Identification of sulfur-oxidizing bacteria from fishponds and their performance to remove hydrogen sulfide under aquarium conditions

Identification of sulfur-oxidizing bacteria from fishponds and their performance to remove hydrogen sulfide under aquarium conditions | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification of sulfur-oxidizing bacteria from fishponds and their performance to remove hydrogen sulfide under aquarium conditions Rana Dashtbin, Nemat Mahmoudi, Hossein Besharati Kollayeh, Blažo Lalevic This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2830694/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Hydrogen sulfide is a highly toxic gas that causes many economic losses in aquaculture ponds. The application of sulfur-oxidizing bacteria (SOB) to remove hydrogen sulfide is an eco-friendly approach. This study aimed to isolate and identify the most efficient SOBs from the sediment of warm-water fish farms. Enrichment and isolation were performed in three different culture media (Starkey, Postgate, and H-3) based on both mineral and organic carbon. Overall, 27 isolates (14 autotrophic and 13 heterotrophic isolates) were purified based on colony and cell morphology differences. Initial screening was performed based on pH decrease. For final screening, the isolates were assessed based on their efficacy in thiosulfate oxidation and the sulfate production on Starkey liquid medium. Among isolated strains, 3 strains of Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) that belonged to the genus Thiobacillus thioparus (identified by 16s rRNA) showed the highest ability in thiosulfate oxidation (413.21, 1362.50, and 4188.03 mg l ‒1 for 14 days) and the highest sulfate production (3350, 2075, and 1600 mg l ‒1 ). In the final phase, the performance of these strains under aquarium conditions showed that Iran 1 and Iran 2 had the highest ability in sulfur oxidation. In conclusion, Iran 1 and 2 strains can be used as effective SOB to remove hydrogen sulfide in fish farms. It is very important to evaluate strains in an appropriate strategy using a combination of different criteria to ensure optimal performance of SOB in farm conditions. Biofertilizer Hydrogen sulfide Sulfur oxidizing bacteria Sustainable aquaculture Thiobacillus thioparus fishpond Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Hydrogen sulfide is a colorless and highly toxic gas that causes many economic losses in aquaculture ponds (Lahav et al. 2004 ). In earthen fishponds, external inputs like feed pellets, fish feces, and fertilizers increase the amount of organic matter compared to the natural environment (Abraham et al. 2004 ). In these conditions, sulfate-reducing bacteria use sulfate as the terminal electron acceptor for the degradation of organic compounds, resulting in the production of H 2 S (Muyzer and Stams 2008 ). H 2 S concentration depends on pH, temperature, and salinity (Datta 2012 ). As the pH of the pond water decreases during the night, the toxicity of hydrogen sulfide increases (Tucker and D’Abramo 2008 ). H 2 S causes problems such as fish mortality (Poulton et al. 2002 ; Lahav et al. 2004 ), a decrease in fish growth (Pillay 2008 ), and high oxygen consumption (Kang et al. 2016 ). Hydrogen sulfide can inhibit Cytochrome-c oxidase activity, the electron transport system, and aerobic respiration, which increases blood lactate and tissue hypoxia (Affonso et al. 2004 ). Field and laboratory studies have also pointed out that hydrogen sulfide is toxic to other aquatic organisms. In this regard, the research of Kuster et al. (2005) showed that the effective concentration of 50% (EC50) of hydrogen sulfide on Vibrio fischeri , Daphnia magna zooplankton, and Scenedesmus vacuolatus microalgae 9, 0.12, and 1.8 mg l ‒1 , respectively. In addition, studies have shown that low levels of hydrogen sulfide have a significant negative effect on nitrification performance (up to 50%) (Joye and Hollibaugh 1995 ; Crab et al. 2007 ). Aeration and water exchange (Howerton, 2001 ; Boyd and Tucker 2012 ), and the addition of lime and other chemical oxidants (hydrogen peroxide, iron hydroxide, potassium permanganate, and coal ash) (Poulton et al. 2002 ; Lahav et al. 2004 ; Asaoka et al. 2009 ; Sun et al. 2014 ) are commonly used to control hydrogen sulfide concentration in earthen fish ponds. Disadvantages of these methods include low efficiency, sudden fish losses, turbidity formation, high energy costs, production of secondary pollutants, need to obtain expensive chemicals, and public health concerns (Duranceau et al. 2010 ; Zhao et al. 2016). The application of sulfur-oxidizing microorganisms (bacteria, actinomyces, fungi, and yeast) to remove hydrogen sulfide in water is one of the new sustainable approaches (Zhao et al. 2016). Among microorganisms, phototrophic and chemotrophic SOB bacteria play a significant role in hydrogen sulfide oxidation (Behera et al. 2014 ). Photolitotrophic sulfide oxidation is carried out by green sulfur bacteria ( Chlorobium ) and purple sulfur bacteria ( Allochromatium and Chromatium ) (Ghosh and Dam 2009 ). Chemolitotrophic oxidation is carried out by colorless sulfur bacteria, such as Thiobacillus , Acidithiobacillus and Achromatuim groups (Behera et al. 2014 ). The previous studies on SOB have mostly focused on their role in different natural ecosystems (Behera et al. 2014 ; Behera et al. 2016 ). Moreover, the use of SOB for the removal of hydrogen sulfide from industrial wastewater has been extensively studied (Mirzaei et al. 2014 ). Despite the advantages of SOB, few studies have been conducted on SOB diversity and their potential for H 2 S oxidation in the warm-water fishpond (Krishnani et al. 2010 ). In addition, literature analyses have shown that one of the most reasons for the inefficiency of biofertilizers in farm conditions was that the components of culture media were not similar to the natural environment. In this regard, some researchers have stated that the strains selected solely by media (solid and liquid) did not provide acceptable results in natural conditions as biofertilizer (Bashan et al. 2013 ). As a solution, it has been recommended that the process of assessing the strains be complemented with additional tests in soil/sediment incubation. Therefore, the aim of this study was, (1) to isolate autochthonous chemolithotrophic SOB strains from fishponds in the different media, (2) to evaluate the ability of these bacteria to thiosulfate oxidation in solid and liquid media, (3) to molecular identification of efficient strains and (4) to evaluate their performance during thiosulfate oxidation in aquarium conditions containing sediment and water of fishpond. In addition, the isolation of autochthonous SOB is advised as they are well adapted to the natural climate of their habitat, thus the introduction of alien species and their adverse environmental effects are prevented. Materials And Methods Study sites and sample collection Sediment samples from six earthen warm-water fishponds were collected with a Van-Veen grab sampler (Hydro-Bios, Germany) located in northern Iran along the southern coast of the Caspian Sea over an area extending from 36° 28´- 36° 42´N and 52° 32´- 52° 59´E. The samples were stored at 4°C in sterile containers until further analysis. Common carp ( Cyprinus carpio ), grass carp ( Ctenopharyngodon idella ), bighead carp ( Hypophthalmichthys nobilis ), and silver carp ( Hypophthalmichthys molitrix ) are the most common species reared in these ponds. Silver carp is the most common species (55-60 percent of the total number of fish). Various animal-based fertilizers (manure and poultry manure) are largely (3.5-5 tons per hectare) used by farmers in the area. Farmers combine chemicals and manure and use them weekly on their land. To increase alkalinity and Ca supply agricultural lime is used continuously. Burnt lime is used when pests appear (especially parasites). The geographic coordinates of sampling points are shown in table 1. Table 1 here Culture media SOB were isolated from samples using three media, Starkey medium (Starkey 1934), Postgate medium (Postgate 1966), and H-3 medium (medium 81. DSMZ) based on both mineral carbon and organic carbon to isolate autotrophic and heterotrophic bacteria, respectively. In mineral-based media, sodium bicarbonate (H-3) and carbon dioxide (Postgate and Starkey) were used as a carbon source; in organic-based media, glucose (Postgate and Starkey) and ammonium ferric citrate (H-3) was used as the carbon source. Moreover, these media were prepared in liquid and solid (2% agar) forms. Thymol blue was used as a pH indicator. In this study, sodium thiosulfate was used as a source of sulfur for the SOBs isolation (Luo et al . 2013; Meyer et al . 2016; Zhoa et al . 2016). Table 2 shows the compositions of these media. Table 2 here Enrichment of SOBs Due to the low population of SOBs, enrichment culture was used to increase SOB counts. For this purpose, one gram of homogenized sediment samples was suspended in 100 ml of Starkey, Postgate, and H-3 liquid media (Makzum et al . 2017). Incubation was performed in a shaker incubator (Comecta, Spain) at 29 ± 1 °C, 100 rpm for 14 days or until turbidity developed, which is related to microbial growth. After incubation, 5% of suspensions were transferred to fresh broth media, and then series dilutions (from 10 -1 to 10 -5 ) were spread on Starkey, Postgate, and H-3 solid media. Inoculated plates were incubated at 29 ± 1 °C for 14 days (Zhou et al . 2007). Isolation and purification of SOBs In this stage, colonies were isolated based on morphological characteristics such as shape, size, color, edge, elevation, and consistency . Colonies that showed constant characteristics (in terms of gram staining and morphology) were considered pure colonies (Brenner et al . 2005). The purified isolates were kept in a solid Starkey medium in the refrigerator (4 °C). Based on the pH changes and the number of isolate Starkey medium was selected for the screening stage. Screening of SOBs In the initial screening, the isolates were assessed based on their efficacy to reduce pH in the medium. In this stage, one loop from each pure colony was inoculated into 50 ml Starkey broth medium with an initial pH value of 7; incubated at 29 ± 1 °C, 100 rpm for 14 days (Zhuang et al . 2017; Ranjit Kumar et al . 2018). The pH value of the medium was determined using a pH meter (Jenway3510, England). In addition, the pH decrease in the Starkey solid medium was measured with bromothymol blue as the color indicator of the pH changes. In the final screening, the selected strains from the previous stage were inoculated into a Starkey broth medium. A strain suspension amounted to 1ml (10 6 CFU ml -1 ) was transferred into 100 ml Starkey broth medium. In this stage, parameters such as the thiosulfate oxidation, sulfate production, and the density of bacteria were assessed (Ohba and Owa, 2005; Ranjit Kumar et al . 2018). Finally, the most efficient isolates were identified using 16S rRNA gene sequencing. Thiosulfate ions concentration was measured by standard iodometric titration after neutralization of the medium with 50% (v/v) acetic acid (Banciu et al . 2008). 10 ml of the sample was transferred to 50 ml Erlenmeyer flasks and then, 1 ml 1% starch solution was added as an indicator. Titration continued using a 10mM iodine solution until blue color appeared. The volume of iodine solution consumption was recorded and afterward consumption amount of thiosulfate ions was obtained. The value of sulfate ions was determined by turbidimetry method using the spectrophotometer (Hach DR1900, USA) at 450 nm wavelength. Moreover, the count of viable cells was reported with the standard plate count method. Genomic DNA extraction and amplification of 16S /rRNA gene from SOB For the extraction of the selected strain genome from gram-negative bacteria CinnaPure DNA kit (SinaClon CO., LTD) was used. To amplify the 16S rDNA gene 1 μl of 27F primer (AGAGTTTGATCMTGGCTCAG), 1 μl of 1492R primer (5΄-GGTTACCTTGTTACGACTT-3΄) (Weisburg et al . 1991), 1 μl of DNTPs, 3 μl of DNA sample, 1 μl of MgCl 2 , and 0.6 μl of Taq DNA polymerase were used. The PCR product was electrophoresed on a 1% agarose gel (Dena Gene Tajhiz, Iran) to ensure the correct amplification of the 16S rRNA gene. The PCR product obtained after purification by the column kit method was used for sequencing using the Sanger method and ABI3730 reading was performed in both directions. Bioneer Company in South Korea performed the nucleotide sequencing. The nucleotide sequence obtained in the raw ab1 file was manually evaluated and modified. For molecular identification of strains, the modified nucleotide sequence was further recorded in the BLASTn search system in the World GenBank (http://www.ncbi.nlm.nih.gov) (Altschul et al . 1997). The sequence of similar species was extracted in Clustalw software using Geneious software (9.1.3). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 6 (Molecular Evolutionary Genetics Analysis) software (Tamura et al . 2013). The final sequence was submitted to GenBank. Evaluation of the more efficient strain in aquarium condition In this phase, sediments from several fishponds were collected and sterilized in the autoclave (model E500, Germany). Aquariums were held at 28±1°C in an environmentally controlled room with a 12‐h light-dark cycle. 1.5 kg of sterilized sediment was spread on the bottom of 6 aquariums (25×25×30 cm) filled with 6 l of sterilized water. As an electron donor source 10 gr of sodium thiosulfate per liter was added. For inoculating, strains FH-21 (Iran 3), FH-13 (Iran 2), and FH-14 (Iran 1) were first cultured in Starkey liquid media at 29 °C, 120rpm for 8 days in the incubator shaker. Strain suspension (density of 10 2 CFU g -1 in sediment) was then inoculated into the aquariums for 3 consecutive days. The aquarium without inoculation was considered the control group. This experiment was carried out in triplicate for 12 days. The value of sulfate ion production, thiosulfate ion consumption, and pH were measured before inoculation (day 0) and then 4, 8, and 12 days after the last inoculation; strain density was measured on day 12. Statistical analysis The one-way analysis of variance (ANOVA) was used to analyze the comparison of sulfate, thiosulfate and pH levels among different strains in media and in the aquariums. Statistical tests were performed using SPSS 20.0. Duncan’s Multiple Range Test was performed to determine differences in the mean of treatments. P value was set at 0.05. Results Enrichment and isolation of SOBs Because of the significant reduction in pH or deposition of sulfur particles, enrichment of SOBs was confirmed in all culture media except Postgate media based on organic carbon. The results showed that most of the changes in pH decrease were related to Starkey media (Table 3). Furthermore, the enriched sediment samples were able to form colonies in the dilutions between 10 -4 -10 -5 ml. Table 3 here Based on colony and cell morphologies differences in each solid media, 27 isolates (14 autotrophic and 13 heterotrophic isolates) were purified, from where 14, 6, and 7 isolates were obtained from Starkey, Postgate, and H-3 media, respectively. These results showed that the use of different media allows for obtaining more isolates. Ability of strains to SOB in Starkey media After the initial evaluation of isolates in Starkey liquid and solid media, only 6 isolates (2 autotrophic and 4 heterotrophic isolates) were able to reduce the pH, which was accompanied by a change in color from green to yellow (Fig. 1), including FH-13, FH-14, FH-15, FH-21, FH-24, and FH-27 (Table 4). Table 4 and Fig. I here The results of the final evaluation (Table 5 and Fig. 2) showed that among isolated strains, 3 strains of Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) had the highest oxidation ability of thiosulfate (413.21, 1362.50, and 4188.03 mg l ‒1 for 14 days, respectively) and sulfate production (3350, 2075, and 1600 mg l ‒1 , respectively). The initial value of thiosulfate and sulfate in Starkey liquid media was 4467 and 1110, respectively. Therefore, these three isolates were selected as the most efficient strains and they were considered for molecular identification and evaluation in aquarium conditions. Fig. 2 and Table 5 here Identification of efficient isolates The results showed that the strains Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21) had 99.70, 100, and 99.80% phylogenetic similarity to the gene sequence of Thiobasillus thioparus with a GenBank accession number HM173634 (Table 6). The phylogenetic tree of SOB strains based on the neighbor-joining method with the tamura-nei model and their close relationship with other SOBs is shown in Fig. 3. Table 6 and Fig. 3 here The phenotypic characteristics of the most efficient isolates indicated that strains create round colonies with a flat, entire edge, with white color for Iran 1 and Iran 2 colonies and cream color for Iran 3 strain. Moreover, strains were rod-shaped and gram-negative (Table 7). Table 7 here Performance of the most efficient isolates in aquarium condition The impact of the selected isolates on pH changes A comparison of the impact of isolates Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21) on the pH in aquariums after 12 days are shown in Fig 4. There were no significant differences in pH value on the fourth day between the biotic treatments and the control group (P>0.05). The pH value in aquariums inoculated with Iran 1 (FH-14) and Iran 2 (FH-13) showed a significant decrease after 8 days compared with the control group and Iran 3 (FH-21) isolates (P >0.05); The lowest value of pH was observed in Iran 1 (FH-14) isolate on the 12 th day, which was significantly different compared to the control group (P >0.05). Fig. 4 here The impact of the selected isolates on the value of sulfate ions The concentration of sulfate ions showed a significant increase in the isolates Iran 1 (FH-14) and Iran 2 (FH-13), compared with the control group on the 8 th day. On the other side, a significant difference in sulfate ion value between Iran 3 and control was not observed. However, the concentration of sulfate ion in the Iran 1 (FH-14) group was highest (P < 0.05) compared with control and other isolates on the 12 th day (Fig. 5). Fig. 5 here The impact of the selected isolates on the concentration of thiosulfate ions The results showed that on the 4 th and 8 th day, the concentration of thiosulfate ions in isolates Iran 1 (FH-14) and Iran 2 (FH-13) were significantly lower than in the control group and isolate Iran 3 (FH-21) (P <0.05). The minimum concentration of the ions was observed in the isolate Iran 1 (FH-14) followed by the isolate Iran 2 (FH-13) (Fig. 6). The lowest value of the thiosulfate was observed in Iran 1 (FH-14) and Iran 2 (FH-13) isolates on the 12 th day, which was significantly different compared to the control group. Fig. 6 here The density of the strains in aquariums on the 12 th day was 2.4 × 10 5 , 2.5 × 10 3 , and 0 CFU/ml for the strains Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21), respectively. Discussion Removal of hydrogen sulfide using biological processes is one of the most important issues in advanced research of the environment and agriculture (Zhuang et al. 2017 ). However, there are a few studies in the field of aquaculture in this regard. In this study, several media were used to isolate SOBs. There are some reasons for the no-growth or weak growth of most microorganisms in culture-dependent methods such as improper pH, incubation periods, and lack of knowledge of nutrient requirements (carbon sources, nitrogen, etc.) (Pulschen et al. 2017 ). Therefore, to obtain more isolates in this study, three media, Starkey, Postgate, and H-3 both mineral carbon-based and organic carbon-based were used. A total of 27 isolates were obtained and purified from the sediment of the warm-water fishpond. Similar to these results, some researchers (Vidyalakshmi and Sridar 2007 ; Rojas-Avelizapa et al. 2013 ; Chaudhary et al. 2017 ) used different media based on mineral and organic carbon-base to the isolation of SOB. Starkey media was selected as the optimal medium because of the changes in pH and the higher number of isolates that appeared on it. Studies on various samples such as soil, water, industrial wastes, and paddy rhizosphere have also shown good performance on the Starkey medium to isolate SOBs (Vidyalakshmi and Sridar 2007 ; Rojas-Avelizapa et al. 2017). In this study, sodium thiosulfate was used as a source of sulfur for the isolation of SOBs due to its stability at neutral pH, water solubility, and non-toxicity in high concentrations (Luo et al. 2013 ; Meyer et al. 2016 ; Zhoa et al. 2016 ). In agreement with this study, some research (Kantachote et al. 2008 ; Hidayat et al. 2017 ) have used sodium thiosulfate as a sulfur substrate for bacterial growth to isolate hydrogen sulfide oxidizing bacteria from the effluent. Enrichment culture is the technique to enhance the population density of a particular group of microorganisms within the total microbial population of a sample. This is achieved by preferentially stimulating the growth of the target group of microorganisms by judicious manipulation of the physiological conditions during the enrichment phase. (Madhuri et al. 2019 ). In this study, due to the low density of SOBs in sediment samples and the lack of colony formation in the solid medium, the enrichment technique was used in Starkey, Postgate, and H-3 liquid mediums. In the present study, after preparing the series dilution from the enriched sediment samples, colonies were formed in dilutions between 10 − 4 – 10 − 5 . This method was also used in most studies on the isolation of SOB. In this regard, Rojas-Avelizapa et al. ( 2013 ) reported that the use of an enrichment method resulted in the isolation of 75 sulfur-oxidizing microorganisms from different environmental samples. Nadella et al. ( 2019 ) used enrichment methods to isolate SOBs from soil samples taken from fish farms. They found that enriched samples could produce colonies in dilutions up to 10 − 5 on a solid culture medium. In the present study, the ability of strains to oxide sulfur was determined based on changes in pH, sulfate ions, and thiosulfate ions. These parameters were considered suitable indicators for sulfur oxidation in most studies (Ohba and Owa 2005 ; Rojas-Avelizapa et al. 2013 ; Sridar et al. 2015 ; Nadella et al. 2019 ). Among 27 isolates, only 6 isolates were able to reduce the pH in the medium. The decrease in pH was probably due to the production of sulfuric acid, which is created during the biological oxidation of thiosulfate to sulfate. This process was also reported in the studies of Suzuki ( 1999 ) and Rojas-Avelizapa et al. ( 2013 ). The results of this study in the final screening showed that Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) isolates had the highest ability to oxidize thiosulfate and produce sulfate ions in the liquid medium. There also was a good relationship between thiosulfate oxidation and sulfate production. For these reasons, these strains were selected as the most efficient strains for sulfur oxidation in screening. In this regard, Rojas-Avelizapa et al. ( 2013 ) showed that from 41 isolates, seven isolates of chemolithoautotrophic SOBs had the highest ability to oxidize sulfur into sulfate (from 190 to 380 mg l ‒1 ) in the modified Starkey medium. In the study of Ohba and Owa ( 2005 ), only three isolates of Thiomonas (N4-1C, N4-1E, and N4-1G) were selected as SOBs due to a decrease of pH from 6.5 to 3.4–4.3 and consumption of almost all thiosulfate ions (1.51–1.60 mM(, which is in the accordance with the results of our study. Many studies in various ecosystems (Cho et al. 1991 ; Vlasceanu et al. 1997 ; Barbosa et al. 2006 ; Muyzer et al. 2013 ; Hutt et al. 2017 ) have reported that these bacteria have the ability to oxidize sulfur compounds including thiosulfate, trithionate, tetrathionate, pentathionate, hexationate, thiocyanate, dithionate, hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, Methane thiol (CH 3 SH), and methyl mercaptan. For a more precise evaluation, the most efficient strains were evaluated in aquarium conditions (containing sediment and water of fishpond) on a larger and more realistic scale. Studies on the isolation and selection of the best SOBs have mostly been based only on a culture medium (Ravichandra et al. 2007 ; Zhoa et al. 2016 ; Ranjit Kumar et al. 2018 ; Nadella et al. 2019 ). However, researchers have stated that the strains selected solely by media (solid and liquid) often are ineffective in natural and field conditions (Bashan et al. 2013 ). The results of this study showed that the weakly efficient strain during the final screening i.e. Iran 1 (FH-14), had the highest oxidation under aquarium conditions. This indicates the importance of using environments similar to the aquaculture ecosystem in selecting the more efficient strains. Overall, aquarium conditions are more similar to the sediment and water of the fishpond in terms of sources and concentrations of carbon, nitrogen, and phosphorus. In conclusion, three more efficient strains of SOBs were isolated and identified from the sediments of warm-water fishponds, all of which belong to the genus Thiobacillus (Iran 1, 2, and 3). Iran 1 and Iran 2 can be used as biofertilizers to remove sulfur compounds from warm-water fish farms due to their optimal performance in the aquarium stage. In this study, we tried to use the autochthonous SOBs from fishpond as a new approach for the decomposition of sulfur compounds, especially hydrogen sulfide. It is advisable to perform this strategy to provide an acceptable indication of the sulfur-oxidizing ability of SOB. Since the conditions in ponds are very complex, further studies on the sulfur-oxidizing ability for SOB in a pilot experiment (small-scale fishponds), should be carried out before application in pond aquaculture. Declarations Acknowledgements Authors are grateful to Dr. Morteza Kamali, Dr. Hossein Kari Dolatabad, Hossein Norani, Khadije Arbabi, Mahnaz Otadi for his helpful assistance. Author contributions RD, NM, HBK and BL conceived and designed research. RD and NM conducted experiments. NM and HBK analyzed data. RD, NM and BL wrote the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript. All authors read and approved the manuscript. Funding This work was supported by Tarbiat Modares University and Soil and Water Research Institute. Conflict of interest The authors report no declarations of interest. 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Name of Samples Regions Geographic coordinate Longitude Latitude S 1 Ferem village- Fereydunkenar 52° 32' 21.6" E 36° 37' 9.3" N S 2 Heydar Kola village- Fereydunkenar 52° 32' 51.8" E 36° 37' 4.2" N S 3 Mirodposht village- Babol 52° 42' 15.4" E 36° 28' 36.5" N S 4 Musa Kola village- Simorgh 52° 45' 17.4" E 36° 33' 19.9" N S 5 Bahnamir city- Babolsar 52° 45' 40.2" E 36° 41' 22.4" N S 6 Seyyed Mahalleh village- Sari 52° 59' 41.9" E 36° 42' 39.6" N Table 2 Composition of SOB media. Chemical Concentration (mineral-based media) Concentration (organic-based media) Starkey Postgate H-3 Starkey Postgate H-3 Macroelements (g/l) Na 2 S 2 O 3 .5H 2 O 10 7 10 10 7 10 (NH 4 )SO 4 1 3.5 - 1 3.5 - (NH 4 )Cl - - 1 - - 1 MgSO 4 .7H 2 O 1.5 0.7 0.5 1.5 0.7 0.5 CaCl 2 .2H 2 O 0.42 0.4 - 0.42 0.4 - KH 2 PO 4 0.5 0.15 2.30 0.5 0.15 2.30 Na 2 HPO 4 .2H 2 O - - 2.9 - - 2.9 KCl 0.7 - - 0.7 - - MnCl 2 .4H 2 O - - 0.003 - - 0.003 NaHCO 3 (5%) - - 10 ml - - Fe(NH4)citrate - - - - - 0.05 glucose - - - 5 5 - Microelements (g/100 ml) EDTA 0.0005 5 0.0005 0.0005 5 0.0005 ZnSO 4 .7H 2 O 0.01 2.2 0.01 0.01 2.2 0.01 FeSO 4 .7H 2 O 0.0002 0.49 0.0002 0.0002 0.49 0.0002 CaCl 2 .2H 2 O - 0.72 - - 0.72 - H 3 BO 3 0.03 - 0.03 0.03 - 0.03 MnCl 2 .4H 2 O 0.003 0.16 0.003 0.003 0.16 0.003 NaMoO 4 .2H 2 O 0.003 - 0.003 0.003 - 0.003 (NH 4 )Mo 7 O 24 .4H 2 O - 0.11 - - 0.11 - CuSO 4 .5H 2 O 0.001 0.16 0.001 0.001 0.16 0.001 CoCl 2 .6H 2 O 0.02 0.16 0.02 0.02 0.16 0.02 NiCl 2 .6 H 2 O 0.002 - 0.002 0.002 - 0.002 Table 3 The pH value in Starkey, Postgate, and H-3 media in the enrichment stage of SOBs after 14 days of incubation. Mean ± SD. The initial pH for the media was 7. Final pH Culture media Carbon source 2.70 ± 0.22 Starkey Mineral carbon 3.65 ±1.10 Postgate 4.20 ± 1.28 H-3 2.67 ± 0.03 Starkey Organic carbon 7.15 ± 0.65 Postgate 3.63 ± 0.54 H-3 Table 4 The pH value created by the selected isolates in Starkey liquid media after 14 days for initial screening. The initial pH for the media was 7. Final pH Isolates Carbon source 7.48 D-1 Mineral 7.37 D-2 7.41 M-3 7.44 M-4 7.47 Mi-11 8.67 FH-12 5.46 FH-13 5.98 FH-14 7.46 FH-5 7.49 FH-6 7.36 FH-7 7.50 FH-8 7.45 FH-9 7.33 FH-10 5.65 FH-15 organic 7.57 FH-16 7.40 FH-17 7.38 FH-18 7.37 Mi-19 7.20 FH-20 6.35 FH-21 7.51 FH-22 7.30 FH-23 5.31 FH-24 7.45 FH-25 7.42 FH-26 6.73 FH-27 Table 5 The concentration of sulfate ion, thiosulfate ion and growth of the selected isolates in Starkey media for final screening after 14 days. Mean ± SD. Sulfur-oxidizing isolates Sulfate concentration (mg l ‒1 ) Thiosulfate concentration (mg l ‒1 ) Isolates’ growth (cfu ml ‒1 ) control 1110 ± 110 e 4467 ± 0 a 0 FH-13 3350 ± 50 a 413.21 ± 33.50 e 3.6 × 10 6 FH-14 1600 ± 100 c 4188.03 ± 55.84 c 5.1 × 10 6 FH-15 1275 ± 25 d 4366.72 ± 11.17 b 2 × 10 4 FH-21 2075 ± 75 b 1362.50 ± 22.32 d 1 × 10 6 FH-24 1200 ± 50 ed 4455.93 ± 11.05 a 0 FH-27 1225 ±25 ed 4411.27 ± 55.72 ab 2.8 × 10 6 Table 6 Comparison of the gene similarity of the selected isolates in this study with standard strains. Description % Identical Sites Accession number Name Thiobacillus thioparus HM173634 99.70 MK828485 Iran 1 (FH-14) Thiobacillus thioparus HM173634 100 MK828486 Iran 2 (FH-13) Thiobacillus thioparus HM173634 99.70 MK828487 Iran 3 (FH-21) Table 7 The phenotypic characteristics of the most efficient isolates. Characters Iran 1 (FH-14) Iran 2 (FH-13) Iran 3 (FH-21) Gram negative negative negative Shape rod rod rod edge entire entire entire elevation flat flat flat color white white Bisque consistency buttery hard buttery Size 1mm 1mm 1mm Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted 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-2830694","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":193100159,"identity":"4e3c1c1e-546d-4916-a51b-359538faa355","order_by":0,"name":"Rana Dashtbin","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rana","middleName":"","lastName":"Dashtbin","suffix":""},{"id":193100160,"identity":"2219ad8f-7d2a-43bd-90ef-41de0f360d20","order_by":1,"name":"Nemat Mahmoudi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYJACZjB5mPkAAwMbXFCCGC1sCaRqOcBjgKwFNzBn4E78XJhjk9h3nOfrhg9ldvn8/AcYP/xgsMjHpcWygXez9MxtaYkzD/NuuznjXLLlzBkJzJI9DBKWDTi0GBzg3SDNu+1w4gagltu8bcwGBjcYGKSBfjHAZQtQy+bfEC08z4Ba6g3szx9g/k1AyzaoLTxsQC2HDQwYEtjw2wJ0jzXvtjTjmYfZzIB+OW4gcSOxzbLHAI+W472bb/Nus5HtO3/42Y0PZdUG/P2HD9/4UVGHUws0UlAAYwPQKJwaRsEoGAWjYBQQAQDwCVQD5wTrswAAAABJRU5ErkJggg==","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Nemat","middleName":"","lastName":"Mahmoudi","suffix":""},{"id":193100161,"identity":"861f839f-0f68-4118-a0c2-a335c5eff1eb","order_by":2,"name":"Hossein Besharati Kollayeh","email":"","orcid":"","institution":"Soil and Water Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hossein","middleName":"Besharati","lastName":"Kollayeh","suffix":""},{"id":193100162,"identity":"9d92d8fe-34a3-40bc-b4a9-f5af3430c55d","order_by":3,"name":"Blažo Lalevic","email":"","orcid":"","institution":"Belgrade University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Blažo","middleName":"","lastName":"Lalevic","suffix":""}],"badges":[],"createdAt":"2023-04-18 08:14:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2830694/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2830694/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36067629,"identity":"a4169d67-0971-4801-ae65-a0959f82bfd1","added_by":"auto","created_at":"2023-04-20 13:58:20","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":45671,"visible":true,"origin":"","legend":"\u003cp\u003eInitial screening of isolates on a Starkey liquid medium (A) and Starkey solid medium (B) with bromothymol blue as the colour indicator of the pH changes.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/220970ca1ea03e8727f2e473.jpg"},{"id":36067631,"identity":"17445c31-8253-48d2-8c60-cc07db3154fa","added_by":"auto","created_at":"2023-04-20 13:58:20","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":66418,"visible":true,"origin":"","legend":"\u003cp\u003ePurified colonies of FH-13 (A), FH-14 (B) and FH-21 isolates (C) on Starkey medium.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/fa974f85e04bed13c76f217f.jpg"},{"id":36068868,"identity":"a0ceedb1-767b-464a-b1fa-0068e09de12d","added_by":"auto","created_at":"2023-04-20 14:06:20","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":72104,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree of the bacteria identified by the neighbor-joining method with the Tamura-Nei model with 1000 repetitions and nodes accuracy factor above 50%.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/0414701e610006654aeb987d.jpg"},{"id":36067632,"identity":"81301a3d-54ba-4643-92e5-9668720bd1a3","added_by":"auto","created_at":"2023-04-20 13:58:20","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":82595,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of isolates Iran 2 (FH-13), Iran 1 (FH-14), and Iran 3 (FH-21) on pH value in the aquarium after 12 days.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/7d535d5375940e57179bf3e9.jpg"},{"id":36068869,"identity":"749424da-4a33-444a-a04a-c47a1544d9f6","added_by":"auto","created_at":"2023-04-20 14:06:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":31703,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of isolates on the concentration of sulfate ion.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/667f81e06faccd1b11ba16e7.jpg"},{"id":36069342,"identity":"74b78d33-6fe6-43af-ad58-8ff0c6b3612f","added_by":"auto","created_at":"2023-04-20 14:14:20","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":33901,"visible":true,"origin":"","legend":"\u003cp\u003eThe impact of isolates on the concentration of thiosulfate ion after 12 days.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/e3060327bebe09fcd1c2dfbf.jpg"},{"id":36434842,"identity":"cd554329-f7d0-4560-ab59-db33c4b84243","added_by":"auto","created_at":"2023-04-28 15:44:42","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":631663,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2830694/v1/dcfc3919-9a62-4be0-a557-88c841969486.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Identification of sulfur-oxidizing bacteria from fishponds and their performance to remove hydrogen sulfide under aquarium conditions","fulltext":[{"header":"Introduction","content":"\u003cp\u003eHydrogen sulfide is a colorless and highly toxic gas that causes many economic losses in aquaculture ponds (Lahav et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In earthen fishponds, external inputs like feed pellets, fish feces, and fertilizers increase the amount of organic matter compared to the natural environment (Abraham et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). In these conditions, sulfate-reducing bacteria use sulfate as the terminal electron acceptor for the degradation of organic compounds, resulting in the production of H\u003csub\u003e2\u003c/sub\u003eS (Muyzer and Stams \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). H\u003csub\u003e2\u003c/sub\u003eS concentration depends on pH, temperature, and salinity (Datta \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). As the pH of the pond water decreases during the night, the toxicity of hydrogen sulfide increases (Tucker and D\u0026rsquo;Abramo \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eH\u003csub\u003e2\u003c/sub\u003eS causes problems such as fish mortality (Poulton et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lahav et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e), a decrease in fish growth (Pillay \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), and high oxygen consumption (Kang et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Hydrogen sulfide can inhibit Cytochrome-c oxidase activity, the electron transport system, and aerobic respiration, which increases blood lactate and tissue hypoxia (Affonso et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Field and laboratory studies have also pointed out that hydrogen sulfide is toxic to other aquatic organisms. In this regard, the research of Kuster et al. (2005) showed that the effective concentration of 50% (EC50) of hydrogen sulfide on \u003cem\u003eVibrio fischeri\u003c/em\u003e, \u003cem\u003eDaphnia magna\u003c/em\u003e zooplankton, and \u003cem\u003eScenedesmus vacuolatus\u003c/em\u003e microalgae 9, 0.12, and 1.8 mg l\u003csup\u003e‒1\u003c/sup\u003e, respectively. In addition, studies have shown that low levels of hydrogen sulfide have a significant negative effect on nitrification performance (up to 50%) (Joye and Hollibaugh \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1995\u003c/span\u003e; Crab et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAeration and water exchange (Howerton, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Boyd and Tucker \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and the addition of lime and other chemical oxidants (hydrogen peroxide, iron hydroxide, potassium permanganate, and coal ash) (Poulton et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Lahav et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Asaoka et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e) are commonly used to control hydrogen sulfide concentration in earthen fish ponds. Disadvantages of these methods include low efficiency, sudden fish losses, turbidity formation, high energy costs, production of secondary pollutants, need to obtain expensive chemicals, and public health concerns (Duranceau et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Zhao et al. 2016).\u003c/p\u003e \u003cp\u003eThe application of sulfur-oxidizing microorganisms (bacteria, actinomyces, fungi, and yeast) to remove hydrogen sulfide in water is one of the new sustainable approaches (Zhao et al. 2016). Among microorganisms, phototrophic and chemotrophic SOB bacteria play a significant role in hydrogen sulfide oxidation (Behera et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Photolitotrophic sulfide oxidation is carried out by green sulfur bacteria (\u003cem\u003eChlorobium\u003c/em\u003e) and purple sulfur bacteria (\u003cem\u003eAllochromatium\u003c/em\u003e and \u003cem\u003eChromatium\u003c/em\u003e) (Ghosh and Dam \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Chemolitotrophic oxidation is carried out by colorless sulfur bacteria, such as \u003cem\u003eThiobacillus\u003c/em\u003e, \u003cem\u003eAcidithiobacillus\u003c/em\u003e and \u003cem\u003eAchromatuim\u003c/em\u003e groups (Behera et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe previous studies on SOB have mostly focused on their role in different natural ecosystems (Behera et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Behera et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Moreover, the use of SOB for the removal of hydrogen sulfide from industrial wastewater has been extensively studied (Mirzaei et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Despite the advantages of SOB, few studies have been conducted on SOB diversity and their potential for H\u003csub\u003e2\u003c/sub\u003eS oxidation in the warm-water fishpond (Krishnani et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In addition, literature analyses have shown that one of the most reasons for the inefficiency of biofertilizers in farm conditions was that the components of culture media were not similar to the natural environment. In this regard, some researchers have stated that the strains selected solely by media (solid and liquid) did not provide acceptable results in natural conditions as biofertilizer (Bashan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As a solution, it has been recommended that the process of assessing the strains be complemented with additional tests in soil/sediment incubation.\u003c/p\u003e \u003cp\u003eTherefore, the aim of this study was, (1) to isolate autochthonous chemolithotrophic SOB strains from fishponds in the different media, (2) to evaluate the ability of these bacteria to thiosulfate oxidation in solid and liquid media, (3) to molecular identification of efficient strains and (4) to evaluate their performance during thiosulfate oxidation in aquarium conditions containing sediment and water of fishpond. In addition, the isolation of autochthonous SOB is advised as they are well adapted to the natural climate of their habitat, thus the introduction of alien species and their adverse environmental effects are prevented.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003eStudy sites and sample collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSediment samples from\u0026nbsp;six\u0026nbsp;earthen warm-water fishponds were collected with a Van-Veen grab sampler\u0026nbsp;(Hydro-Bios, Germany)\u0026nbsp;located in\u0026nbsp;northern Iran\u0026nbsp;along the southern\u0026nbsp;coast of the\u0026nbsp;Caspian Sea over an area extending from 36\u0026deg; 28\u0026acute;- 36\u0026deg; 42\u0026acute;N and 52\u0026deg; 32\u0026acute;- 52\u0026deg; 59\u0026acute;E.\u0026nbsp;The samples were stored at 4\u0026deg;C in sterile containers until further analysis. Common carp (\u003cem\u003eCyprinus carpio\u003c/em\u003e), grass carp (\u003cem\u003eCtenopharyngodon idella\u003c/em\u003e), bighead carp (\u003cem\u003eHypophthalmichthys nobilis\u003c/em\u003e), and silver carp (\u003cem\u003eHypophthalmichthys molitrix\u003c/em\u003e) are the most common species reared in these ponds. Silver carp is the most common species (55-60 percent of the total number of fish). Various animal-based fertilizers (manure and poultry manure) are largely (3.5-5 tons per hectare) used by farmers in the area. Farmers combine chemicals and manure and use them weekly on their land. To increase alkalinity and Ca supply agricultural lime is used continuously. Burnt lime is used when pests appear (especially parasites). The geographic coordinates\u0026nbsp;of sampling points are shown\u0026nbsp;in table 1.\u003c/p\u003e\n\u003cp\u003eTable 1 here\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCulture media\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSOB were isolated from samples using three media, Starkey medium (Starkey 1934), Postgate medium (Postgate 1966), and H-3 medium (medium 81. DSMZ) based on both mineral carbon and organic carbon to isolate autotrophic and heterotrophic bacteria, respectively. In mineral-based media, sodium bicarbonate (H-3) and carbon dioxide (Postgate and Starkey) were used as a carbon source; in organic-based media, glucose (Postgate and Starkey) and ammonium ferric citrate (H-3) was used as the carbon source. Moreover, these media were prepared in liquid and solid (2% agar) forms. Thymol blue was used as a pH indicator. In this study, sodium thiosulfate was used as a source of sulfur for the SOBs isolation (Luo et al\u003cem\u003e.\u003c/em\u003e 2013; Meyer et al\u003cem\u003e.\u003c/em\u003e 2016; Zhoa et al\u003cem\u003e.\u003c/em\u003e 2016). Table 2 shows the compositions of these media.\u003c/p\u003e\n\u003cp\u003eTable 2 here\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnrichment of SOBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDue to the low population of SOBs, enrichment culture was used to increase SOB counts. For this purpose,\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eone gram of homogenized sediment samples was suspended in 100 ml of Starkey, Postgate, and H-3 liquid media (Makzum et al\u003cem\u003e.\u003c/em\u003e 2017). Incubation was performed in a shaker incubator (Comecta, Spain) at 29 \u0026plusmn; 1 \u0026deg;C, 100 rpm for 14 days or until turbidity developed, which is related to microbial growth. After incubation, 5% of suspensions were transferred to fresh broth media, and then series dilutions (from 10\u003csup\u003e-1\u003c/sup\u003e to 10\u003csup\u003e-5\u003c/sup\u003e) were spread on Starkey, Postgate, and H-3 solid media.\u0026nbsp;Inoculated\u0026nbsp;plates were incubated at 29 \u0026plusmn; 1 \u0026deg;C for 14 days (Zhou et al\u003cem\u003e.\u003c/em\u003e 2007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIsolation and purification of SOBs\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this stage, colonies were isolated based on morphological characteristics such as shape, size, color, edge, elevation, and consistency\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e Colonies that showed constant characteristics (in terms of\u0026nbsp;gram staining and morphology) were considered pure colonies (Brenner et al\u003cem\u003e.\u003c/em\u003e 2005). The purified isolates were kept in a solid Starkey medium in the refrigerator (4 \u0026deg;C). Based on the pH changes and the number of isolate Starkey medium was selected for the screening stage.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening of SOBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the initial screening, the isolates were assessed based on their efficacy to reduce pH in the medium. In this stage, one loop from each pure colony was inoculated into 50 ml Starkey broth medium with an initial pH value of 7; incubated at 29 \u0026plusmn; 1 \u0026deg;C, 100 rpm for 14 days (Zhuang et al\u003cem\u003e.\u003c/em\u003e 2017; Ranjit\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eKumar\u0026nbsp;et al\u003cem\u003e.\u003c/em\u003e 2018). The pH value of the medium was determined using a pH meter (Jenway3510, England). In addition, the pH decrease in the Starkey solid medium was measured with bromothymol blue as the color indicator of the pH changes.\u003c/p\u003e\n\u003cp\u003eIn the final screening, the selected strains from the previous stage were inoculated into a Starkey broth medium. A strain suspension amounted to 1ml (10\u003csup\u003e6\u003c/sup\u003e CFU ml\u003csup\u003e-1\u003c/sup\u003e) was transferred into 100 ml Starkey broth medium. In this stage, parameters such as the thiosulfate oxidation, sulfate production, and the density of bacteria were assessed (Ohba\u0026nbsp;and\u0026nbsp;Owa, 2005; Ranjit\u0026nbsp;Kumar et al\u003cem\u003e.\u003c/em\u003e 2018). Finally, the most efficient isolates were identified using 16S rRNA gene sequencing.\u003c/p\u003e\n\u003cp\u003eThiosulfate ions concentration was measured by standard iodometric titration after neutralization of the medium with 50% (v/v) acetic acid (Banciu et al\u003cem\u003e.\u003c/em\u003e 2008). 10 ml of the sample was transferred to 50 ml Erlenmeyer flasks and then, 1 ml 1% starch solution was added as an indicator. Titration continued using a 10mM iodine solution until blue color appeared. The volume of iodine solution consumption was recorded and afterward consumption amount of thiosulfate ions was obtained. The value of sulfate ions was determined by turbidimetry method using the spectrophotometer (Hach DR1900, USA) at 450 nm wavelength. Moreover, the count of viable cells was reported with the standard plate count method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenomic DNA extraction and amplification of 16S /rRNA gene from SOB\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFor the extraction of the\u0026nbsp;selected strain\u0026nbsp;genome from gram-negative bacteria CinnaPure DNA\u0026nbsp;kit (SinaClon CO., LTD) was used. To amplify the\u0026nbsp;16S rDNA\u0026nbsp;gene\u0026nbsp;1 \u0026mu;l of 27F primer (AGAGTTTGATCMTGGCTCAG), 1 \u0026mu;l of 1492R primer (5΄-GGTTACCTTGTTACGACTT-3΄)\u0026nbsp;(Weisburg et al\u003cem\u003e.\u003c/em\u003e 1991),\u0026nbsp;1 \u0026mu;l of\u0026nbsp;DNTPs, 3 \u0026mu;l of DNA sample, 1 \u0026mu;l of MgCl\u003csub\u003e2\u003c/sub\u003e, and 0.6 \u0026mu;l of Taq DNA polymerase were used.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eThe PCR product was electrophoresed on a 1% agarose gel (Dena Gene Tajhiz, Iran) to ensure the correct amplification of the 16S rRNA gene. The PCR product obtained after purification by the column kit method was used for sequencing using the Sanger method and ABI3730 reading was performed in both directions.\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eBioneer Company in South Korea performed the nucleotide sequencing. The nucleotide sequence obtained in the raw ab1 file was manually evaluated and modified. For molecular identification of strains, the modified nucleotide sequence was further recorded in the BLASTn search system in the World GenBank\u0026nbsp;(http://www.ncbi.nlm.nih.gov) (Altschul et al\u003cem\u003e.\u003c/em\u003e 1997).\u0026nbsp;The sequence of similar species was extracted in Clustalw software using Geneious software (9.1.3). The phylogenetic tree was constructed by the neighbor-joining method using the MEGA 6 (Molecular Evolutionary Genetics Analysis) software (Tamura et al\u003cem\u003e.\u003c/em\u003e 2013). The final sequence was submitted to GenBank.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of the more efficient strain in aquarium condition\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this phase, sediments from several fishponds were collected and sterilized in the autoclave (model E500, Germany). Aquariums were held at 28\u0026plusmn;1\u0026deg;C in an environmentally controlled room with a 12‐h light-dark cycle. 1.5 kg of sterilized sediment was spread on the bottom of 6 aquariums (25\u0026times;25\u0026times;30 cm) filled with 6 l of sterilized water. As an electron donor source 10 gr of sodium thiosulfate per liter was added.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFor inoculating,\u0026nbsp;strains FH-21 (Iran 3), FH-13 (Iran 2), and FH-14 (Iran 1) were first cultured in Starkey liquid media at 29 \u0026deg;C, 120rpm for 8 days in the incubator shaker. Strain suspension (density of 10\u003csup\u003e2\u003c/sup\u003e CFU g\u003csup\u003e-1\u003c/sup\u003e in sediment) was then inoculated into the aquariums for 3 consecutive days. The aquarium without inoculation was considered the control group. This experiment was carried out in triplicate for 12 days. The value of sulfate ion production, thiosulfate ion consumption, and pH were measured before inoculation\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003e(day 0) and then 4, 8, and 12 days after the last inoculation; strain density was measured on day 12.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe one-way analysis of variance (ANOVA) was used to analyze the comparison of sulfate, thiosulfate and pH levels among different strains in media and in the aquariums. Statistical tests were performed using SPSS 20.0. Duncan\u0026rsquo;s Multiple Range Test was performed to determine differences in the mean of treatments. \u003cem\u003eP\u0026nbsp;\u003c/em\u003evalue was set at 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEnrichment and isolation of SOBs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBecause of the significant reduction in pH or deposition of sulfur particles, enrichment of SOBs was confirmed in all culture media except Postgate media based on organic carbon. The results showed that most of the changes in pH decrease were related to Starkey media (Table 3). Furthermore, the enriched sediment samples were able to form colonies in the dilutions between 10\u003csup\u003e-4\u003c/sup\u003e-10\u003csup\u003e-5\u003c/sup\u003e ml.\u003c/p\u003e\n\u003cp\u003eTable 3 here\u003c/p\u003e\n\u003cp\u003eBased on colony and cell morphologies differences in each solid media, 27 isolates (14 autotrophic and 13 heterotrophic isolates) were purified, from where 14, 6, and 7 isolates were obtained from Starkey, Postgate, and H-3 media, respectively.\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eThese results showed that the use of different media allows for obtaining more isolates.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAbility of strains to SOB in Starkey media\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter the initial evaluation of isolates in Starkey liquid and solid media, only 6 isolates (2 autotrophic and 4 heterotrophic isolates) were able to reduce the pH, which was accompanied by a change in color from green to yellow (Fig. 1), including FH-13, FH-14, FH-15, FH-21, FH-24, and FH-27 (Table 4). \u003c/p\u003e\n\u003cp\u003eTable 4 and Fig. I here\u003c/p\u003e\n\u003cp\u003eThe results of the final evaluation (Table\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e5 and Fig. 2) showed that among isolated strains, 3 strains\u003cem\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/em\u003eof Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) had the highest oxidation ability of thiosulfate (413.21, 1362.50, and 4188.03 mg l\u003csup\u003e‒1\u003c/sup\u003e for 14 days, respectively) and sulfate production (3350, 2075, and 1600 mg l\u003csup\u003e‒1\u003c/sup\u003e, respectively). The initial value of thiosulfate and sulfate in Starkey liquid media was 4467 and 1110, respectively. Therefore, these three isolates were selected as the most efficient strains and they were considered for molecular identification and evaluation in aquarium conditions.\u003c/p\u003e\n\u003cp\u003eFig. 2 and Table 5 here\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eIdentification of efficient isolates\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that the strains Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21) had 99.70, 100, and 99.80% phylogenetic similarity to the gene sequence of \u003cem\u003eThiobasillus thioparus\u003c/em\u003e with a GenBank accession number HM173634 (Table 6). The phylogenetic tree of SOB strains based on the neighbor-joining method with the tamura-nei model and their close relationship with other SOBs is shown in Fig. 3. \u003c/p\u003e\n\u003cp\u003eTable 6 and Fig. 3 here\u003c/p\u003e\n\u003cp\u003eThe phenotypic characteristics of the most efficient isolates indicated that strains create round colonies with a flat, entire edge, with white color for Iran 1 and Iran 2 colonies and cream color for Iran 3 strain. Moreover, strains were rod-shaped and gram-negative (Table 7).\u003c/p\u003e\n\u003cp\u003eTable 7 here\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003ePerformance of the most efficient isolates in aquarium condition \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impact of the selected isolates on pH changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA comparison of the impact of isolates Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21) on the pH in aquariums after 12 days are shown in Fig 4.\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eThere were no significant differences in pH value on the fourth day between the biotic treatments and the control group (P\u0026gt;0.05). The pH value in aquariums inoculated with Iran 1 (FH-14) and Iran 2 (FH-13) showed a significant decrease after 8 days compared with the control group and Iran 3 (FH-21) isolates (P \u0026gt;0.05); The lowest value of pH was observed in Iran 1 (FH-14) isolate on the 12\u003csup\u003eth\u003c/sup\u003e day, which was significantly different compared to the control group (P \u0026gt;0.05).\u003c/p\u003e\n\u003cp\u003eFig. 4 here\u003c/p\u003e\n\u003cp\u003e\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impact of the selected isolates on the value of sulfate ions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of sulfate ions showed a significant increase in the isolates Iran 1 (FH-14) and Iran 2 (FH-13), compared with the control group on the 8\u003csup\u003eth\u003c/sup\u003e day. On the other side, a significant difference in sulfate ion value between Iran 3 and control was not observed. However, the concentration of sulfate ion in the Iran 1 (FH-14) group was highest (P \u0026lt; 0.05) compared with control and other isolates on the 12\u003csup\u003eth\u003c/sup\u003e day (Fig. 5). \u003c/p\u003e\n\u003cp\u003eFig. 5 here\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe impact of the selected isolates on the concentration of thiosulfate ions \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results showed that on the 4\u003csup\u003eth\u003c/sup\u003e and 8\u003csup\u003eth\u003c/sup\u003e day, the concentration of thiosulfate ions in isolates Iran 1 (FH-14) and Iran 2 (FH-13) were significantly lower than in the control group and isolate Iran 3 (FH-21) (P \u0026lt;0.05). The minimum concentration of the ions was observed in the isolate Iran 1 (FH-14) followed by the isolate Iran 2 (FH-13) (Fig.\u003cspan dir=\"RTL\"\u003e \u003c/span\u003e6).\u003cspan dir=\"RTL\"\u003e \u003c/span\u003eThe lowest value of the thiosulfate was observed in Iran 1 (FH-14) and Iran 2 (FH-13) isolates on the 12\u003csup\u003eth\u003c/sup\u003e day, which was significantly different compared to the control group.\u003c/p\u003e\n\u003cp\u003eFig. 6 here\u003c/p\u003e\n\u003cp\u003eThe density of the strains in aquariums on the 12\u003csup\u003eth\u003c/sup\u003e day was 2.4 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, 2.5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e, and 0 CFU/ml for the strains Iran 1 (FH-14), Iran 2 (FH-13), and Iran 3 (FH-21), respectively.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRemoval of hydrogen sulfide using biological processes is one of the most important issues in advanced research of the environment and agriculture (Zhuang et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). However, there are a few studies in the field of aquaculture in this regard.\u003c/p\u003e \u003cp\u003eIn this study, several media were used to isolate SOBs. There are some reasons for the no-growth or weak growth of most microorganisms in culture-dependent methods such as improper pH, incubation periods, and lack of knowledge of nutrient requirements (carbon sources, nitrogen, etc.) (Pulschen et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, to obtain more isolates in this study, three media, Starkey, Postgate, and H-3 both mineral carbon-based and organic carbon-based were used. A total of 27 isolates were obtained and purified from the sediment of the warm-water fishpond. Similar to these results, some researchers (Vidyalakshmi and Sridar \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rojas-Avelizapa et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chaudhary et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) used different media based on mineral and organic carbon-base to the isolation of SOB.\u003c/p\u003e \u003cp\u003eStarkey media was selected as the optimal medium because of the changes in pH and the higher number of isolates that appeared on it. Studies on various samples such as soil, water, industrial wastes, and paddy rhizosphere have also shown good performance on the Starkey medium to isolate SOBs (Vidyalakshmi and Sridar \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Rojas-Avelizapa et al. 2017).\u003c/p\u003e \u003cp\u003eIn this study, sodium thiosulfate was used as a source of sulfur for the isolation of SOBs due to its stability at neutral pH, water solubility, and non-toxicity in high concentrations (Luo et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Meyer et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Zhoa et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In agreement with this study, some research (Kantachote et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Hidayat et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have used sodium thiosulfate as a sulfur substrate for bacterial growth to isolate hydrogen sulfide oxidizing bacteria from the effluent.\u003c/p\u003e \u003cp\u003eEnrichment culture is the technique to enhance the population density of a particular group of microorganisms within the total microbial population of a sample. This is achieved by preferentially stimulating the growth of the target group of microorganisms by judicious manipulation of the physiological conditions during the enrichment phase. (Madhuri et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). In this study, due to the low density of SOBs in sediment samples and the lack of colony formation in the solid medium, the enrichment technique was used in Starkey, Postgate, and H-3 liquid mediums. In the present study, after preparing the series dilution from the enriched sediment samples, colonies were formed in dilutions between 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e \u0026ndash; 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e. This method was also used in most studies on the isolation of SOB. In this regard, Rojas-Avelizapa et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) reported that the use of an enrichment method resulted in the isolation of 75 sulfur-oxidizing microorganisms from different environmental samples. Nadella et al. (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) used enrichment methods to isolate SOBs from soil samples taken from fish farms. They found that enriched samples could produce colonies in dilutions up to 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e on a solid culture medium.\u003c/p\u003e \u003cp\u003eIn the present study, the ability of strains to oxide sulfur was determined based on changes in pH, sulfate ions, and thiosulfate ions. These parameters were considered suitable indicators for sulfur oxidation in most studies (Ohba and Owa \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Rojas-Avelizapa et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Sridar et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Nadella et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong 27 isolates, only 6 isolates were able to reduce the pH in the medium. The decrease in pH was probably due to the production of sulfuric acid, which is created during the biological oxidation of thiosulfate to sulfate. This process was also reported in the studies of Suzuki (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) and Rojas-Avelizapa et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of this study in the final screening showed that Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) isolates had the highest ability to oxidize thiosulfate and produce sulfate ions in the liquid medium. There also was a good relationship between thiosulfate oxidation and sulfate production. For these reasons, these strains were selected as the most efficient strains for sulfur oxidation in screening. In this regard, Rojas-Avelizapa et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) showed that from 41 isolates, seven isolates of chemolithoautotrophic SOBs had the highest ability to oxidize sulfur into sulfate (from 190 to 380 mg l\u003csup\u003e‒1\u003c/sup\u003e) in the modified Starkey medium. In the study of Ohba and Owa (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2005\u003c/span\u003e), only three isolates of \u003cem\u003eThiomonas\u003c/em\u003e (N4-1C, N4-1E, and N4-1G) were selected as SOBs due to a decrease of pH from 6.5 to 3.4\u0026ndash;4.3 and consumption of almost all thiosulfate ions (1.51\u0026ndash;1.60 mM(, which is in the accordance with the results of our study. Many studies in various ecosystems (Cho et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1991\u003c/span\u003e; Vlasceanu et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Barbosa et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Muyzer et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Hutt et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) have reported that these bacteria have the ability to oxidize sulfur compounds including thiosulfate, trithionate, tetrathionate, pentathionate, hexationate, thiocyanate, dithionate, hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, Methane thiol (CH\u003csub\u003e3\u003c/sub\u003eSH), and methyl mercaptan.\u003c/p\u003e \u003cp\u003eFor a more precise evaluation, the most efficient strains were evaluated in aquarium conditions (containing sediment and water of fishpond) on a larger and more realistic scale. Studies on the isolation and selection of the best SOBs have mostly been based only on a culture medium (Ravichandra et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Zhoa et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Ranjit Kumar et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Nadella et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). However, researchers have stated that the strains selected solely by media (solid and liquid) often are ineffective in natural and field conditions (Bashan et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The results of this study showed that the weakly efficient strain during the final screening i.e. Iran 1 (FH-14), had the highest oxidation under aquarium conditions. This indicates the importance of using environments similar to the aquaculture ecosystem in selecting the more efficient strains. Overall, aquarium conditions are more similar to the sediment and water of the fishpond in terms of sources and concentrations of carbon, nitrogen, and phosphorus.\u003c/p\u003e \u003cp\u003eIn conclusion, three more efficient strains of SOBs were isolated and identified from the sediments of warm-water fishponds, all of which belong to the genus \u003cem\u003eThiobacillus\u003c/em\u003e (Iran 1, 2, and 3). Iran 1 and Iran 2 can be used as biofertilizers to remove sulfur compounds from warm-water fish farms due to their optimal performance in the aquarium stage. In this study, we tried to use the autochthonous SOBs from fishpond as a new approach for the decomposition of sulfur compounds, especially hydrogen sulfide. It is advisable to perform this strategy to provide an acceptable indication of the sulfur-oxidizing ability of SOB. Since the conditions in ponds are very complex, further studies on the sulfur-oxidizing ability for SOB in a pilot experiment (small-scale fishponds), should be carried out before application in pond aquaculture.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e Authors are grateful to Dr. Morteza Kamali, Dr. Hossein Kari Dolatabad,\u0026nbsp;Hossein Norani,\u0026nbsp;Khadije\u0026nbsp;Arbabi, Mahnaz Otadi\u0026nbsp;for his helpful assistance.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u0026nbsp;\u003c/strong\u003eRD, NM, HBK and BL conceived and designed research. RD and NM conducted experiments. NM and HBK analyzed data. RD, NM and BL wrote the manuscript. All authors provided critical feedback and helped shape the research, analysis and manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by Tarbiat Modares University and Soil and Water Research Institute.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u0026nbsp;\u003c/strong\u003eThe authors report no declarations of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed consent\u003c/strong\u003e Informed consent was obtained from all individual participants included in the study.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbraham TJ, Ghosh S, Nagesh TS, Sasmal D (2004) Distribution of bacteria involved in nitrogen and sulphur cycles in shrimp culture systems of west Bengal, India. 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Indian J Biotechnol 14: 72\u0026ndash;80\u003cspan dir=\"RTL\"\u003e.\u003c/span\u003e \u003c/li\u003e\n\u003cli\u003eStarkey RL (1935) Products of the oxidation of thiosulfate by bacteria in mineral media. J gen physiol 18: 325-349. https://doi.org/10.1085/jgp.18.3.325.\u003c/li\u003e\n\u003cli\u003eSuzuki I (1999) Oxidation of inorganic sulfur compounds: chemical and enzymatic reactions. Can J Microbiol 45: 97-105. https://doi.org/10.1139/w98-223.\u003c/li\u003e\n\u003cli\u003eSun J, Zhou J, Shang C, Kikkert GA (2014) Removal of aqueous hydrogen sulfide by granular ferric hydroxide\u0026mdash;Kinetics, capacity and reuse. Chemosphere 117: 324-329. https://doi.org/10.1016/j.chemosphere.2014.07.086.\u003c/li\u003e\n\u003cli\u003eTamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30: 2725\u0026ndash;2729. https://doi.org/10.1093/molbev/mst197.\u003c/li\u003e\n\u003cli\u003eTucker CS, D\u0026rsquo;Abramo LR (2008) Managing high pH in freshwater ponds. Southern Regional Aquaculture Center.\u003c/li\u003e\n\u003cli\u003eVidyalakshmi R, Sridar R (2007) Isolation and characterization of sulphur oxidizing bacteria. J Cult Collect 5: 73-77.\u003c/li\u003e\n\u003cli\u003eVlasceanu L, Popa R, Kinkle BK (1997) Characterization of \u003cem\u003eThiobacillus thioparus\u003c/em\u003e LV43 and its distribution in a chemoautotrophically based groundwater ecosystem. Appl Environ Microbiol 63: 3123-3127.\u003c/li\u003e\n\u003cli\u003eWeisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J bacteriol 173: 697-703. https://doi.org/ 10.1128/jb.173.2.697-703.1991.\u003c/li\u003e\n\u003cli\u003eZhoa YG, Zheng Y, Tian W, Bai J, Feng G, Guo L, Gao M (2016) Enrichment and immobilization of sulfide removal microbiota applied for environmental biological remediation of aquaculture area. Environ Pollut 214: 307-313. https://doi.org/10.1016/j.envpol.2016.03.028.\u003c/li\u003e\n\u003cli\u003eZhou QG, Bo F, Bo ZH, Xi L, Jian G, Fei LF, Hua CX (2007) Isolation of a strain of acidithiobacillus caldus and its role in bioleaching of chalcopyrite. World J Microbiol Biotechnol 23: 1217-1225. https://doi.org/10.1007/s11274-007-9350-6.\u003c/li\u003e\n\u003cli\u003eZhuang R, Lou Y, Qiu X, Zhao Y, Qian D, Yan X, Qian L (2017) Identification of a yeast strain able to oxidize and remove sulfide high efficiently. Appl Microbiol Biotechnol 101: 391-400. https://doi.org/10.1007/s00253-016-7852-6.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable\u0026nbsp;1 Geographic coordinate\u0026nbsp;of sampling points.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eName of Samples\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eRegions\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" width=\"40.10989010989011%\"\u003e\n \u003cp\u003eGeographic coordinate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"47.48858447488585%\"\u003e\n \u003cp\u003eLongitude\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"52.51141552511415%\"\u003e\n \u003cp\u003eLatitude\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eS\u003csub\u003e1\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eFerem village- Fereydunkenar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 32\u0026apos; 21.6\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 37\u0026apos; 9.3\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eS\u003csub\u003e2\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eHeydar Kola village- Fereydunkenar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 32\u0026apos; 51.8\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 37\u0026apos; 4.2\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eS\u003csub\u003e3\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eMirodposht village- Babol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 42\u0026apos; 15.4\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 28\u0026apos; 36.5\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eS\u003csub\u003e4\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eMusa Kola village- Simorgh\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 45\u0026apos; 17.4\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 33\u0026apos; 19.9\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp\u003eS\u003csub\u003e5\u003c/sub\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eBahnamir city- Babolsar\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 45\u0026apos; 40.2\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 41\u0026apos; 22.4\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"20.32967032967033%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eS\u003csub\u003e6\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"39.56043956043956%\"\u003e\n \u003cp\u003eSeyyed Mahalleh village- Sari\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.047619047619047%\"\u003e\n \u003cp\u003e52\u0026deg; 59\u0026apos; 41.9\u0026quot; E\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"21.062271062271062%\"\u003e\n \u003cp\u003e36\u0026deg; 42\u0026apos; 39.6\u0026quot; N\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;2\u0026nbsp;Composition of SOB media.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" width=\"642\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"2\" rowspan=\"2\" width=\"34.05909797822706%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eChemical\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" width=\"32.19284603421462%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eConcentration (mineral-based media)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"3\" valign=\"top\" width=\"33.748055987558324%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eConcentration (organic-based media)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"14.893617021276595%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;Starkey\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.31205673758865%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003ePostgate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.494089834515368%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;H-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.130023640661939%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eStarkey\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.73049645390071%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003ePostgate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.43971631205674%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;H-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"12\" width=\"15.109034267912772%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMacroelements (g/l)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.003115264797508%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eNa\u003csub\u003e2\u003c/sub\u003eS\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.813084112149532%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.74766355140187%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.526479750778817%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.968847352024921%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.682242990654206%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.149532710280374%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e(NH\u003csub\u003e4\u003c/sub\u003e)SO\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e(NH\u003csub\u003e4\u003c/sub\u003e)Cl\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMgSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eKH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eNa\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eKCl\u003c/span\u003e\u003c/p\u003e\n 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dir=\"LTR\"\u003eMnCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n 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dir=\"LTR\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFe(NH4)citrate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.05\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eglucose\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e-\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"11\" width=\"15.109034267912772%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMicroelements (g/100 ml)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"19.003115264797508%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eEDTA\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.813084112149532%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"10.74766355140187%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.526479750778817%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"9.968847352024921%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.682242990654206%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.149532710280374%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0005\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eZnSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.01\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFeSO\u003csub\u003e4\u003c/sub\u003e.7H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.0002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCaCl\u003csub\u003e2\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.72\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.72\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eH\u003csub\u003e3\u003c/sub\u003eBO\u003csub\u003e3\u003c/sub\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMnCl\u003csub\u003e2\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eNaMoO\u003csub\u003e4\u003c/sub\u003e.2H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.003\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e(NH\u003csub\u003e4\u003c/sub\u003e)Mo\u003csub\u003e7\u003c/sub\u003eO\u003csub\u003e24\u003c/sub\u003e.4H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCuSO\u003csub\u003e4\u003c/sub\u003e.5H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.001\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCoCl\u003csub\u003e2\u003c/sub\u003e.6H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.02\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"22.38532110091743%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eNiCl\u003csub\u003e2\u003c/sub\u003e.6 H\u003csub\u003e2\u003c/sub\u003eO\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.559633027522937%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"12.660550458715596%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.577981651376147%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"11.743119266055047%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.761467889908257%\"\u003e\n \u003cp dir=\"RTL\"\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.311926605504587%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0.002\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;3\u0026nbsp;The pH value in Starkey, Postgate, and H-3 media in the enrichment stage of SOBs after 14 days of incubation.\u0026nbsp;Mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SD.\u0026nbsp;The initial pH for the media was 7.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" dir=\"rtl\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"34.75728155339806%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFinal\u0026nbsp;pH\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCulture media\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCarbon source\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"34.75728155339806%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.70\u0026nbsp;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;0.22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eStarkey\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMineral carbon\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"51.585014409221905%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3.65\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;\u0026plusmn;1.10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"48.414985590778095%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003ePostgate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"51.585014409221905%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e4.20\u0026nbsp;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;1.28\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"48.414985590778095%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eH-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"34.75728155339806%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.67\u0026nbsp;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026plusmn; 0.03\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eStarkey\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"3\" width=\"32.62135922330097%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eOrganic carbon\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"51.585014409221905%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.15\u0026nbsp;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026plusmn; 0.65\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"48.414985590778095%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003ePostgate\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"51.585014409221905%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3.63\u0026nbsp;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026plusmn;\u003c/span\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;0.54\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"48.414985590778095%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eH-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable\u0026nbsp;4\u0026nbsp;The pH value created by the\u0026nbsp;selected isolates in Starkey liquid media\u003cspan dir=\"RTL\"\u003e\u0026nbsp;\u003c/span\u003eafter 14 days for initial screening.\u0026nbsp;The initial pH for the media was 7.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\" dir=\"rtl\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFinal pH\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIsolates\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"41.801385681293304%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCarbon source\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.48\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eD-1\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"14\" width=\"41.801385681293304%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMineral\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eD-2\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.41\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eM-3\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.44\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eM-4\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.47\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMi-11\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e8.67\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-12\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5.98\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.46\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-5\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.49\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-6\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.36\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-7\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.50\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-8\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-9\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.33\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-10\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5.65\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"29.099307159353348%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"13\" width=\"41.801385681293304%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eorganic\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.57\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-16\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.40\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-17\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.38\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-18\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.37\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMi-19\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-20\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e6.35\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.51\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-22\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.30\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-23\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5.31\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.45\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-25\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e7.42\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-26\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e6.73\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"50%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-27\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;5 The concentration of sulfate ion, thiosulfate ion and growth of the selected isolates in Starkey media for final screening after 14 days. Mean\u0026nbsp;\u0026plusmn;\u0026nbsp;SD.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eSulfur-oxidizing isolates\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eSulfate concentration (mg l\u003csup\u003e‒1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eThiosulfate concentration (mg l\u003csup\u003e‒1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIsolates\u0026rsquo; growth\u003c/span\u003e\u003c/p\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp; (cfu ml\u003csup\u003e‒1\u003c/sup\u003e)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003econtrol\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1110 \u0026plusmn; 110\u003csup\u003ee\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e4467 \u0026plusmn; 0\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-13\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3350 \u0026plusmn; 50\u003csup\u003ea\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e413.21 \u0026plusmn; 33.50 \u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e3.6 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-14\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1600 \u0026plusmn; 100\u003csup\u003ec\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e4188.03 \u0026plusmn; 55.84\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e5.1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-15\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1275 \u0026plusmn; 25\u003csup\u003ed\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e4366.72 \u0026plusmn; 11.17\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-21\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2075 \u0026plusmn; 75\u003csup\u003eb\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e1362.50 \u0026plusmn; 22.32\u003csup\u003ed\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-24\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1200 \u0026plusmn; 50\u003csup\u003eed\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e4455.93 \u0026plusmn; 11.05\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e0\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"27.419354838709676%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eFH-27\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.655913978494624%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e1225 \u0026plusmn;25\u003csup\u003eed\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.448028673835125%\"\u003e\n \u003cp\u003e4411.27 \u0026plusmn; 55.72\u003csup\u003eab\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"23.476702508960575%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e2.8 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable\u0026nbsp;6 Comparison of the gene similarity of the selected isolates in this study with standard strains.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.153846153846153%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eDescription\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.564102564102566%\"\u003e\n \u003cp dir=\"RTL\"\u003e%\u0026nbsp;\u003cspan dir=\"LTR\"\u003eIdentical Sites\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.273504273504273%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eAccession number\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.00854700854701%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eName\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.153846153846153%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cem\u003e\u003cspan dir=\"LTR\"\u003eThiobacillus thioparus\u003c/span\u003e\u003c/em\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;HM173634\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.564102564102566%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e99.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.273504273504273%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMK828485\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.00854700854701%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 1 (FH-14)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.153846153846153%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cem\u003e\u003cspan dir=\"LTR\"\u003eThiobacillus thioparus\u003c/span\u003e\u003c/em\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;HM173634\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.564102564102566%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e100\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.273504273504273%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMK828486\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.00854700854701%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 2 (FH-13)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"26.153846153846153%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cem\u003e\u003cspan dir=\"LTR\"\u003eThiobacillus thioparus\u003c/span\u003e\u003c/em\u003e\u003cspan dir=\"LTR\"\u003e\u0026nbsp;HM173634\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"22.564102564102566%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003e99.70\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"24.273504273504273%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eMK828487\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"27.00854700854701%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 3 (FH-21)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003eTable 7 The phenotypic characteristics of the most efficient isolates.\u003c/p\u003e\n \u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.12676056338028%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eCharacters\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 1 (FH-14)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.169014084507044%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 2 (FH-13)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eIran 3 (FH-21)\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.12676056338028%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eGram\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003enegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.169014084507044%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003enegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003enegative\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.12676056338028%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eShape\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003erod\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.169014084507044%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003erod\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003erod\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.12676056338028%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eedge\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eentire\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"28.169014084507044%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eentire\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.35211267605634%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eentire\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"21.12676056338028%\"\u003e\n \u003cp dir=\"RTL\"\u003e\u003cspan dir=\"LTR\"\u003eelevation\u003c/span\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" 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\u003c/table\u003e\n\u003c/div\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"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":"Biofertilizer, Hydrogen sulfide, Sulfur oxidizing bacteria, Sustainable aquaculture, Thiobacillus thioparus, fishpond ","lastPublishedDoi":"10.21203/rs.3.rs-2830694/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2830694/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHydrogen sulfide is a highly toxic gas that causes many economic losses in aquaculture ponds. The application of sulfur-oxidizing bacteria (SOB) to remove hydrogen sulfide is an eco-friendly approach. This study aimed to isolate and identify the most efficient SOBs from the sediment of warm-water fish farms. Enrichment and isolation were performed in three different culture media (Starkey, Postgate, and H-3) based on both mineral and organic carbon. Overall, 27 isolates (14 autotrophic and 13 heterotrophic isolates) were purified based on colony and cell morphology differences. Initial screening was performed based on pH decrease. For final screening, the isolates were assessed based on their efficacy in thiosulfate oxidation and the sulfate production on Starkey liquid medium. Among isolated strains, 3 strains\u003cem\u003e \u003c/em\u003eof Iran 2 (FH-13), Iran 3 (FH-21), and Iran 1 (FH-14) that belonged to the genus \u003cem\u003eThiobacillus thioparus\u003c/em\u003e (identified by 16s rRNA) showed the highest ability in thiosulfate oxidation (413.21, 1362.50, and 4188.03 mg l\u003csup\u003e‒1\u003c/sup\u003e for 14 days) and the highest sulfate production (3350, 2075, and 1600 mg l\u003csup\u003e‒1\u003c/sup\u003e). In the final phase, the performance of these strains under aquarium conditions showed that Iran 1 and Iran 2 had the highest ability in sulfur oxidation. In conclusion, Iran 1 and 2 strains can be used as effective SOB to remove hydrogen sulfide in fish farms. It is very important to evaluate strains in an appropriate strategy using a combination of different criteria to ensure optimal performance of SOB in farm conditions.\u003c/p\u003e","manuscriptTitle":"Identification of sulfur-oxidizing bacteria from fishponds and their performance to remove hydrogen sulfide under aquarium conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-20 13:58:16","doi":"10.21203/rs.3.rs-2830694/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":"3c5d8621-5d3c-40e9-b35b-6c867080fd45","owner":[],"postedDate":"April 20th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-05-01T09:59:27+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-20 13:58:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2830694","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2830694","identity":"rs-2830694","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}