were mostly bacilli except for DJ31, DE16, DE42, and DE52 which were cocci (as shown in Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). Of all bacterial isolates, DJ36, DJ44, DE17, DE21, DE41, and DE51 were found to have hemolysis activities. Thus, they were not used for further experiments.\u003c/p\u003e\n \u003cdiv class=\"Section3\" id=\"Sec16\"\u003e\n \u003ch2\u003eEvaluation of arsenite resistance and accumulation by bacterial cells\u003c/h2\u003e\n \u003cp\u003eTo examining potential arsenite resistivity of isolates, minimum inhibition concentrations (MICs) were determined and recorded in Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. Overall, bacterial strains isolated from Dongjin mining site exhibited greater abilities to tolerate arsenite over those from contaminated areas in Duckum, which might be due to higher concentrations of total arsenic in general in Dongjin soil samples. Among these strains, DJ22 and DJ41 thrived the best in the presence of arsenite with ability to sustain their growth in the presence of over 20 mM of sodium arsenite. Interestingly, these two strains had several similar characteristics: smooth, yellow colonies; Gram-positive, and cellular size of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(0.5\\times 0.6 \\mu m\\)\u003c/span\u003e\u003c/span\u003e. Strains DJ25 and DE12 also exhibited excellent resistance ability, with MIC values of 20 mM and 18 mM, respectively. Gram-staining of these two strains results showed the presence of spore formation, an attribute of several bacteria to resist extreme external conditions. Microscopic observation of several strains revealed capsular material surrounding these bacteria, which could be one of the reasons for their high resistance to arsenic. All strains that were able to tolerate above 4 mM of arsenite were used for further experiments.\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab2\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eMinimum inhibition concentrations (MICs) of arsenite for bacterial isolates\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eIsolates\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ11\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ21\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ22\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ23\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ24\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ25\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ26\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ28\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eDJ31\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC (mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ37\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDJ42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC (mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026gt;\u0026thinsp;20\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e12\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eIsolates\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE42\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eDE53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003eMIC (mM)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003eArsenite accumulating abilities of isolated strains are illustrated in Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e. Isolate DJ24 showed the greatest arsenic accumulation ability (0.247 mg/g dry biomass). Results of MICs for arsenite and arsenic accumulation suggested that bacterial response toward arsenite varied among isolates. Particularly, strains DJ22 and DJ41 with the highest resistant abilities were among the bacteria that accumulated the lowest amount of arsenic, which indicated that these strains had mechanisms other than accumulation to survive in the presence of arsenite. Isolate DE12, which possessed high arsenic resistant and accumulating abilities (0.236 mg/g dry biomass) was then selected for species identification.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec17\"\u003e\n \u003ch2\u003eIdentification of selected bacterial strains\u003c/h2\u003e\n \u003cp\u003eBased on results of 16S rRNA gene sequence and phylogenetic analysis, DE12 was designated as a species beling to \u003cem\u003eBacillus\u003c/em\u003e genera and shared a high similarity with \u003cem\u003eBacillus megaterium\u003c/em\u003e as shown in Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e. Detailed characterization of DE12 was also performed to help assure its identification and to explore its bioctechnologically important traits. Table \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e displays results for particular biochemical tests of strain DE12 and other bacteria identified as \u003cem\u003eBacillus megaterium\u003c/em\u003e from previous studies. It also describes biochemical characteristics of \u003cem\u003eBacillus megaterium\u003c/em\u003e in Bergey\u0026rsquo;s Manual (Whitman \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). Physiological characteristics of strain DE12 were found to be similar to those of other bacteria listed. In addition, DE12 was found to be a Gram-positive bacterium with an average size of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(1.5 \\times 2.3 \\mu m\\)\u003c/span\u003e\u003c/span\u003e. It was able to form spores, a dormant form of bacteria to resist physical and chemical influences. There has been no evidence of pathogenic traits of this species. Moreover, \u003cem\u003eBacillus megaterium\u003c/em\u003e has been claimed to be of economic importance due to its ability to produce several crucial enzymes (Sura and Hiremath \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e), Therefore, strain DE12 was chosen to be the bacterium for biofertilizer formation.\u003c/p\u003e\u0026nbsp;\u003ctable border=\"1\" id=\"Tab3\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003ePhysiological characteristics of strain DE12 and \u003cem\u003eBacillus megaterium\u003c/em\u003e in other studies.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eReference\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSize (\u0026micro;m)\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eSpore\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGram\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eCat\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eAra\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLac\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eMan\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eGlu\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eVP\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eOrn\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eLys\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eArg\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThis study (DE12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1.5 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e 2.3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eSura and Hiremath \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\ge\\)\u003c/span\u003e\u003c/span\u003e0.9 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e (2\u0026ndash;4)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAndriani et al. \u003cspan class=\"CitationRef\"\u003e2017\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eBergey\u0026rsquo;s Manual\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e(1.2\u0026ndash;1.5) \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e (2\u0026ndash;5)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u003cstrong\u003e-\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"13\"\u003e+: positive reaction; -: negative reaction\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"Section3\" id=\"Sec18\"\u003e\n \u003ch2\u003eAdsorption capacity of carrier\u003c/h2\u003e\n \u003cp\u003eAlthough agricultural wastes, which are carbonaceous materials, are usually used for biochar formation to enhance the adsorption capacity of pollutants, production cost of biochar could make it harder for biofertilizer to be applied in a large scale in paddy field. In this study, experiment was conducted only on raw materials. Rice straw was chosen for its high adsorption capacity to be the carrier to form biofertilizer together with strain DE12. Coconut husk exhibited the lowest adsorption capacity at 0.016 mg/kg among studied materials. Adsorption capacity of coconut husk in this study was lower than that of coconut fiber in the study of Nashine and Tembhurkar (\u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) at over 0.03 mg As (III)/ g adsorbent with the same adsorbent dose (10 g/L) but higher initial arsenite concentration (0.380 mg/L) (Nashine and Tembhurkar \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Rice straw was found to have higher adsorption capacity (0.104\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008 mg/g) than rice husk (0.066\u0026thinsp;\u0026plusmn;\u0026thinsp;0.009 mg/g). This could be explained by differences in chemical compositions between rice straw and rice husk. Lignin and silica are two components presented in significant amounts in these parts of rice plant. Both of these components can reduce the binding between accessible functional groups on rice husks and rice straw surfaces and adsorbate ions/molecules (Chakraborty et al. \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). According to a previous study, lignin content in rice husk amounted up to 22.5%, which was significantly higher than the lignin content in rice straw (13.5%) (Rosado et al. \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). Silica was also found to be more abundant in rice husk (93%) than in rice straw (82%) (Damanhuri et al. \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Therefore, the presence of high content of lignin and silica can hinder rice husk from becoming a potential adsorbent material.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003ch3\u003eBiofertilizer Formation And Plant Analysis\u003c/h3\u003e\n\u003cp\u003eStrain DE12 and rice straw were selected as components of biofertilizer. Biofertilizer is generally recommended to be free from contaminants and should contain a microbial load of approximately 10\u003csup\u003e7\u003c/sup\u003e cells per gram carrier to achieve the best support for plant growth (Sethi and Adhikary \u003cspan class=\"CitationRef\"\u003e2012\u003c/span\u003e). For measurements made at 7 days after incubation, the formulation contained \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(1.5 \\times {10}^{9}\\)\u003c/span\u003e\u003c/span\u003e CFU/g, which could be applied to the field. This result is also similar to previous studies on biofertilizer preparation containing \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(13 \\times {10}^{8}\\)\u003c/span\u003e\u003c/span\u003e CFU/g and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(19 \\times {10}^{8}\\)\u003c/span\u003e\u003c/span\u003e CFU/g of \u003cem\u003ePseudomonas moraviensis\u003c/em\u003e and \u003cem\u003eB. cereus\u003c/em\u003e, respectively (Hassan and Bano \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). For the BF-CS group, 10 % (w/w) biofertilozer wa introduced to mixed soil and left in shade for 2 days before being used for cultivation.\u003c/p\u003e\n\u003cp\u003ePlant samples were collected after particular cultivation time. The difference observed between length of plants in cultivated group was not significant. The study of Egodawatta et al. (\u003cspan class=\"CitationRef\"\u003e2018\u003c/span\u003e) has shown a poor relationship between both length and biomass (in root and shoot) of water spinach and exposure to As concentration under 100 mg/kg in natural contaminated soils. Codling (\u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e) has also reported an insignificant reduction in lettuce yield when lettuce is cultivated in naturally As-contaminated soil (133\u0026ndash;153 mg As/kg).\u003c/p\u003e\n\u003cp\u003eOverall, As was accumulated the most in roots of all three cultivated plant types. High arsenic and metal concentrations are usually expected in roots because of their direct contact with these elements. Concentrations of toxic elements are generally decreased sharply from roots to shoots and stems. Kumwimba et al. (\u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e) have studied As adsorption in five lettuce cultivars and found that the average As concentration in roots was 12\u0026ndash;31 times higher than that in shoots (Kumwimba et al. \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e), which is quite similar to the present study (9\u0026ndash;25 times for lettuce). These results indicate a role of roots in restricting the transport of these compounds from soil to aerial parts of these plants.\u003c/p\u003e\n\u003cp\u003eIn addition, cultivating leafy vegetables in soil with elevated concentrations of potentially toxic elements can result in alarming levels of these compounds in shoot and root samples. Contents of arsenic in CS group were 6\u0026ndash;18 times higher than those in FS group which was cultivated in non-contaminated soil. In addition, China has released National Food Safety Standard of Maximum Levels of Contaminants in Foods and set limit for As at 0.5 mg/kg in vegetables (Clever and Jie \u003cspan class=\"CitationRef\"\u003e2014\u003c/span\u003e). Results showed that concentrations of these elements in all samples cultivated in CS group (cultivated in contaminated soil) exceeded this standard. Therefore, these vegetables are not recommended for fresh consumption.\u003c/p\u003e\n\u003cp\u003eInterestingly, As contents in edible parts of sweet basil harvested after 60 days were comparable to those in water spinach, which was cultivated in contaminated soil for 40 days. Amounts of arsenic compounds accumulated in plant biomasses of these two plants were also noticeably higher than those in lettuce. This could be due to the promoted arsenic accumulation under flooding condition of water spinach. In this study, arsenic contents in shoots and roots of CS group were 5.08 mg/kg and 84.73 mg/kg, respectively. These levels were quite similar to aresnic content in shoots of water spinach at 5.18 mg/kg dry weight when cultivated in a flooding area with soil containing 72.1 mgAs/kg in a previous study of Liao et \u003cem\u003eal.\u003c/em\u003e (2021). The study also reported that arsenic content in water spinach cultured in aerobic condition with the same soil was 1.57 mg/kg. This result demonstrated that higher As concentration in pore water of flooding soil could increase the accumulation of As in roots and shoots compared to that in aerobic soil.\u003c/p\u003e\n\u003cp\u003eIn this study, arsenic contents in BF-CS were lower than those in CS, meaning that inoculation of biofertilizer could reduce arsenic accumulation in studied plants. However, the efficiency of biofertilizer in alleviating arsenic accumulation was different among cultivating plants. For lettuce, As concentrations in shoots and roots of BF-CS group (1.29 mg/kg and 27.86 mg/kg) were slightly lower than those of the CS group (1.56 mg/kg and 38.18 mg/kg). Accordingly, the application of biofertilizer appeared to bring little benefit in alleviating As accumulation lettuce. For sweet basil, inoculation of biofertilizer reduced 34.1% of As uptake in edible parts of sweet basil, from 5.16 mg/kg (CS group) to 3.40 mg/kg (BF-CS group). The presence of biofertilizer in water spinach shared the same efficiency as sweet basil, reducing arsenic content in shoots from 5.07 mg/kg (CS group) to 3.34 mg/kg (BF-CS group). Although the introduction of biofertilizer into cultivating soil did diminish up to one third of arsenic level in edible parts of these plants, it was unable to meet the standard for safe consumption. Therefore, screening for more desirable carriers for better bacterial survival support and arsenic adsorption should be carried out to raise the efficiency of biofertilizer.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eStrain DE12, which was identified as \u003cem\u003eBacillus megaterium\u003c/em\u003e, showed great abilities to resist and accumulate arsenite among strains isolated from arsenic contaminated soil in South Korea. Results of adsorption capacitiy determination revealed that rice straw was able to adsorb the highest amount of arsenite than other studied adsorbent. The combination of this carrier and strain DE12 was able to reduce arsenic contents in edible parts of lettuce, water spinach, and sweet basil compared to the control group. Application of this biofertilizer has potential to produce biofertilizer that can help reduce arsenic accumulation in other crop plants cultivated in paddy soil with elevated arsenic levels. However, further studies are needed to determine interactions among bacteria, carrier, and As species in both submerged and aerobic soil as well as to improve the performance of carrier. In addition, investigations in cultivation especially in field trials are required to evaluate the efficiency of the biofertilizer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgement\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2021R1A2C1094272).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAndriani, Y., Rochima, E., Safitri, R., \u0026amp; Rahayuningsih, S. R. (2017). 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Tracing lead pollution sources in abandoned mine areas using stable Pb isotope ratios. \u003cem\u003eEnvironmental Monitoring and Assessment\u003c/em\u003e, \u003cem\u003e186\u003c/em\u003e(2), 781\u0026ndash;789. https://doi.org/10.1007/s10661-013-3416-8\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"arsenic, accumulation, bacteria, biofertilizer, Bacillus megaterium, soil remediation","lastPublishedDoi":"10.21203/rs.3.rs-2164650/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2164650/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElevated levels of arsenic in crop plants have been found in various regions worldwide, especially where agricultural soils have been affected by arsenic-enriched aquifer and human activities including mining, smelting, pesticide application, and so forth. Given the highly toxic nature of arsenic, remediation should be carried out immediately to reduce this potentially toxic element transport from soil to crop plants. This study focused on the utilization of biofertilizer which is a combination of arsenic-accumulating microorganisms and adsorbent (carrier) in order to achieve high efficiency of arsenic immobilization and ability to apply in the field. Thirty-two bacterial strains were isolated from 9 soil samples collected from Dongjin and Duckum mining areas in Korea using nutrient medium amended with 2 mM sodium arsenite. Among isolates, strain DE12 identified as \u003cem\u003eBacillus megaterium\u003c/em\u003e exhibited the greatest arsenic accumulation capacity (0.236 mg/g dry biomass) and ability to resist up to 18 mM arsenite. Among three agricultural waste adsorbents studied, rice straw was proved to have higher adsorption capacity (0.104 mg/g) than rice husk and corn husk. Therefore, rice straw was chosen to be the carrier to form biofertilizer together with strain DE12. Inoculation of biofertilizer in soil showed reduction of arsenic content in edible part of lettuce, water spinach, and sweet basil by 17.5%, 34.1%, and 34,1%, respectively compared to control group. The use of biofertilizer may open up the potential application in the field for other food plants.\u003c/p\u003e","manuscriptTitle":"Mitigation of Arsenic Accumulation in Crop Plants Using Biofertilizer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-10-18 16:32:38","doi":"10.21203/rs.3.rs-2164650/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e5573258-df3d-4a4d-8246-147f8f8cfd31","owner":[],"postedDate":"October 18th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-03-21T08:14:08+00:00","versionOfRecord":{"articleIdentity":"rs-2164650","link":"https://doi.org/10.1007/s11356-024-32825-w","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-03-18 08:14:08","publishedOnDateReadable":"March 18th, 2024"},"versionCreatedAt":"2022-10-18 16:32:38","video":"","vorDoi":"10.1007/s11356-024-32825-w","vorDoiUrl":"https://doi.org/10.1007/s11356-024-32825-w","workflowStages":[]},"version":"v1","identity":"rs-2164650","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2164650","identity":"rs-2164650","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}