Growth in Ever-Increasing Acidity Condition Enhanced the Adaption and Bioleaching of Leptospirlium Ferriphilum

Growth in Ever-Increasing Acidity Condition Enhanced the Adaption and Bioleaching of Leptospirlium Ferriphilum | 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 Growth in Ever-Increasing Acidity Condition Enhanced the Adaption and Bioleaching of Leptospirlium Ferriphilum Ronghui Liu, Hongbo Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-528164/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Low pH could eliminate the jarosite accumulation and improve the interfacial reaction rate during bioleaching process. However, a great challenge existed between microbial activity and bioleaching ability in low pH conditions. This study demonstrated that the adaption and bioleaching of Leptospirilum ferriphilum could be improved after long term adaptive evolution under acidity condition. It was found that the acidity adapted strain showed robust activity in wider pH, high concentration of ferrous iron and lower temperature. Although the enhancement for heavy metal tolerance was limited, the tolerance for MgSO 4 , Na 2 SO 4 and organic matter was great. More importantly, both pyrite and printed circuit boards bioleaching revealed the higher bioleaching ability of the acid-resistant strain. These adaptation and bioleaching details provided an available approach for the improvement of bioleaching techniques. General Microbiology Leptospirilum ferriphilum bioleaching low pH adaptation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Acidophiles were the most efficient community on the metals bioleaching from sulfide minerals. Recent disclosures demonstrated that these microorganisms still conducted crucial function for metals extraction from solid waste such as waste printed wiring board (Xia et al., 2017), electroplating sludge (Zhou et al., 2019) and coal fly ash (Fan et al., 2019). However, the fickle bioleaching environment such as fluctuated pH and the increased metal ion was detrimental to the growth of microorganisms and thus weakened the biological effects, which resulted in low leaching efficiency. In order to enhance the bioleaching efficiency, our previous studies developed an extreme acid tolerance consortium through three steps adaptive evolution (Liu et al., 2019b) and subsequent research revealed that the obtained consortia could extract near 100% metals from waste resin powder at high pulp density (Liu et al., 2020). For adaptation, the synergetic interaction in this consortium plays an important role in the stable maintenance of function in wider conditions (Liu et al., 2019b). However, the adaptive evolutionary community on the effect of a single microorganism for environmental adaptations is unknown. An in-depth analysis of these problems can not only deepen the understanding of the characteristics of the adaptive evolutionary communities but also contribute to the further development and utilization of adaptive evolutionary strains. In this study, the main strain in the extreme acid tolerance community was isolated with a low pH gradient dilution method. Comparative investigation of original and adapted strain on growth at wider pH, temperature, concentration of metal ion, 9K basic salt and organic matters were explored. After that, the bioleaching capacity of isolated strain in wider conditions was illustrated. These findings preliminary revealed the strain evolution characteristics of adaptation and provide some new insight to the adaptive evolution of acidophiles. 2. Materials And Methods 2.1 Medium and strains Leptospirillum ferriphilum DX-m was isolated from the main bio-heap of Dexing copper mine and reserved by our laboratory. The extreme acid adaptive community used in this study was obtained through a modified adaptive evolution process by our laboratory (Liu et al., 2019b). The medium used for the culture of community and pure culture was described as previous reports (Liu et al., 2019b; Liu et al., 2019c). 2.2 Isolation of the evolved strain Previous studies revealed the extreme acid adaptive community mainly composed of Sulfobacillus spp., Leptospirillum spp. and Ferroplasma spp.(Liu et al., 2019b). During isolating process, consideration of the indispensability of organic matter for the quick growth of both Sulfobacillus spp. and Ferroplasma spp.(Zhang et al., 2015; Zhou et al., 2015), selective liquid medium without the addition of organic matter was used to achieve the separation of L. ferriphilum . In order to prevent the interference of some non-acid-resistant mutants or reverting mutants, a low pH strategy was employed during the gradient dilution process. Simply, the obtained adaptive evolutionary community was inoculated into a shaking flask containing a selective medium (9K inorganic salt, 350 mM Fe 2+ and pH 0.7) after gradient dilution. After 85% ferrous iron was oxidized to ferric iron, cells were collected by centrifugation and DNA was extracted by the DNA extraction kit for further molecular identification by PCR and 16S sequencing. The common and specific primers used in this study were same as previous studies (Liu et al., 2019b; Ma et al., 2018). 2.3 Growth characteristic of starting and evolved strains on different biochemical parameters conditions The effects of biochemical parameters on the growth of microorganisms are extremely important for the application of acidophiles (Shiers et al., 2016). This experiment explored effects of ferrous iron concentration (5-30 gL -1 ), pH (0.6-1.8), temperature (25-45°C) and 9K basic salt ( (NH 4 ) 2 SO 4 : 05-3 gL -1 ; K 2 HPO 4 : 0.05-0.5 gL -1 ; KCl: 0-0.01 gL -1 ) on the growth of starting strain and evolved strain under high and low pH condition. During the experiment, samples were taken at an interval to determine the iron oxidation rate and biomass. 2.4 Tolerance experiment of starting strain and evolved strain During the solid waste leaching process, heavy metals, magnesium ions, sodium ions and organic matter generally increased significantly (Liu et al., 2020), which notably affected the bioleaching efficiency. The tolerance of microorganisms to these factors becomes the key to accelerate metals extraction. This study explored different concentrations of magnesium sulfate (1-20 gL -1 ), sodium sulfate (1-20 gL -1 ) and yeast extract (0-1 gL -1 ) on the effects of iron oxidation and biomass production of starting and evolved strains. As for tolerance test of heavy metals, 1 and 5g L -1 Ni、Cr、Cu and Co was adopted. The medium used in these assays was 9K medium (initial ferrous ion 10 gL -1 ), and the culture conditions were 45°C and 180 rpm. During the experiment, samples were taken at an interval to determine the changes in ferrous ions. After complete oxidation, the average iron oxidation rate was calculated and the biomass was measured. All experiments were performed in triplicates. 2.5 Bioleaching tests Bioleaching of waste printed circuit boards and pyrite at different conditions was carried out to evaluate the leaching efficiency of evolved strains. The minerals used in this were present as our previous description (Liu et al., 2019a; Liu et al., 2019b; Liu et al., 2020). Bioleaching experiments were performed in shake flask, and the factors of pH (1.6 and 1.0) and temperature (30°C and 45°C) was taken into consideration. Pyrite bioleaching assays were present as following: 2% pulp density, 10 days. For PCB bioleaching, the condition was 10% pulp density, 1g/L ferrous iron and 3 days. The 9 K basic salt medium and 10% bacterial inoculation were performed in these experiments. During bioleaching process, bioleaching tests with starting strain were set as the control, and samples were taken at an interval to determine the change in chemical parameters. 2.6 Analytical methods Total cells in the supernatant were determined by direct counts (blood cells counting chambers: XB-K-25, QiuJing, Shanghai, China; Microscope: BX-41TF, Olympus, Tokyo, Japan). The total iron and ferrous iron were determined by a spectrophotometer with the o-phenanthroline (TGI, Shanghai, China) spectrophotometry assay (1500, Thermo, USA). The Cu contents in solutions were analyzed using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima 5300 DV, PerkinElmer Instrument). The pH of the supernatant was measured with a pH meter (PH-3S) (pHS-3C, Leici, Shanghai, China). The data analysis was performed by SPSS version 24.0 (SPSS Inc., Chicago, IL, USA). 3 Results And Discussions 3.1 Characteristic of acid tolerance strain Analysis of the 16S rDNA sequences and construction of the phylogenetic tree based on neighbor-joining analysis were performed for molecular identification of isolated strains. The partial 16S rDNA sequence of isolated strains displayed 100% identity with corresponding sequences of Leptospirilum ferriphilum DX-m (Fig. 1). And the isolated strain was therefore named Leptospirilum ferriphilum DX-m-ALE. Surface groups analysis based on FTIR revealed that the peak intensity in 1080 cm -1 、1545 cm -1 、1640 cm -1 and 2080 cm -1 increased, while in 1190 cm -1 、1290 cm -1 、1470 cm -1 、1570 cm -1 and 3000 cm -1 decreased (Fig. 2). These data indicating more polysaccharide like substances, -C-O-C-, -C=C- and heterocyclic compound in Leptospirilum ferriphilum DX-m-ALE. 3.2 Iron oxidation and biomass production of Leptospirilum ferriphilum DX-m-ALE in wider conditions As illustrated in Fig 3A, the iron oxidation capacity of Leptospirilum ferriphilum DX-m gradually decreased with the decrease of pH value and no biological iron ion oxidation was observed at pH 0.6. These results indicated serious inhibition of acid on the iron oxidation of Leptospirilum ferriphilum (Zhang et al., 2010). In comparison, Leptospirilum ferriphilum DX-m-ALE completely oxidized 10 g/L iron within 48 hours in the range of pH 0.6-1.8. Meanwhile, the production of biomass showed the same trend with ferrous iron oxidation (Fig.3B). These results indicating that the acid resistance of the evolved strain Leptospirilum ferriphilum DX-m-ALE was improved. The effect of temperature on iron oxidation of both the starting strain Leptospirilum ferriphilum DX-m and the evolving strain Leptospirilum ferriphilum DX-m-ALE was presented in Fig 3C. In the case of pH 1.6, the iron oxidation rate for the evolved strain was 250 mg L -1 h -1 at 30℃, while that of the starting strain was 80 mg L -1 h -1 . Even the temperature was at 25℃, the evolved strain showed 150 mg L -1 h -1 iron oxidation rate. It is obvious that the evolved strain had stronger adaptability at low temperature. In the case of pH 0.7 , the evolved strain showed the maximum iron oxidation rate of 350 mgL -1 h -1 at 45℃. However, the iron oxidation rate in low pH culture was slightly lower than that in high pH culture when pH below 37℃. For biomass production, the lower the temperature, the less biomass. It is not hard to conclude that the adaptive evolutionary strain Leptospirilum ferriphilum DX-m-ALE showed a wider range of temperature adaptability and could maintain this superiority under the low pH environment, but the double stress of low temperature and acidity still affects the activity of the evolved strain. The combined effect of ferrous iron and pH on the iron oxidation for Leptospirilum ferriphilum DX-m-ALE and Leptospirilum ferriphilum DX-m was also determined (Fig 3D-F). At pH 1.6, both strains oxidized all ferrous iron within 48 hours below 5-10 g/L Fe 2+ . However, high concentrations of ferrous iron seriously inhibit the growth of starting strain. In the case of 30g/L Fe 2+ , little ferrous iron was observed to be biologically oxidized with 72 hours. In contrast, the evolved strain completely oxidized ferrous iron to ferric iron. These results demonstrated the evolved strain has a greater tolerance to Fe 3+ compared to the starting strain. At pH 0.7, the evolved strain showed higher iron oxidation ability when the ferrous iron concentration below 20 g/L, which may due to Leptospirilum ferriphilum under low pH need more energy to resist acid stress (Matsumoto et al., 2004). However, dual stress of acid and high concentration of iron ions still inhibited the iron oxidation function of the evolved strain Leptospirilum ferriphilum DX-m-ALE, the time to oxidize 30 gL -1 Fe 2+ extended to 84 hours. Based on the above results, it is concluded that Leptospirilum ferriphilum DX-m-ALE showed greater capacity for ferrous iron oxidation under different conditions 3.3 The adaptation for the basic salt in 9K medium During the bioleaching process, the constituent lack of 9K basic salt seriously inhibited the growth of microorganisms, but the excessive chemicals in 9Kmeidum not only caused energy waste but also lead to the production of secondary precipitates and limited the growth of cells (Gramp et al., 2008). The reasonable component and concentration of basic salt is allimportant for the application of bioleaching techniques. It was observed that the iron oxidation capacity and biomass production of the evolved Leptospirilum ferriphilum in different concentration of basic salt was different from the starting strain. Little influence on the iron oxidation rate of the starting strain was observed at 1-3 g/L ammonium salt, and the high concentration of ammonium salt may not be beneficial to the growth of starting strain (Fig. 4A). In comparison, the evolved strain showed a higher iron oxidation rate in the wider concentration of ammonium sulfate. In addition, the culture of the evolved L. ferriphilum in the low pH condition showed similar trends. These results indicated little dependency on ammonium salt concentration for starting strain and evolved strain, but the evolved strain showed higher growth capacity under different ammonium salt concentrations. The negligible effect of KCl on iron oxidation rate was observed for both evolved and starting strains (Fig. 4B). Even in the absence of potassium chloride, the iron oxidation rate achieved for 250 mgL -1 h -1 . It was noted that the high concentration of potassium chloride improved the biomass production. Particularly in the case of pH 0.7, the higher concentration of potassium chloride, the more biomass was detected. This is possible that a higher concentration potassium ion could improve the acid resistance stress of microorganisms(Guan and Liu, 2020). It is demonstrated that the evolved strain could adapt wide range of KCl and thus would improve the growth. The concentration of K 2 HPO 4 had a great influence on iron oxidation and biomass of strains, and the effect of K 2 HPO 4 on the iron oxidation capacity for starting strain and evolution was similar (Fig 4C). The maximum iron oxidation rate and biomass was obtained at 0.2g/L K 2 HPO 4 . However, the evolved strain showed higher iron oxidation rate at pH 0.7. And the biomass gradually increased with the increase of the concentration of K 2 HPO 4 . This may because more potassium ions improved the acidity resistance of microorganisms (Baker-Austin and Dopson, 2007) and low pH eliminated the inhibition effect of jarosite on cells. Therefore, the 0.2 g L -1 K 2 HPO 4 is more suitable for the culture of the strain at high pH, but the increase of K 2 HPO 4 concentration at low pH is beneficial to increase the growth rate of microorganisms. In all, the evolved strain could grow in a wider range of 9K basic salt. 3.4 The tolerance for high concentration salts, organic matter and heavy metals After acid adaptation, L. ferriphilum showed the improved tolerance for higher concentrations of magnesium sulfate (Fig.5A). And the average iron oxidation rate of evolved L. ferriphilum reached 341 mg L -1 h -1 under 10 g/L MgSO 4 condition. Even at 20 g/L MgSO 4 , the average iron oxidation of 310 mg L -1 h -1 was observed. In contrast, the average iron oxidation rate of starting L. ferriphilum reached the maximum of 291 mg L -1 h -1 at 5 g L -1 MgSO 4 . At pH 0.7, the iron oxidation rate of the evolved L. ferriphilum still showed 320 mg L -1 h -1 at 20 gL -1 MgSO 4 . It was worthy to note that the higher concentration of MgSO 4 , the smaller the biomass produced by both the evolved strain and the starting strain, indicating that high concentration of MgSO 4 is detrimental to the production of biomass, previous studies also demonstrated that high concentration of Mg 2+ reduced the biofilm quantity (Tang et al., 2018).Therefore, the enhanced iron oxidation capacity may due to high ionic conductivity environment that improved electron transfer (Li et al., 2014). The improved tolerance for high concentration Na 2 SO 4 were also observed (Fig. 5B). However, the iron oxidation rate and biomass decreased gradually with the increase of sodium sulfate concentration. In case of 20 gL -1 , the iron oxidation rate of the starting strain declined to 250 mg L -1 h -1 , while the iron oxidation rate of the evolved strain maintained to 308 mgL -1 h -1 . Accordingly, the enhanced performance on acid resistance was beneficial for the improvement of adaptation for sulfate. During bioleaching process, high concentration of sodium sulfate seriously limited the bioleaching efficiency through the formation of Fe(III)-precipitates (Liu et al., 2018) and inhibition effect for microorganisms (Bevilaqua et al., 2013), the adaptation of L. ferriphilum in high concentration of sodium sulfate and low pH conditions indicated significant synergetic relationship between acid tolerance and environmental adaptation. In the absence of organic matter and high pH, the iron oxidation rate of the starting L. ferriphilum was 270 mg L -1 h -1 and that of the evolved L. ferriphilum was 325 mgL -1 h -1 (Fig 5C) However, the iron oxidation rate of the starting strain gradually decreased and only 163 mg L -1 h -1 was observed under 1g/L organic matter condition. Meanwhile, the total biomass decreased to 0.6×10 8 cells/mL. These results indicated that the organic matter greatly inhibited the iron oxidation and microbial growth of the starting strain. In contrast, the iron oxidation rate of the evolved strain maintained over 280 mg L - h -1 at same range of organic matter. Especially at low pH condition, the 310 mg L -1 h -1 IOR and 1.7×10 8 cells/mL biomass was observed. These results indicated that, although organic compounds still had a strong inhibitory effect on the evolving strains, the performance on adaptation was improved. Therefore, adaptive evolution may be an available strategy to improve adaptation of L. ferriphilum for organic matter. Previous research revealed that the addition of galactose in the medium can significantly improve the adhesion performance of EPS, strengthen the adsorption effect of strain (Aguirre et al., 2018), the evolutionary strain of L. ferriphilum increase in sensitivity to organic matter for the application of the bacteria also provides a new train of thought. Besides, no double inhibition effect of organic matter and acidity also revealed the probability of a similar mechanism of tolerance for acidity and organic matter. Great challenges for bioleaching deriving from the inhibition effect of heavy metals, such as Ni, Cu, Co and Cr seriously limited iron oxidation and cell yields of microorganism. After adaptation, this phenomenon is still serious (Fig 5D-F). In the case of nickel-containing medium, it was observed that the iron oxidation rate of both the starting strain and the evolving strain declined to 138 mg L -1 h -1 at pH 1.6, indicating that nickel ion had a serious inhibitory effect on the growth of both the evolving strain and the originating strain, and no great difference in the nickel tolerance was observed between the evolving strain and the starting strain, indicating that only the improvement of acidity tolerance can’t realize the adaptation for nickel ion. In contrast, the evolved L. ferriphilum showed 166 mgL -1 h -1 IOR at pH 0.7. Although nickel ion still has serious influence on the growth of microorganism inhibition, the IOR increased by 24.48% compared to the high pH cultivation. The reason for the enhanced nickel ions resistance at low condition may due to acid activated the nickel operon (Tian et al., 2007), implying that the nickel resistance may due to the transcriptional control. In the presence of Cu and Co ions, it took 72 hours for both the starting strain and the evolved strain to completely oxidize 10g/L ferrous iron at pH 1.6, and 96 hours for the evolved strain in pH 0.7. The results indicated the double inhibitory effect of these two metals for L. ferriphilum . It is demonstrated the resistance pathway of the strains to these two metals was not consistent with the acid-resisting pathway. No obvious ferrous iron oxidation and biomass increase were observed in the presence of Cr ions, indicating that Cr not only inhibited the iron oxidation activity of the strain but also inhibited the proliferation of the bacteria. It is concluded that the adaptability to heavy metals was not improved after acid adaptation. Therefore, it is necessary for the improvement of adaptation to heavy metals before the industrial application of acidity adapted L. ferriphilum . 3.5 Bioleaching of metals from PCB and pyrite The dissolution of pyrite is observed to vary greatly under bioleaching by starting and evolved strain (Fig. 6A). At 45°C, the leaching efficiency of the evolved L. ferriphilum is observed to be 58% at pH 1.6. In comparison, that of the evolved L. ferriphilum displayed superior pyrite leaching at pH 1.0, the soluble iron achieved for 8.43 g/L (79.52%). When the temperature of the leach environment was 30°C, the low pH leaching performance of the evolved L. ferriphilum was inferior to the case of pH 1.6, suggesting that double stress of low temperature and acidity was detrimental to the bioleaching activity of the evolved strain. Meanwhile, the release of iron ion favored the production of acidity (Fig. 6B). All in all, the evolved L. ferriphilum would be advantageous to leach pyrite for the generation of H 2 SO 4 and Fe 3+ . As for PCB bioleaching, no notable difference was observed at pH 1.6 for the cases of the evolved and starting strain (Fig. 6C). The Cu extraction efficiency reached 83% and 88%, respectively. Similar with pyrite bioleaching, low temperature bioleaching environment showed the lesser metal extraction. Notable observation indicated that bioleaching at low pH seriously inhibited the dissolution of Cu. Our previous studies demonstrated that acid catalyst coupling bioleaching could significantly enhance the Cu extraction from PCBs(Liu et al., 2020), together revealed the importance of heavy metal resistance for the application of L. ferriphilum . After adaptation, the acidity adapted L. ferriphilum realized the maximum Cu extraction at pH 1.0 (Fig. 6D), agreeing with previous results that low pH bioleaching eliminated the jarosite barrier and improved the interfacial reaction (Liu et al., 2020). In this regard, it demonstrated that extreme acid tolerance microorganisms would be more promising for the development of bioleaching techniques. 4 Conclusions In this study, L. ferriphilum that could survive in pH 0.7 was obtained from an adaptive evolution community. It showed the enhanced adaptability to acidity, temperature and iron ions. Meanwhile, its growth is slightly affected by the wider range of 9K inorganic salt component, and no notable difference in growth was observed at low concentration of ammonium sulfate, dipotassium hydrogen phosphate and potassium chloride. Although the growth of the evolved L. ferriphilum was inhibited by high concentration of organic matter and salt ions to some extent, its tolerance was significantly improved. However, heavy metals still showed a serious inhibitory effect on the growth and iron oxidation of acidity adapted L. ferriphilum under the condition of low pH. Compared with starting strain, the evolved L. ferriphilum also demonstrated stronger leaching ability before and after adaptation. These findings revealed adaptive evolution of extreme acid tolerance driving the adaptation of acidophiles and thus provided an available approach for the improvement of bioleaching. Declarations Funding This work was supported by the National key R & D program Task, China (2017YFD0801304). The Heavy Metal Pollution Control Project of WEEE Dismantling Industry in Qingyuan （Phase II & III ） (No. PM-hx020-201610-0309). Competing interests Ronghui Liu and Hongbo Zhou declare that they have no conflict of interest. Availability of data and materials All data generated or analyzed during this study are included in this published article. Authors' contributions RHL carried out the research work and prepared the manuscript. HBZ performed the manuscript preparation and editing of the manuscript. Consent for publication All authors have given their consent to publish this research article. Acknowledgements This work was supported by the National key R & D program Task, China (2017YFD0801304).The Heavy Metal Pollution Control Project of WEEE Dismantling Industry in Qingyuan （Phase II & III ） (No. PM-hx020-201610-0309). References Aguirre P, Guerrero K, Sanchez-Rodriguez A, Gentina JC, Schippers A (2018) Making sticky cells: effect of galactose and ferrous iron on the attachment of Leptospirillum ferrooxidans to mineral surfaces. Res Microbiol Baker-Austin C, Dopson M (2007) Life in acid: pH homeostasis in acidophiles. 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Chemosphere 232:345–355 Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 24 May, 2021 Reviewers invited by journal 24 May, 2021 Editor invited by journal 24 May, 2021 Editor assigned by journal 17 May, 2021 First submitted to journal 13 May, 2021 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-528164","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":28898487,"identity":"8917b341-0bd0-4a55-bf81-11f659401709","order_by":0,"name":"Ronghui Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYDACCeaGAwwV/+T4GRgbiNXCCNRy5oCxZAMpWhgY2w4kbjhArLvMZzc2HuZtu5O4+fzhtgc/GOzyCGqRuXOw4TDPuWfG224kthv2MCQXE3aXRCJQSxmz7LYbjG0SPAwHEhuI08LGzLi5/2Cb5B/itbQdVtzAkNgmTbQtB+ecSTOWuAHUImOQTIyW5MMf3lTYyPH3H38m+abCjrAWNGBAovpRMApGwSgYBdgBABTSQqOTATEnAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9755-854X","institution":"Southern University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Ronghui","middleName":"","lastName":"Liu","suffix":""},{"id":28898488,"identity":"6826d95e-5beb-447a-9db3-19a6a509fff9","order_by":1,"name":"Hongbo Zhou","email":"","orcid":"","institution":"Central South University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hongbo","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2021-05-15 13:47:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-528164/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-528164/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":9611081,"identity":"f45e2fbf-8c8c-45cb-bc4b-9928608d2edf","added_by":"auto","created_at":"2021-05-26 14:24:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":44673,"visible":true,"origin":"","legend":"Phylogenetic tree analysis of the isolated L. ferriphilum","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/5526dc9a079064e6a46bff6f.png"},{"id":9610857,"identity":"b48812b1-e8e1-4f76-bd97-90d2bb15113e","added_by":"auto","created_at":"2021-05-26 14:21:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":25434,"visible":true,"origin":"","legend":"FTIR analysis of L. ferriphilum DX-m and L. ferriphilum DX-m-ALE","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/04cbf2934bbc6f40155fa2e7.png"},{"id":9610854,"identity":"37a60507-1af1-478f-a595-d2f03d6a420e","added_by":"auto","created_at":"2021-05-26 14:21:25","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":66905,"visible":true,"origin":"","legend":"The growth of starting and evolved strain under different biochemical factors condition (Solid: Fe2+ or IOR; Open: Biomass; A: Culturing L. ferriphilum DX-m at different pH value; B: Culturing L. ferriphilum DX-m-ALE at different pH value; C: IOR and biomass production for L. ferriphilum DX-m and L. ferriphilum DX-m-ALE at different temperature (L.f 1.6: Culturing L. ferriphilum DX-m at pH 1.6; ALE L.f-1.6: Culturing L. ferriphilum DX-m-ALE at pH 1.6; ALE L.f-0.7: Culturing L. ferriphilum DX-m-ALE at pH 0.7); D: IOR and biomass production for L. ferriphilum DX-m at pH 1.6 and different concentration of ferrous iron; E: IOR and biomass production for L. ferriphilum DX-m-ALE at pH 1.6 and different concentration of ferrous iron; F: IOR and biomass production for L. ferriphilum DX-m-ALE at pH 0.7 and different concentration of ferrous iron)","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/16c27ad5cf2d40864eb4aa8d.png"},{"id":9611079,"identity":"b458b201-2331-4cfe-8095-dafc634ed018","added_by":"auto","created_at":"2021-05-26 14:24:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":34786,"visible":true,"origin":"","legend":"Iron oxidation capacity and biomass production of starting and evolved strain under different concentrations of 9K basic salt (Solid: IOR; Open: Biomass; L.f 1.6: Culturing L. ferriphilum DX-m at pH 1.6; ALE L.f-1.6: Culturing L. ferriphilum DX-m-ALE at pH 1.6; ALE L.f-0.7: Culturing L. ferriphilum DX-m-ALE at pH 0.7; A: (NH4)SO4; B: KCl; C: K2HPO4)","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/3f19bad88ade668049e918d5.png"},{"id":9610859,"identity":"0988a050-cc38-4132-88b2-34188ecd0565","added_by":"auto","created_at":"2021-05-26 14:21:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":49276,"visible":true,"origin":"","legend":"Iron oxidation capacity and biomass production of starting and evolved strain under high concentrations salt, organic matter and heavy metals conditions (Solid: IOR or Fe2+; Open: Biomass; A: MgSO4; B: Na2SO4; C: organic matter; D: The culture of L. ferriphilum DX-m with heavy metals at pH 1.6; E: The culture of L. ferriphilum DX-m-ALE with heavy metals at pH 1.6; F: The culture of L. ferriphilum DX-m-ALE with heavy metals at pH 0.7)","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/af32c3430ec10a64dcb40e30.png"},{"id":9611080,"identity":"79244664-01b3-4c51-be78-1616d9e909c2","added_by":"auto","created_at":"2021-05-26 14:24:26","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":53578,"visible":true,"origin":"","legend":"the biochemical parameters during pyrite and PCB bioleaching process (Colum: metals concentration; Line: dissolution efficiency; A: the extracted total iron from pyrite. B: Changes of Ph during pyrite bioleaching; C: PCB bioleaching by L. ferriphilum DX-m and L. ferriphilum DX-m-ALE; D: PCB bioleaching by adapted L. ferriphilum DX-m and adapted L. ferriphilum DX-m-ALE)","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/e70f6078106e6f8846e45b8c.png"},{"id":15672685,"identity":"0287fde9-2b6c-461e-b91d-d1224629647a","added_by":"auto","created_at":"2021-11-18 14:13:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":434972,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-528164/v1/3465fc38-4597-42a5-8777-428b75f25a0c.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eGrowth in Ever-Increasing Acidity Condition Enhanced the Adaption and Bioleaching of \u003cem\u003eLeptospirlium Ferriphilum\u003c/em\u003e\u003c/p\u003e","fulltext":[{"header":"1.\tIntroduction","content":"\u003cp\u003eAcidophiles were the most efficient community on the metals bioleaching from sulfide minerals. Recent disclosures demonstrated that these microorganisms still conducted crucial function for metals extraction from solid waste such as waste printed wiring board (Xia et al., 2017), electroplating sludge (Zhou et al., 2019) and coal fly ash (Fan et al., 2019). However, the fickle bioleaching environment such as fluctuated pH and the increased metal ion was detrimental to the growth of microorganisms and thus weakened the biological effects, which resulted in low leaching efficiency.\u003c/p\u003e\n\u003cp\u003eIn order to enhance the bioleaching efficiency, our previous studies developed an extreme acid tolerance consortium through three steps adaptive evolution (Liu et al., 2019b) and subsequent research revealed that the obtained consortia could extract near 100% metals from waste resin powder at high pulp density (Liu et al., 2020). For adaptation, the synergetic interaction in this consortium plays an important role in the stable maintenance of function in wider conditions (Liu et al., 2019b). However, the adaptive evolutionary community on the effect of a single microorganism for environmental adaptations is unknown. An in-depth analysis of these problems can not only deepen the understanding of the characteristics of the adaptive evolutionary communities but also contribute to the further development and utilization of adaptive evolutionary strains.\u003c/p\u003e\n\u003cp\u003eIn this study, the main strain in the extreme acid tolerance community was isolated with a low pH gradient dilution method. Comparative investigation of original and adapted strain on growth at wider pH, temperature, concentration of metal ion, 9K basic salt and organic matters were explored. After that, the bioleaching capacity of isolated strain in wider conditions was illustrated. These findings preliminary revealed the strain evolution characteristics of adaptation and provide some new insight to the adaptive evolution of acidophiles.\u003c/p\u003e"},{"header":"2. Materials And Methods","content":"\u003cp\u003e\u003cem\u003e2.1 Medium and strains \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLeptospirillum ferriphilum \u003c/em\u003eDX-m was isolated from the main bio-heap of Dexing copper mine and reserved by our laboratory. The extreme acid adaptive community used in this study was obtained through a modified adaptive evolution process by our laboratory (Liu et al., 2019b). The medium used for the culture of community and pure culture was described as previous reports (Liu et al., 2019b; Liu et al., 2019c).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.2 Isolation of the evolved strain\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies revealed the extreme acid adaptive community mainly composed of \u003cem\u003eSulfobacillus \u003c/em\u003espp., \u003cem\u003eLeptospirillum\u003c/em\u003e spp. and \u003cem\u003eFerroplasma\u003c/em\u003e spp.(Liu et al., 2019b). During isolating process, consideration of the indispensability of organic matter for the quick growth of both\u003cem\u003e Sulfobacillus\u003c/em\u003e spp. and \u003cem\u003eFerroplasma\u003c/em\u003e spp.(Zhang et al., 2015; Zhou et al., 2015), selective liquid medium without the addition of organic matter was used to achieve the separation of \u003cem\u003eL. ferriphilum\u003c/em\u003e. In order to prevent the interference of some non-acid-resistant mutants or reverting mutants, a low pH strategy was employed during the gradient dilution process. Simply, the obtained adaptive evolutionary community was inoculated into a shaking flask containing a selective medium (9K inorganic salt, 350 mM Fe\u003csup\u003e2+\u003c/sup\u003e and pH 0.7) after gradient dilution. After 85% ferrous iron was oxidized to ferric iron, cells were collected by centrifugation and DNA was extracted by the DNA extraction kit for further molecular identification by PCR and 16S sequencing. The common and specific primers used in this study were same as previous studies (Liu et al., 2019b; Ma et al., 2018).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.3 Growth characteristic of starting and evolved strains on different biochemical parameters conditions \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of biochemical parameters on the growth of microorganisms are extremely important for the application of acidophiles (Shiers et al., 2016). This experiment explored effects of ferrous iron concentration (5-30 gL\u003csup\u003e-1\u003c/sup\u003e), pH (0.6-1.8), temperature (25-45\u0026deg;C) and 9K basic salt ( (NH\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e : 05-3 gL\u003csup\u003e-1\u003c/sup\u003e; K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e: 0.05-0.5 gL\u003csup\u003e-1\u003c/sup\u003e; KCl: 0-0.01 gL\u003csup\u003e-1\u003c/sup\u003e) on the growth of starting strain and evolved strain under high and low pH condition. During the experiment, samples were taken at an interval to determine the iron oxidation rate and biomass.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.4 Tolerance experiment of starting strain and evolved strain\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDuring the solid waste leaching process, heavy metals, magnesium ions, sodium ions and organic matter generally increased significantly (Liu et al., 2020), which notably affected the bioleaching efficiency. The tolerance of microorganisms to these factors becomes the key to accelerate metals extraction. This study explored different concentrations of magnesium sulfate (1-20 gL\u003csup\u003e-1\u003c/sup\u003e), sodium sulfate (1-20 gL\u003csup\u003e-1\u003c/sup\u003e) and yeast extract (0-1 gL\u003csup\u003e-1\u003c/sup\u003e) on the effects of iron oxidation and biomass production of starting and evolved strains. As for tolerance test of heavy metals, 1 and 5g L\u003csup\u003e-1\u003c/sup\u003e Ni、Cr、Cu and Co was adopted. The medium used in these assays was 9K medium (initial ferrous ion 10 gL\u003csup\u003e-1\u003c/sup\u003e), and the culture conditions were 45\u0026deg;C and 180 rpm. During the experiment, samples were taken at an interval to determine the changes in ferrous ions. After complete oxidation, the average iron oxidation rate was calculated and the biomass was measured. All experiments were performed in triplicates.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.5 Bioleaching tests \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBioleaching of waste printed circuit boards and pyrite at different conditions was carried out to evaluate the leaching efficiency of evolved strains. The minerals used in this were present as our previous description (Liu et al., 2019a; Liu et al., 2019b; Liu et al., 2020). Bioleaching experiments were performed in shake flask, and the factors of pH (1.6 and 1.0) and temperature (30\u0026deg;C and 45\u0026deg;C) was taken into consideration. Pyrite bioleaching assays were present as following: 2% pulp density, 10 days. For PCB bioleaching, the condition was 10% pulp density, 1g/L ferrous iron and 3 days. The 9 K basic salt medium and 10% bacterial inoculation were performed in these experiments. During bioleaching process, bioleaching tests with starting strain were set as the control, and samples were taken at an interval to determine the change in chemical parameters.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2.6 Analytical methods \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTotal cells in the supernatant were determined by direct counts (blood cells counting chambers: XB-K-25, QiuJing, Shanghai, China; Microscope: BX-41TF, Olympus, Tokyo, Japan). The total iron and ferrous iron were determined by a spectrophotometer with the o-phenanthroline (TGI, Shanghai, China) spectrophotometry assay (1500, Thermo, USA). The Cu contents in solutions were analyzed using an inductively coupled plasma-optical emission spectrometer (ICP-OES) (Optima 5300 DV, PerkinElmer Instrument). The pH of the supernatant was measured with a pH meter (PH-3S) (pHS-3C, Leici, Shanghai, China). The data analysis was performed by SPSS version 24.0 (SPSS Inc., Chicago, IL, USA).\u003c/p\u003e"},{"header":"3 Results And Discussions ","content":"\u003cp\u003e\u003cem\u003e3.1 Characteristic of acid tolerance strain\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAnalysis of the 16S rDNA sequences and construction of the phylogenetic tree based on neighbor-joining analysis were performed for molecular identification of isolated strains. The partial 16S rDNA sequence of isolated strains displayed 100% identity with corresponding sequences of \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m (Fig. 1). And the isolated strain was therefore named \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE. Surface groups analysis based on FTIR revealed that the peak intensity in 1080 cm\u003csup\u003e-1\u003c/sup\u003e、1545 cm\u003csup\u003e-1\u003c/sup\u003e、1640 cm\u003csup\u003e-1\u003c/sup\u003e and 2080 cm\u003csup\u003e-1\u003c/sup\u003e increased, while in 1190 cm\u003csup\u003e-1\u003c/sup\u003e、1290 cm\u003csup\u003e-1\u003c/sup\u003e、1470 cm\u003csup\u003e-1\u003c/sup\u003e、1570 cm\u003csup\u003e-1\u003c/sup\u003e and 3000 cm\u003csup\u003e-1\u003c/sup\u003e decreased (Fig. 2). These data indicating more polysaccharide like substances, -C-O-C-, -C=C- and heterocyclic compound in \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.2 Iron oxidation and biomass production of Leptospirilum ferriphilum DX-m-ALE in wider conditions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs illustrated in Fig 3A, the iron oxidation capacity of \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m gradually decreased with the decrease of pH value and no biological iron ion oxidation was observed at pH 0.6. These results indicated serious inhibition of acid on the iron oxidation of \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e (Zhang et al., 2010). In comparison, \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE completely oxidized 10 g/L iron within 48 hours in the range of pH 0.6-1.8. Meanwhile, the production of biomass showed the same trend with ferrous iron oxidation (Fig.3B). These results indicating that the acid resistance of the evolved strain \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE was improved.\u003c/p\u003e\n\u003cp\u003eThe effect of temperature on iron oxidation of both the starting strain \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m and the evolving strain \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE was presented in Fig 3C. In the case of pH 1.6, the iron oxidation rate for the evolved strain was 250 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at 30℃, while that of the starting strain was 80 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e. Even the temperature was at 25℃, the evolved strain showed 150 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1 \u003c/sup\u003eiron oxidation rate. It is obvious that the evolved strain had stronger adaptability at low temperature. In the case of pH 0.7 , the evolved strain showed the maximum iron oxidation rate of 350 mgL\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at 45℃. However, the iron oxidation rate in low pH culture was slightly lower than that in high pH culture when pH below 37℃. For biomass production, the lower the temperature, the less biomass. It is not hard to conclude that the adaptive evolutionary strain \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE showed a wider range of temperature adaptability and could maintain this superiority under the low pH environment, but the double stress of low temperature and acidity still affects the activity of the evolved strain.\u003c/p\u003e\n\u003cp\u003eThe combined effect of ferrous iron and pH on the iron oxidation for \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE and \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m was also determined (Fig 3D-F). At pH 1.6, both strains oxidized all ferrous iron within 48 hours below 5-10 g/L Fe\u003csup\u003e2+\u003c/sup\u003e. However, high concentrations of ferrous iron seriously inhibit the growth of starting strain. In the case of 30g/L Fe\u003csup\u003e2+\u003c/sup\u003e, little ferrous iron was observed to be biologically oxidized with 72 hours. In contrast, the evolved strain completely oxidized ferrous iron to ferric iron. These results demonstrated the evolved strain has a greater tolerance to Fe\u003csup\u003e3+\u003c/sup\u003e compared to the starting strain. At pH 0.7, the evolved strain showed higher iron oxidation ability when the ferrous iron concentration below 20 g/L, which may due to \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e under low pH need more energy to resist acid stress (Matsumoto et al., 2004). However, dual stress of acid and high concentration of iron ions still inhibited the iron oxidation function of the evolved strain \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE, the time to oxidize 30 gL\u003csup\u003e-1\u003c/sup\u003e Fe\u003csup\u003e2+\u003c/sup\u003e extended to 84 hours. Based on the above results, it is concluded that \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e DX-m-ALE showed greater capacity for ferrous iron oxidation under different conditions\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.3 The adaptation for the basic salt in 9K medium\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eDuring the bioleaching process, the constituent lack of 9K basic salt seriously inhibited the growth of microorganisms, but the excessive chemicals in 9Kmeidum not only caused energy waste but also lead to the production of secondary precipitates and limited the growth of cells (Gramp et al., 2008). The reasonable component and concentration of basic salt is allimportant for the application of bioleaching techniques. It was observed that the iron oxidation capacity and biomass production of the evolved \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e in different concentration of basic salt was different from the starting strain.\u003c/p\u003e\n\u003cp\u003eLittle influence on the iron oxidation rate of the starting strain was observed at 1-3 g/L ammonium salt, and the high concentration of ammonium salt may not be beneficial to the growth of starting strain (Fig. 4A). In comparison, the evolved strain showed a higher iron oxidation rate in the wider concentration of ammonium sulfate. In addition, the culture of the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e in the low pH condition showed similar trends. These results indicated little dependency on ammonium salt concentration for starting strain and evolved strain, but the evolved strain showed higher growth capacity under different ammonium salt concentrations.\u003c/p\u003e\n\u003cp\u003eThe negligible effect of KCl on iron oxidation rate was observed for both evolved and starting strains (Fig. 4B). Even in the absence of potassium chloride, the iron oxidation rate achieved for 250 mgL\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e. It was noted that the high concentration of potassium chloride improved the biomass production. Particularly in the case of pH 0.7, the higher concentration of potassium chloride, the more biomass was detected. This is possible that a higher concentration potassium ion could improve the acid resistance stress of microorganisms(Guan and Liu, 2020). It is demonstrated that the evolved strain could adapt wide range of KCl and thus would improve the growth.\u003c/p\u003e\n\u003cp\u003eThe concentration of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e had a great influence on iron oxidation and biomass of strains, and the effect of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e on the iron oxidation capacity for starting strain and evolution was similar (Fig 4C). The maximum iron oxidation rate and biomass was obtained at 0.2g/L K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. However, the evolved strain showed higher iron oxidation rate at pH 0.7. And the biomass gradually increased with the increase of the concentration of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e. This may because more potassium ions improved the acidity resistance of microorganisms (Baker-Austin and Dopson, 2007) and low pH eliminated the inhibition effect of jarosite on cells. Therefore, the 0.2 g L\u003csup\u003e-1\u003c/sup\u003e K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e is more suitable for the culture of the strain at high pH, but the increase of K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e concentration at low pH is beneficial to increase the growth rate of microorganisms. In all, the evolved strain could grow in a wider range of 9K basic salt.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3.4 The tolerance for high concentration salts, organic matter and heavy metals \u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAfter acid adaptation, \u003cem\u003eL. ferriphilum\u003c/em\u003e showed the improved tolerance for higher concentrations of magnesium sulfate (Fig.5A). And the average iron oxidation rate of evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e reached 341 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e under 10 g/L MgSO\u003csub\u003e4\u003c/sub\u003e condition. Even at 20 g/L MgSO\u003csub\u003e4\u003c/sub\u003e, the average iron oxidation of 310 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e was observed. In contrast, the average iron oxidation rate of starting \u003cem\u003eL. ferriphilum\u003c/em\u003e reached the maximum of 291 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at 5 g L\u003csup\u003e-1 \u003c/sup\u003eMgSO\u003csub\u003e4\u003c/sub\u003e. At pH 0.7, the iron oxidation rate of the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e still showed 320 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at 20 gL\u003csup\u003e-1\u003c/sup\u003e MgSO\u003csub\u003e4\u003c/sub\u003e. It was worthy to note that the higher concentration of MgSO\u003csub\u003e4\u003c/sub\u003e, the smaller the biomass produced by both the evolved strain and the starting strain, indicating that high concentration of MgSO\u003csub\u003e4\u003c/sub\u003e is detrimental to the production of biomass, previous studies also demonstrated that high concentration of Mg\u003csup\u003e2+\u003c/sup\u003e reduced the biofilm quantity (Tang et al., 2018).Therefore, the enhanced iron oxidation capacity may due to high ionic conductivity environment that improved electron transfer (Li et al., 2014).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The improved tolerance for high concentration Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e were also observed (Fig. 5B). However, the iron oxidation rate and biomass decreased gradually with the increase of sodium sulfate concentration. In case of 20 gL\u003csup\u003e-1\u003c/sup\u003e, the iron oxidation rate of the starting strain declined to 250 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e, while the iron oxidation rate of the evolved strain maintained to 308 mgL\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e. Accordingly, the enhanced performance on acid resistance was beneficial for the improvement of adaptation for sulfate. During bioleaching process, high concentration of sodium sulfate seriously limited the bioleaching efficiency through the formation of Fe(III)-precipitates (Liu et al., 2018) and inhibition effect for microorganisms (Bevilaqua et al., 2013), the adaptation of \u003cem\u003eL. ferriphilum\u003c/em\u003e in high concentration of sodium sulfate and low pH conditions indicated significant synergetic relationship between acid tolerance and environmental adaptation.\u003c/p\u003e\n\u003cp\u003eIn the absence of organic matter and high pH, the iron oxidation rate of the starting \u003cem\u003eL. ferriphilum \u003c/em\u003ewas 270 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e and that of the evolved \u003cem\u003eL. ferriphilum \u003c/em\u003ewas 325 mgL\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e (Fig 5C) However, the iron oxidation rate of the starting strain gradually decreased and only 163 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e was observed under 1g/L organic matter condition. Meanwhile, the total biomass decreased to 0.6\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL. These results indicated that the organic matter greatly inhibited the iron oxidation and microbial growth of the starting strain. In contrast, the iron oxidation rate of the evolved strain maintained over 280 mg L\u003csup\u003e-\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at same range of organic matter. Especially at low pH condition, the 310 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e IOR and 1.7\u0026times;10\u003csup\u003e8\u003c/sup\u003e cells/mL biomass was observed. These results indicated that, although organic compounds still had a strong inhibitory effect on the evolving strains, the performance on adaptation was improved. Therefore, adaptive evolution may be an available strategy to improve adaptation of\u003cem\u003e L. ferriphilum\u003c/em\u003e for organic matter. Previous research revealed that the addition of galactose in the medium can significantly improve the adhesion performance of EPS, strengthen the adsorption effect of strain (Aguirre et al., 2018), the evolutionary strain of \u003cem\u003eL. ferriphilum \u003c/em\u003eincrease in sensitivity to organic matter for the application of the bacteria also provides a new train of thought. Besides, no double inhibition effect of organic matter and acidity also revealed the probability of a similar mechanism of tolerance for acidity and organic matter.\u003c/p\u003e\n\u003cp\u003eGreat challenges for bioleaching deriving from the inhibition effect of heavy metals, such as Ni, Cu, Co and Cr seriously limited iron oxidation and cell yields of microorganism. After adaptation, this phenomenon is still serious (Fig 5D-F). In the case of nickel-containing medium, it was observed that the iron oxidation rate of both the starting strain and the evolving strain declined to 138 mg L\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e at pH 1.6, indicating that nickel ion had a serious inhibitory effect on the growth of both the evolving strain and the originating strain, and no great difference in the nickel tolerance was observed between the evolving strain and the starting strain, indicating that only the improvement of acidity tolerance can\u0026rsquo;t realize the adaptation for nickel ion. In contrast, the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e showed 166 mgL\u003csup\u003e-1\u003c/sup\u003eh\u003csup\u003e-1\u003c/sup\u003e IOR at pH 0.7. Although nickel ion still has serious influence on the growth of microorganism inhibition, the IOR increased by 24.48% compared to the high pH cultivation. The reason for the enhanced nickel ions resistance at low condition may due to acid activated the nickel operon (Tian et al., 2007), implying that the nickel resistance may due to the transcriptional control. In the presence of Cu and Co ions, it took 72 hours for both the starting strain and the evolved strain to completely oxidize 10g/L ferrous iron at pH 1.6, and 96 hours for the evolved strain in pH 0.7. The results indicated the double inhibitory effect of these two metals for \u003cem\u003eL. ferriphilum\u003c/em\u003e. It is demonstrated the resistance pathway of the strains to these two metals was not consistent with the acid-resisting pathway. No obvious ferrous iron oxidation and biomass increase were observed in the presence of Cr ions, indicating that Cr not only inhibited the iron oxidation activity of the strain but also inhibited the proliferation of the bacteria. It is concluded that the adaptability to heavy metals was not improved after acid adaptation. Therefore, it is necessary for the improvement of adaptation to heavy metals before the industrial application of acidity adapted \u003cem\u003eL. ferriphilum\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e3.5 \u003cem\u003eBioleaching of metals from PCB and pyrite\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe dissolution of pyrite is observed to vary greatly under bioleaching by starting and evolved strain (Fig. 6A). At 45\u0026deg;C, the leaching efficiency of the evolved\u003cem\u003e L. ferriphilum\u003c/em\u003e is observed to be 58% at pH 1.6. In comparison, that of the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e displayed superior pyrite leaching at pH 1.0, the soluble iron achieved for 8.43 g/L (79.52%). When the temperature of the leach environment was 30\u0026deg;C, the low pH leaching performance of the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e was inferior to the case of pH 1.6, suggesting that double stress of low temperature and acidity was detrimental to the bioleaching activity of the evolved strain. Meanwhile, the release of iron ion favored the production of acidity (Fig. 6B). All in all, the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e would be advantageous to leach pyrite for the generation of H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and Fe\u003csup\u003e3+\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eAs for PCB bioleaching, no notable difference was observed at pH 1.6 for the cases of the evolved and starting strain (Fig. 6C). The Cu extraction efficiency reached 83% and 88%, respectively. Similar with pyrite bioleaching, low temperature bioleaching environment showed the lesser metal extraction. Notable observation indicated that bioleaching at low pH seriously inhibited the dissolution of Cu. Our previous studies demonstrated that acid catalyst coupling bioleaching could significantly enhance the Cu extraction from PCBs(Liu et al., 2020), together revealed the importance of heavy metal resistance for the application of \u003cem\u003eL. ferriphilum\u003c/em\u003e. After adaptation, the acidity adapted \u003cem\u003eL. ferriphilum\u003c/em\u003e realized the maximum Cu extraction at pH 1.0 (Fig. 6D), agreeing with previous results that low pH bioleaching eliminated the jarosite barrier and improved the interfacial reaction (Liu et al., 2020). In this regard, it demonstrated that extreme acid tolerance microorganisms would be more promising for the development of bioleaching techniques.\u003c/p\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eIn this study, \u003cem\u003eL. ferriphilum\u003c/em\u003e that could survive in pH 0.7 was obtained from an adaptive evolution community. It showed the enhanced adaptability to acidity, temperature and iron ions. Meanwhile, its growth is slightly affected by the wider range of 9K inorganic salt component, and no notable difference in growth was observed at low concentration of ammonium sulfate, dipotassium hydrogen phosphate and potassium chloride. Although the growth of the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e was inhibited by high concentration of organic matter and salt ions to some extent, its tolerance was significantly improved. However, heavy metals still showed a serious inhibitory effect on the growth and iron oxidation of acidity adapted \u003cem\u003eL. ferriphilum\u003c/em\u003e under the condition of low pH. Compared with starting strain, the evolved \u003cem\u003eL. ferriphilum\u003c/em\u003e also demonstrated stronger leaching ability before and after adaptation. These findings revealed adaptive evolution of extreme acid tolerance driving the adaptation of acidophiles and thus provided an available approach for the improvement of bioleaching.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National key R \u0026amp; D program Task, China (2017YFD0801304). The Heavy Metal Pollution Control Project of WEEE Dismantling Industry in Qingyuan （Phase II \u0026amp; III ） (No. PM-hx020-201610-0309).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRonghui Liu and Hongbo Zhou declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRHL carried out the research work and prepared the manuscript. HBZ performed the manuscript preparation and editing of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have given their consent to publish this research article.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National key R \u0026amp; D program Task, China (2017YFD0801304).The Heavy Metal Pollution Control Project of WEEE Dismantling Industry in Qingyuan （Phase II \u0026amp; III ） (No. PM-hx020-201610-0309).\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAguirre P, Guerrero K, Sanchez-Rodriguez A, Gentina JC, Schippers A (2018) Making sticky cells: effect of galactose and ferrous iron on the attachment of Leptospirillum ferrooxidans to mineral surfaces. Res Microbiol\u003c/p\u003e\n\u003cp\u003eBaker-Austin C, Dopson M (2007) Life in acid: pH homeostasis in acidophiles. Trends Microbiol 15(4):165\u0026ndash;171\u003c/p\u003e\n\u003cp\u003eBevilaqua D et al (2013) Effect of Na-chloride on the bioleaching of a chalcopyrite concentrate in shake flasks and stirred tank bioreactors. Hydrometallurgy 138:1\u0026ndash;13\u003c/p\u003e\n\u003cp\u003eFan X-l et al (2019) Extraction of Al and Ce from coal fly ash by biogenic Fe\u003csup\u003e3+\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e. Chem Eng J 370:1407\u0026ndash;1424\u003c/p\u003e\n\u003cp\u003eGramp JP, Jones FS, Bigham JM, Tuovinen OH (2008) Monovalent cation concentrations determine the types of Fe(III) hydroxysulfate precipitates formed in bioleach solutions. Hydrometallurgy 94(1\u0026ndash;4):29\u0026ndash;33\u003c/p\u003e\n\u003cp\u003eGuan NZ, Liu L (2020) Microbial response to acid stress: mechanisms and applications. Appl Microbiol Biot 104(1):51\u0026ndash;65\u003c/p\u003e\n\u003cp\u003eLi X, Mercado R, Berlinger S, Banta S, West AC (2014) Engineering Acidithiobacillus ferrooxidans Growth Media for Enhanced Electrochemical Processing. Aiche J 60(12):4008\u0026ndash;4013\u003c/p\u003e\n\u003cp\u003eLiu F et al (2018) Significance of jarosite dissolution from the biooxidized pyrite surface on further biooxidation of pyrite. Hydrometallurgy 176:33\u0026ndash;41\u003c/p\u003e\n\u003cp\u003eLiu R, Chen J, Zhou W, Cheng H, Zhou H (2019a) Insight to the early-stage adsorption mechanism of moderately thermophilic consortia and intensified bioleaching of chalcopyrite. Biochem Eng J 144:40\u0026ndash;47\u003c/p\u003e\n\u003cp\u003eLiu R et al (2019b) Enhancing microbial community performance on acid resistance by modified adaptive laboratory evolution. Bioresource Technol 287:121416\u003c/p\u003e\n\u003cp\u003eLiu R et al (2019c) Selective removal of cobalt and copper from Fe (III)-enriched high-pressure acid leach residue using the hybrid bioleaching technique. J Hazard Mater\u003c/p\u003e\n\u003cp\u003eLiu RH, Wang W, Zhou WB, Cheng HN, Zhou HB (2020) Acid catalysis coupling bioleaching for enhancement of metals removal from waste resin powder. Journal of Cleaner Production, 247\u003c/p\u003e\n\u003cp\u003eMa L, Wang X, Liu X, Wang S, Wang H (2018) Intensified bioleaching of chalcopyrite by communities with enriched ferrous or sulfur oxidizers. Bioresour Technol 268:415\u0026ndash;423\u003c/p\u003e\n\u003cp\u003eMatsumoto M, Ohishi H, Benno Y (2004) H+-ATPase activity in Bifidobacterium with special reference to acid tolerance. Int J Food Microbiol 93(1):109\u0026ndash;113\u003c/p\u003e\n\u003cp\u003eShiers DW, Collinson DM, Watling HR (2016) Life in heaps: a review of microbial responses to variable acidity in sulfide mineral bioleaching heaps for metal extraction. Res Microbiol 167(7):576\u0026ndash;586\u003c/p\u003e\n\u003cp\u003eTang D et al., 2018. Mg2 + reduces biofilm quantity in Acidithiobacillus ferrooxidans through inhibiting Type IV pili formation. FEMS Microbiol. Lett., 365(4)\u003c/p\u003e\n\u003cp\u003eTian J et al (2007) Nickel-resistant determinant from Leptospirillum ferriphilum. Appl Environ Microb 73(7):2364\u0026ndash;2368\u003c/p\u003e\n\u003cp\u003eXia MC et al (2017) Recycling of metals from pretreated waste printed circuit boards effectively in stirred tank reactor by a moderately thermophilic culture. J Biosci Bioeng 123(6):714\u0026ndash;721\u003c/p\u003e\n\u003cp\u003eZhang LJ et al (2015) Synergetic effects of Ferroplasma thermophilum in enhancement of copper concentrate bioleaching by Acidithiobacillus caldus and Leptospirillum ferriphilum. Biochem Eng J 93:142\u0026ndash;150\u003c/p\u003e\n\u003cp\u003eZhang RY et al (2010) A new strain Leptospirillum ferriphilum YTW315 for bioleaching of metal sulfides ores. T Nonferr Metal Soc 20(1):135\u0026ndash;141\u003c/p\u003e\n\u003cp\u003eZhou D et al (2015) Expression of Critical Sulfur- and Iron-Oxidation Genes and the Community Dynamics During Bioleaching of Chalcopyrite Concentrate by Moderate Thermophiles. Curr Microbiol 71(1):62\u0026ndash;69\u003c/p\u003e\n\u003cp\u003eZhou WB et al (2019) Cleaner utilization of electroplating sludge by bioleaching with a moderately thermophilic consortium: A pilot study. Chemosphere 232:345\u0026ndash;355\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Leptospirilum ferriphilum, bioleaching, low pH, adaptation","lastPublishedDoi":"10.21203/rs.3.rs-528164/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-528164/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLow pH could eliminate the jarosite accumulation and improve the interfacial reaction rate during bioleaching process. However, a great challenge existed between microbial activity and bioleaching ability in low pH conditions. This study demonstrated that the adaption and bioleaching of \u003cem\u003eLeptospirilum ferriphilum\u003c/em\u003e could be improved after long term adaptive evolution under acidity condition. It was found that the acidity adapted strain showed robust activity in wider pH, high concentration of ferrous iron and lower temperature. Although the enhancement for heavy metal tolerance was limited, the tolerance for MgSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e and organic matter was great. More importantly, both pyrite and printed circuit boards bioleaching revealed the higher bioleaching ability of the acid-resistant strain. These adaptation and bioleaching details provided an available approach for the improvement of bioleaching techniques.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Growth in Ever-Increasing Acidity Condition Enhanced the Adaption and Bioleaching of Leptospirlium Ferriphilum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-05-26 14:21:24","doi":"10.21203/rs.3.rs-528164/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-05-24T05:40:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-05-24T04:50:00+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"International Microbiology","date":"2021-05-24T04:29:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-05-17T04:38:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Microbiology","date":"2021-05-13T11:45:35+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-microbiology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"intm","sideBox":"Learn more about [International Microbiology](https://www.springer.com/journal/10123)","snPcode":"10123","submissionUrl":"https://submission.nature.com/new-submission/10123/3","title":"International Microbiology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0728e872-7ea2-45b9-a9e4-f5cdd2ec375e","owner":[],"postedDate":"May 26th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":4581518,"name":"General Microbiology"}],"tags":[],"updatedAt":"2021-12-03T07:33:24+00:00","versionOfRecord":[],"versionCreatedAt":"2021-05-26 14:21:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-528164","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-528164","identity":"rs-528164","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}