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Apr 2019

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A Microbial Bioassay for Direct Contact Assessment of Soil Toxicity Based on Oxygen Consumption of Sulfur Oxidizing Bacteria
基于硫氧化细菌耗氧的直接接触土壤毒性的微生物检定方法   

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Abstract

A new direct contact assessment of soil toxicity using sulfur oxidizing bacteria (SOB) is proposed for analyzing the toxicity of soils. The proposed method is based on the ability of SOB to oxidize elemental sulfur to sulfuric acid in the presence of oxygen. Since sulfate ions are produced from sulfur by SOB oxidation activity, changes in electrical conductivity (EC) serve as a proxy to assess toxicity in water. However, in soil medium, EC values are not reliable due to the adsorption of SO42- ions by soils. Here, we suggest a new parameter which measures oxygen consumption by SOB for 6 hours to assess soil toxicity by using a lubricated glass syringe method. The proposed method is rapid, simple, cost- effective as well as sensitive and capable of assessing direct contact soil toxicity.

Keywords: Toxicity assessment (毒性测定), Sulfur oxidizing bacteria (硫氧化细菌), Soil toxicity assessment (土壤毒性测定), Microbial assay (微生物检定法), Whole cell toxicity (全细胞毒性), Lubricated glass syringe method (润滑玻璃注射器方法), Oxygen consumption (耗氧量)

Background

Currently, the most prevalent technologies used in detecting toxic chemicals are gas chromatography (GC), high liquid chromatography (HPLC), and atomic absorption spectroscopy (AAS) that provide accurate measurements by applying different detection principles. However, these well-established methods require both skilled personnel and expensive equipment and cannot practically measure all the toxic chemicals in soil (Brouwer, 1991; Eom et al., 2019a). In contrast, bioassays have been one of the most useful technologies for the detection of environmental toxicity. Bioassays depend on changes in the physiological responses of living organisms to toxic chemicals. Ecotoxicological tests (ET) more precisely identify the cumulative and synergistic effects of toxic contaminants even if they fail to clearly identify all toxic chemicals (Sisinno et al., 2007).

Several toxicity bioassays are based on measurements of growth inhibition, oxygen uptake, colony formation, or luminescence for screening toxicants in industrial effluents, sediments, and soils (Selivanovskaya et al., 2010). To date, few studies exist for direct contact assessment of toxicants in soil medium. To evaluate soil toxicity, both liquid phase (soil elutriates) and solid phase bioassays (direct contact tests) are commonly used (Hubálek et al., 2007). In liquid-phase bioassays, test organisms are exposed to the elutriate of toxicants previously in solid-phase after dissolution in water or organic solvents (Gälli et al., 1994; Tarradellas et al., 1996; Maxam et al., 2000). This approach provides limited information on solids-associated toxicity because, using aqueous elutriates, the elutriating process cannot accommodate the complexity of the solid-phase of soil (Ronnpagel et al., 1995; Selivanovskaya et al., 2010). Moreover, partial dissolution of toxicants in soil or synergistic effects between toxicants and an extractant can possibly underestimate or overestimate the toxicity of contaminants in soil (Ronnpagel et al., 1995; Tarradellas et al., 1996; Selivanovskaya et al., 2010).

On the other hand, direct contact toxicity tests can measure the total toxic response of diverse types of contaminants in a soil sample. A direct contact bioassay could enables the determination of actual toxicity of contaminants in a highly dynamic and complex system (soil or sediments) much better than aqueous elutriates of solids (Ronnpagel et al., 1995).

Recently, SOB bioassays have been successfully employed in water, wastewater, soil toxicity detection and assessment (Oh et al., 2011; Van Ginkel et al., 2011; Gurung and Oh, 2013; Ahmed et al., 2019, Eom et al., 2019a). Most toxicity assessment studies have been carried out in aqueous phase while few studies have investigated soil toxicity by SOB (Gurung and Oh, 2013; Ahmed et al., 2019, Eom et al., 2019b). SOB are chemoautotrophic bacteria which grow as a biofilm on the surface of elemental sulfur particles. They have the ability to oxidize sulfur (electron donor) to sulfuric acid in the presence of oxygen (electron acceptor) as shown in Eq. 1 (Oh et al., 2011; Hassan et al., 2013).



During oxidation, SOB consumes molecular oxygen and produce sulfate (SO42-) and two protons (H+). Production of H+ acidifies the medium, thereby reducing pH and increasing EC (Oh et al., 2011). Upon introduction of a toxic contaminant into the SOB reactors in aqueous media, SOB growth and oxidation becomes inhibited resulting in lower increase of EC (Hassan et al., 2013). However, the heterogeneous nature of soils and their ability to adsorb sulfate ions prevents EC in soils from increasing, making the use of EC an unreliable indicator of soil toxicity assessment using SOB. Our proposed method introduces the direct contact assessment of soil toxicity using oxygen consumption by SOB during a defined period of incubation as a new parameter.

Materials and Reagents

  1. 0.5 and 2.0 mm testing sieve (Chunggye Sanggong sa, Seoul, Korea)
  2. Sulfur granules (MIDAS-SG, Miwon Commercial Co. Ltd, Korea)
  3. 10 ml glass syringe (Truth, Top Syringe Mfg Co (P) Ltd, India)
  4. 12 L plastic container (Influent and effluent tanks)
  5. 1.3 L fabricated acrylic container (0.13 m x 0.13 m x 0.15 m)
  6. 6 DRAM CLR round glass vials 20-400TD (KD brand, catalog number: 324020-2385)
  7. Stainless steel wire test tube rack (model: TRS-2350, catalog number: ROI-11-590)
  8. 20 MMX.100 PTFE Teflon rubber stoppers (857518197L-610050-20, USA)
  9. Open top screwcaps (Daihan Scientific, WH.W240506 cap, screw, 8-425)
  10. Plastic weighing dish (Lab Korea, B17-132-034-1815-002)
  11. CuSO4·5H2O (Sigma-Aldrich, catalog number: 209198-100G)
  12. (NH4)6Mo7O24·4H2O (Daejung, catalog number: 1073-4475)
  13. FeSO4·7H2O (Sigma-Aldrich, catalog number: 7782-63-0)
  14. NH4Cl (Junsei, catalog number: 18075-0350)
  15. KCl (Daejung, catalog number: 6566-4400)
  16. NaH2PO4 (Daejung, catalog number: 7615-4400)
  17. Na2HPO4 (Daejung, catalog number: 7613-4400)
  18. NaHCO3 (Daejung, catalog number: 7566-4400)
  19. Yeast extract (Bacto, catalog number: 212750)
  20. CaCl2·2H2O (Sigma-Aldrich, catalog number: C3881-500G)
  21. MnCl2·4H2O (Daejung, catalog number: 5526-4400)
  22. ZnSO4·7H2O (Daejung, catalog number: 8607-4400)
  23. K2Cr2O7 (Daejung, catalog number: 6572-4400)
  24. CoCl2·6H2O
  25. Dish washing detergent
  26. Synthetic medium (see Recipes)

Equipment

  1. Adjustable volume micropipettes, 0-200 μl and 100-1,000 μl (Biorad Industries, USA)
  2. Refrigerated low temperature BOD incubator (JSI Industries, Korea JSBI-150C)
  3. Shaking water bath (Lab Companion BS-31 (55 L), model: SKU: V018. AAH44311K)
  4. Digital precise water bath (Daihan Scientific, Korea WB-11)
  5. Electrical conductivity meter (Lutron, model: YK-2005CD)
  6. Digital electronic scale (Scharwz, model: SCH1812S)
  7. Air pump (PhilGreen, model: BT-6500)
  8. 20-30 cm flexible air sparger for the sulfur master culture reactor (SMCR)
  9. Peristatic pump (Techno, Lab system, model: PP-150D; POOLIM. CO, Korea)
  10. Cisa BA 200N electromagnetic digital sieve shaker (Cisa Cedaceria Industrial S.L, Spain)
  11. MasterFlex peristaltic tubing

Software

  1. Sigmaplot (Systat software Inc, https://systatsoftware.com/products/sigmaplot/)
  2. Toxicalc (Tidepool Scientific Software, https://tidepool-scientific.com/ToxCalc/ToxCalc.html)

Procedure

  1. Sulfur master culture reactor (SMCR)
    1. The SMCR is maintained to provide consistent and repeatable SOB cultures attached to sulfur particles for the soil toxicity tests.
    2. Prepare a 1.3 L SMCR made of acrylic with a working capacity of 0.6 L in an incubator maintained at 38 °C.
    3. Place 500 ml of 0.5-2 mm sulfur particles in the SMCR (Figure 1A) filled with 600 ml of synthetic medium (composition is given in Recipes) and maintain at a temperature of 38 °C.
    4. Introduce air into the SMCR using a flexible air diffuser and a air pump at a flow rate of 2-3 L/min and operate the reactor in fed-batch mode in the incubator. Influent and the air pump should also be placed in the incubator to maintain the temperature of 38 ± 1 °C.
    5. Use 10 ml of aerobic return activated sludge from a wastewater treatment plant as the initial inoculum.
    6. Feed the SMCR using synthetic medium (38 °C) at a cycle of 5 min (20 ml/min) and 2 h 55 min of batch reaction mode.
    7. Alternatively, the SMCR can be operated manually using a 1 L beaker by feeding 100 ml and wasting 100 ml of the medium twice daily.
    8. Activity of SOB in the SMCR can be determined by monitoring changes in EC. An increase in EC indicates SOB growth in the SMCR. Before using the SMCR for toxicity tests, it should be operated continuously for more than 5 days in fed-batch mode.


      Figure 1. Sulfur master culture reactor (A) and toxicity test vials (B)

  2. Soil collection
    1. Remove all the gravel, concrete, and plant debris present at the sampling site.
    2. Collect the soil from a depth of 0-30 cm using a shovel or stainless steel spoon.
    3. Completely fill the soil samples in labeled containers or bags to remove any headspace and immediately transfer to the laboratory.
    4. Pass soil samples through a 2-mm sieve. Store the sieved sample in tightly sealed plastic containers until used for toxicity tests.

  3. Soil toxicity tests
    1. Place 1 g soil samples in 25 ml flat-bottomed glass vials equipped with plastic caps and Teflon-lined rubber stoppers.
    2. Add 5 ml NMB medium to the glass vials placed in a water bath set at 38 °C to maintain media temperature of 38 °C (Figure 2A).
    3. When the medium temperature reach to 38 °C, carefully transfer 10 g SOB attached sulfur particles into the glass vials kept in the water bath set at 38 °C (Figure 2A).
    4. A headspace volume of 15.0 ml is allotted for oxygen (Figure 1B). Purge the headspace with pure oxygen for 6 s.
    5. Cap the vials immediately with plastic caps and Teflon-lined rubber stoppers. Immediately transfer these prepared vials into a shaking water bath set at 38 °C and agitate at 90 rpm for 10 min.
    6. After that, briefly remove the vials from the shaking water bath. Equilibrate the pressures by inserting a 26 G needle into the Teflon rubber stoppers for 5 s and immediately re-incubate in the shaking water bath.
    7. Run two control samples: one control sample with unpolluted soil; the other, without soil (i.e., NMB media and sulfur particles contain SOB).
    8. Run the controls and test samples in triplicate. Strictly maintain the temperature (38 °C) throughout the process of sample preparation as SOB activity is temperature dependent.
    9. After 6 h incubation and agitation in the shaking water bath, measure oxygen consumption using the lubricated glass syringe method. Briefly, oxygen volume consumed by SOB can be measured by movement of the glass plunger into the syringe’s barrel. Before measuring the oxygen volume consumed by SOB in each vial, the plunger and the barrel of the glass syringe is lubricated using an aqueous solution comprised of two drops of dish liquid detergent in 100 ml of distilled water. After lubricating the syringe, the plunger is set at the 10 ml mark and the needle is then inserted parallel to the ground through the Teflon rubber stopper into the test vial. The plunger is allowed to move into the barrel of the syringe and equilibrate between the atmospheric pressures. The value on the syringe corresponds to the amount of oxygen consumed by SOB, i.e., a decrease in oxygen in the head space of the test vial (Figure 2B).
      Note: The amount of oxygen consumption in the water control should exceed 5 ml after 6 h incubation to reliably confirm toxicity in soil.


      Figure 2. Test vials containing contaminated soil, sulfur particles, and medium kept in a water bath (A) and oxygen consumption measurement by a 10 ml glass syringe (B)

Data analysis

A decrease in the oxygen consumed in the headspace of each test vial is determined by the glass syringe method. The inhibitory effect of the tested toxic chemicals on SOB activity in soil is determined by Eq. 2.



The results from the polluted soils are given in Figure 3 which clearly shows less oxygen consumption than from unpolluted soil (control). Determined inhibitions (%) for A and B samples are 89.4% ± 4.2 and 99.4% ± 0.97, respectively.


Figure 3. Oxygen consumption for the polluted soil samples

Recipes

  1. Synthetic medium
    1. Nutrient mineral buffer solution
      3.1 g/L NH4Cl
      1.3 g/L KCl
      4.22 g/L NaH2PO4
      2.75 g/L Na2HPO4
    2. Trace mineral solution
      7.34 mg/L CaCl2·2H2O
      5 mg/L FeSO4·7H2O
      2.5 mg/L MnCl2·4H2O
      2.5 mg/L ZnSO4·7H2O
      0.5 mg/L (NH4)6Mo7O24·4H2O
      0.5 mg/L CoCl2·6H2O
      0.2 mg/L CuSO4·5H2O

    The following components are added after 100x dilution of the nutrient mineral buffer solution (Recipe 1a) with distilled water (pH 6.8-7.0):
    200 mg/L NaHCO3
    5 ml/L trace mineral solution (Recipe 1b)
    50 mg/L yeast extract

Acknowledgments

This study is supported by Korea Ministry of Environment as “The SEM project : 2018002450001”

Competing interests

The authors declared no conflicts of interest.

References

  1. Ahmed, N., Ok, Y. S., Jeon, B. H., Kim, J. R., Chae, K. J. and Oh, S. E. (2019). Assessment of benzene, toluene, ethyl-benzene, and xylene (BTEX) toxicity in soil using sulfur-oxidizing bacterial (SOB) bioassay. Chemosphere 220: 651-657.
  2. Brouwer, H. (1991). Testing for chemical toxicity using bacteria: An undergraduate laboratory experiment. J Chem Educ 68(8): 695.
  3. Eom, H., Hwang, J. H., Hassan, S. H. A., Joo, J. H., Hur, J. H., Chon, K., Jeon, B. H., Song, Y. C., Chae, K. J. and Oh, S. E. (2019a). Rapid detection of heavy metal-induced toxicity in water using a fed-batch sulfur-oxidizing bacteria (SOB) bioreactor. J microbiol methods 161, 35-42.
  4. Eom, H., Ashun, E.,Toor, U. A. and Oh, S. E. (2019b). A solid-phase direct contact bioassay using sulfur-oxidizing bacteria (SOB) to evaluate toxicity of soil contaminated with heavy metals. Sensors and Actuators B: Chemical (in press).
  5. Gälli, R., Munz, C. D. and Scholtz, R. (1994). Evaluation and application of aquatic toxicity tests: use of the Microtox test for the prediction of toxicity based upon concentrations of contaminants in soil. Hydrobiologia 273(3): 179-189.
  6. Gurung, A. and Oh, S. E. (2013). Use of sulfur-oxidizing bacteria for assessment of chromium-contaminated soil. Environ Earth Sci 70(1): 139-143.
  7. Hassan, S. H., Van Ginkel, S. W. and Oh, S. E. (2013). Effect of organics and alkalinity on the sulfur oxidizing bacteria (SOB) biosensor. Chemosphere 90(3): 965-970.
  8. Hubálek, T., Vosáhlová, S., Matějů, V., Kováčová, N. and Novotný, Č. (2007). Ecotoxicity monitoring of hydrocarbon-contaminated soil during bioremediation: a case study. Arch Environ Contam Toxicol 52(1): 1-7.
  9. Maxam, G., Rila, J. P., Dott, W. and Eisentraeger, A. (2000). Use of bioassays for assessment of water-extractable ecotoxic potential of soils. Ecotoxicol Environ Saf 45(3): 240-246.
  10. Oh, S. E., Hassan, S. H. A. and Van Ginkel, S. W. (2011). A novel biosensor for detecting toxicity in water using sulfur-oxidizing bacteria. Sensor Actuat B-Chem 154(1): 17-21.
  11. Ronnpagel, K., Liss, W. and Ahlf, W. (1995). Microbial bioassays to assess the toxicity of solid-associated contaminants. Ecotoxicol Environ Saf 31(2): 99-103.
  12. Selivanovskaya, S., Galitskaya, P., Schnell, S. and Hung, Y. T. (2010). A comparison of microbial contact bioassay with conventional elutriate assays for evaluation of wastes hazard. Int J Environ Waste Manage 6(1-2), 183-196.
  13. Sisinno, C. L. S., Rizzo, A. C. L., Bulus, M. R. M., Rocha, D. A., Soriano, A. U., Vital, R. L. and Moreira, J. C. (2007). Application of ecotoxicological tests in a preliminary evaluation of soils treated on bioreactor. J Braz Soc Ecotoxicol 2(2): 157-161.
  14. Tarradellas, J., Bitton, G. and Rossel, D. (1996). Soil ecotoxicology. CRC press.
  15. Van Ginkel, S. W., Hassan, S. H., Ok, Y. S., Yang, J. E., Kim, Y.-S. and Oh, S. E. (2011). Detecting oxidized contaminants in water using sulfur-oxidizing bacteria. Environ Sci Technol 45(8): 3739-3745.

简介

提出了一种使用硫氧化细菌(SOB)对土壤毒性进行直接接触评估的新方法,用于分析土壤毒性。所提出的方法是基于SOB在氧气存在下将元素硫氧化为硫酸的能力。由于硫酸盐离子是通过SOB氧化活性从硫中产生的,因此电导率(EC)的变化可用来评估水中的毒性。然而,在土壤介质中,由于土壤中SO 4 2-离子的吸附,EC值并不可靠。在这里,我们提出了一个新参数,该参数可通过使用润滑玻璃注射器方法测量6小时SOB的耗氧量来评估土壤毒性。所提出的方法是快速,简单,具有成本效益的并且灵敏的并且能够评估直接接触土壤的毒性。
【背景】目前,用于检测有毒化学物质的最普遍技术是气相色谱(GC),高效液相色谱(HPLC)和原子吸收光谱(AAS),它们通过应用不同的检测原理可以提供准确的测量结果。但是,这些行之有效的方法既需要熟练的技术人员又需要昂贵的设备,并且无法实际测量土壤中的所有有毒化学物质(Brouwer,1991; Eom et al。,2019a)。相反,生物测定法已经成为检测环境毒性的最有用技术之一。生物测定取决于活生物体对有毒化学物质的生理反应的变化。生态毒理学测试(ET)更精确地确定了有毒污染物的累积和协同效应,即使它们无法清楚地识别出所有有毒化学物质也是如此(Sisinno et al。,2007)。

几种毒性生物测定法是基于生长抑制,氧气吸收,菌落形成或发光的测量结果,用于筛选工业废水,沉积物和土壤中的有毒物质(Selivanovskaya et al。,2010)。迄今为止,很少有研究直接评估土壤介质中的有毒物质。为了评估土壤毒性,通常使用液相(土壤洗出液)和固相生物测定(直接接触测试)(Hubálek等人,2007)。在液相生物测定法中,待测生物先溶解于水或有机溶剂中,然后在固相中暴露于有毒物质的淘洗液中(Gälli等人,1994; Tarradellas等人。,1996; Maxam et al。,2000)。这种方法提供的有关固体相关毒性的信息有限,因为使用水性淘析物,淘选过程无法适应土壤固相的复杂性(Ronnpagel等人,1995; Selivanovskaya等人。等,2010年)。此外,有毒物质在土壤中的部分溶解或有毒物质与萃取剂之间的协同作用可能会低估或高估土壤中污染物的毒性(Ronnpagel等人,1995; Tarradellas等人。,1996; Selivanovskaya et al。,2010)。

另一方面,直接接触毒性测试可以测量土壤样品中各种污染物的总毒性反应。直接接触生物测定法可以确定高度动态和复杂系统(土壤或沉积物)中污染物的实际毒性,远胜于固体水溶液的水洗液(Ronnpagel等人,1995)。

最近,SOB生物测定已成功用于水,废水,土壤毒性的检测和评估(Oh等,2011; Van Ginkel等,2011; Gurung和哦,2013年;艾哈迈德等人,2019年,Eom 等人,2019a)。大多数毒性评估研究都是在水相中进行的,而很少有研究通过SOB研究土壤毒性(Gurung和Oh,2013年; Ahmed等人,2019年,Eom等人。 ,2019b)。SOB是化学自养细菌,在元素硫颗粒的表面上以生物膜的形式生长。它们具有在氧气(电子受体)存在下将硫(电子给体)氧化成硫酸的能力,如等式1所示。1(Oh et al。,2011; Hassan et al。,2013)。

关键字:毒性测定, 硫氧化细菌, 土壤毒性测定, 微生物检定法, 全细胞毒性, 润滑玻璃注射器方法, 耗氧量



SOB在氧化过程中会消耗分子氧,并生成硫酸盐(SO 4 2-)和两个质子(H + )。H + 的产生会酸化培养基,从而降低pH值并增加EC(Oh et al。,2011)。在向水介质中的SOB反应器中引入有毒污染物后,SOB的生长和氧化受到抑制,导致EC的增加降低(Hassan et al。,2013)。但是,土壤的异质性及其吸附硫酸根离子的能力阻止了土壤中EC的增加,这使EC的使用成为使用SOB评估土壤毒性的不可靠指标。我们提出的方法引入了土壤接触毒性的直接接触评估,该评估是在一定的培养时间内使用SOB消耗的氧气作为新参数。

材料和试剂

  1. 0.5和2.0毫米测试筛(Chunggye Sanggong sa,首尔,韩国)
  2. 硫磺颗粒(MIDAS-SG,韩国美原商业有限公司)
  3. 10 ml玻璃注射器(印度Truth,Top Syringe Mfg Co(P)Ltd)
  4. 12 L塑料容器(进水和出水槽)
  5. 1.3升制成的丙烯酸容器(0.13 mx 0.13 mx 0.15 m)
  6. 6个DRAM CLR圆形玻璃小瓶20-400TD(KD品牌,目录号324020-2385)
  7. 不锈钢丝试管架(型号:TRS-2350,目录号:ROI-11-590)
  8. 20 MMX.100 PTFE铁氟龙橡胶塞(857518197L-610050-20,USA)
  9. 开顶螺帽(Daihan Scientific,WH.W240506螺帽,螺钉,8-425)
  10. 塑料秤盘(韩国实验室,B17-132-034-1815-002)
  11. CuSO 4 ·5H 2 O (Sigma-Aldrich,目录号:209198-100G)
  12. (NH 4 ) 6 Mo 7 O 24 ·4H 2 O (大荣,目录号:1073-4475)
  13. FeSO 4 ·7H 2 O(Sigma-Aldrich,目录号:7782-63-0)
  14. NH4Cl(纯正,目录号:18075-0350)
  15. KCl(大荣,目录号:6566-4400)
  16. NaH 2 PO 4 (大中,目录号:7615-4400)
  17. Na2HPO 4 (大中,目录号:7613-4400)
  18. NaHCO3(大田,目录号:7566-4400)
  19. 酵母提取物(酵母菌,目录号:212750)
  20. CaCl 2 ·2H 2 O(Sigma-Aldrich,目录号:C3881-500G)
  21. MnCl 2 ·4H 2 O(大中,目录号:5526-4400)
  22. ZnSO 4 ·7H 2 O(大中,目录号:8607-4400)
  23. K2Cr2O7(大荣,目录号:6572-4400)
  24. CoCl 2 ·6H 2 O
  25. 洗碗精
  26. 合成培养基(请参见食谱)

设备

  1. 可调体积的微量移液器,0-200μl和100-1,000μl(美国Biorad Industries)
  2. 冷藏低温BOD培养箱(JSI Industries,韩国JSBI-150C)
  3. 摇动水浴(Lab Companion BS-31(55 L),型号:SKU:V018。AAH44311K)
  4. 数字精密水浴锅(大韩科学,韩国WB-11)
  5. 电导率仪(路创,型号:YK-2005CD)
  6. 数字电子秤(Scharwz,型号:SCH1812S)
  7. 气泵(PhilGreen,型号:BT-6500)
  8. 用于硫磺主培养反应器(SMCR)的20-30 cm柔性空气喷雾器
  9. 蠕动泵(Techno,实验室系统,型号:PP-150D;韩国POOLIM。CO)
  10. Cisa BA 200N电磁数字筛振动筛(西班牙Cisa Cedaceria Industrial SL)
  11. MasterFlex蠕动管

软件

  1. Sigmaplot(Systat软件公司, https://systatsoftware.com/products/sigmaplot/ )
  2. Toxicalc(Tidepool科学软件, https://tidepool-scientific.com/ToxCalc/ToxCalc.html)

程序

  1. 硫主培养反应器(SMCR)
    1. 维护SMCR可以为土壤毒性测试提供附着在硫颗粒上的一致且可重复的SOB培养物。
    2. 在保持在38°C的培养箱中,准备工作容量为0.6 L的1.3 L由丙烯酸制成的SMCR。
    3. 将500 ml 0.5-2 mm的硫颗粒放入装有600 ml合成培养基(配方中给出)的SMCR中(图1A),并保持在38°C的温度下。
    4. 使用柔性空气扩散器和气泵以2-3 L / min的流速将空气引入SMCR,并在培养箱中以分批进料模式运行反应器。进水口和气泵也应放置在培养箱中,以保持38±1°C的温度。
    5. 使用来自废水处理厂的10毫升需氧返回活性污泥作为初始接种物。
    6. 使用合成培养基(38°C)以5分钟(20 ml / min)和2小时55分钟的分批反应模式循环喂入SMCR。
    7. 或者,可以使用1 L烧杯手动操作SMCR,方法是每天两次喂入100 ml培养基并浪费100 ml培养基。
    8. 可以通过监测EC的变化来确定SMCR中SOB的活性。EC的增加表明SMCR中SOB的增长。在使用SMCR进行毒性测试之前,应以补料分批方式连续运行5天以上。


      图1.硫磺主培养反应器(A)和毒性测试瓶(B)

  2. 土壤收集
    1. 清除采样现场存在的所有砾石,混凝土和植物残渣。
    2. 使用铲子或不锈钢勺子从0-30厘米的深度收集土壤。
    3. 将土壤样品完全装入带标签的容器或袋子中,以除去任何顶部空间,并立即转移到实验室。
    4. 将土壤样品通过2毫米筛。将过筛的样品保存在密封的塑料容器中,直到用于毒性测试为止。
  3. 土壤毒性测试
    1. 将1克土壤样品放入配有塑料盖和特氟龙衬里橡胶塞的25毫升平底玻璃瓶中。
    2. 将5 ml NMB培养基加到置于38°C水浴中的玻璃瓶中,以保持38°C的培养基温度(图2A)。
    3. 当介质温度达到38°C时,小心地将10 g SOB附着的硫颗粒转移到保持在38°C水浴中的玻璃小瓶中(图2A)。
    4. 为氧气分配了15.0 ml的液上空间(图1B)。用纯氧吹扫顶部空间6秒钟。
    5. 立即用塑料盖和衬有特氟龙的橡胶塞子盖住小瓶。立即将这些准备好的小瓶转移到设置在38°C的振荡水浴中,并以90 rpm的速度搅拌10分钟。
    6. 之后,将小瓶从振荡水浴中取出。通过将26 G的针头插入Teflon橡胶塞中5秒钟来平衡压力,然后立即在振荡水浴中重新孵育。
    7. 运行两个对照样品:一个未污染土壤的对照样品;另一种是没有土壤(即NMB介质和硫颗粒中含有SOB)。
    8. 一式三份运行对照和测试样品。由于SOB活性与温度有关,因此在整个样品制备过程中都严格保持温度(38°C)。
    9. 在振荡水浴中孵育和搅拌6小时后,使用润滑玻璃注射器方法测量氧气消耗量。简而言之,SOB消耗的氧气量可以通过将玻璃柱塞移入注射器针筒中来测量。在测量每个小瓶中SOB消耗的氧气量之前,使用在100毫升蒸馏水中的两滴洗碗液洗涤剂组成的水溶液对玻璃注射器的柱塞和针筒进行润滑。润滑注射器后,将柱塞设置在10 ml标记处,然后将针平行于地面通过特氟龙橡胶塞插入测试瓶中。允许柱塞移动到注射器的针筒中,并在大气压之间达到平衡。注射器上的值对应于SOB消耗的氧气量,即测试瓶顶部空间的氧气减少(图2B)。
      注意:温育6小时后,水分控制中的耗氧量应超过5 ml,以可靠地确认对土壤的毒性。


      图2.测试小瓶,其中包含污染的土壤,硫颗粒和保存在水浴中的培养基(A),并通过10毫升玻璃注射器(B)测量耗氧量

数据分析

通过玻璃注射器方法确定在每个测试瓶的顶部空间中消耗的氧气的减少。由等式确定所测试的有毒化学物质对土壤中SOB活性的抑制作用。2.



图3给出了污染土壤的结果,该结果显然显示出比未污染土壤(对照)的耗氧量少。A和B样品的测定抑制率(%)分别为89.4%±4.2和99.4%±0.97。


图3.污染土壤样品的耗氧量

菜谱

  1. 合成培养基
    1. 营养矿物质缓冲溶液
      3.1 g / L NH 4 Cl
      1.3 g / L氯化钾
      4.22 g / L NaH 2 PO 4
      2.75 g / L Na 2 HPO 4
    2. 微量矿物质溶液
      7.34 mg / L CaCl 2 ·2H 2 O
      5 mg / L FeSO 4 ·7H 2 O
      2.5 mg / L MnCl 2 ·4H 2 O
      2.5 mg / L ZnSO 4 ·7H 2 O
      0.5 mg / L(NH 4 ) 6 Mo 7 O 24 ·4H 2 O
      0.5 mg / L CoCl 2 ·6H 2 O
      0.2 mg / L CuSO 4 ·5H 2 O

用蒸馏水(pH 6.8-7.0)将营养矿物质缓冲溶液(配方1a)稀释100倍后,添加以下成分:
200 mg / L NaHCO 3
5 ml / L微量矿物质溶液(配方1b)
50 mg / L酵母提取物

致谢

这项研究得到了韩国环境省“ SEM项目:2018002450001”的支持

利益争夺

作者宣称没有利益冲突。

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引用:Ashun, E., Toor, U. A., Eom, H. and Oh, S. (2020). A Microbial Bioassay for Direct Contact Assessment of Soil Toxicity Based on Oxygen Consumption of Sulfur Oxidizing Bacteria. Bio-protocol 10(1): e3470. DOI: 10.21769/BioProtoc.3470.
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