Microsoft Word - 02Revised.doc CHEMICAL ENGINEERINGTRANSACTIONS VOL. 55, 2016 A publication of The Italian Association of Chemical Engineering Online at www.aidic.it/cet Guest Editors:Tichun Wang, Hongyang Zhang, Lei Tian Copyright © 2016, AIDIC Servizi S.r.l., ISBN978-88-95608-46-4; ISSN 2283-9216 Selection of Heavy Metal Zinc Adsorption Strains in Contaminated Soil Zhuo Yang Hebei University of Environmental Engineering, Qinghuangdao, Hebei 066000, China. yangzhuo315566@126.com This test focuses on the screening and identification of heavy metal zinc adsorption strains in contaminated soil. Four strains with high zinc adsorption capacity are obtained by collecting contaminated soil samples, enrichment cultivation, strain isolation, and determination of adsorption rate. The strains are named ZnA-4, ZnE-3, ZnH-2 and ZnL-2 respectively. ZnH-2 strain boasts a zinc adsorption rate of 48.30%, higher than that of any other strain, followed by ZnL-2 with an adsorption rate of 42.95%. With conventional bacteria identification method, the four strains are preliminarily indentified as Bacillus.spp based on the two aspects of colony morphology & characteristics and physiological & biochemical tests. The functional strains are potential remediation agents for in-situ fixation of contaminated soil. 1. Introduction As a micronutrient necessary for the growth of animals and plants, Zinc plays an important role in regulating human immune function, maintaining the normal physiological function of the body, promoting the normal development of children, and treating of anorexia and malnutrition (Medina et al., 2005; Zhu et al., 2005; Zhou et al., 2007). With the rapid development of mining of lead, zinc and other mineral resources, metal smelting, fuel production, recent years has seen zinc and other heavy metals entering the soil environment through a variety of ways, causing serious heavy metal pollution to soil (Zhang et al., 2012). Crop failure is commonplace on zinc-contaminated soil as plants absorb zinc from the soil. To make matters worse, the zinc absorbed by the plants would pass up the food chain and ultimately endanger human health. As a result, the quality of the soil and the safety of agricultural products are of paramount importance to human health (Wang and Wei, 1995). The traditional control methods of heavy metal pollution are chemical precipitation, ion exchange, electrolysis, and membrane separation. However, traditional methods have the disadvantages of high cost, high energy consumption, and the likelihood of secondary pollution. In contrast, microbial adsorption technology, a recent and effective method for removing heavy metals from soil, has low operating cost, and no influence on soil fertility and metabolic activity, and can prevent damages to human health and environment by pollutants transfer. Thus, the technology attracts extensive attention from researchers over the years (He 2012). Aiming at improve bacteria resources for biotechnological treatment of zinc-contaminated soil and lay a scientific foundation for further research, this paper selects and isolates the bacterial strains with high efficiency in zinc adsorption, and identifies the species of the strains with strong adsorption capacity. 2. Materials and methods 2.1 Experiment instruments The following instruments are used in the experiment: MLS-3020 autoclave by Sanyo; ZHWY-211B double- deck shaker by Shanghai Zhicheng Analytical Instrument Co., Ltd.; SPX-250 intelligent biochemical incubator by Ningbo Haishu Saifu Experimental Instrument Plant; electronic balance (Adventurer) by Ohaus USA; benchtop by Suzhou Antai Airtech Co., Ltd; BC/BD-325 cryogenic refrigerator by Haier; benchtop by Sanyo; optical microscope by Nikon; 4°C refrigerator (MDF-382E) by Sanyo; 752-type ultraviolet and visible spectrophotometer by Spectrum Shanghai; TGL-16M high-speed centrifuge by Hunan Saitexiang Instrument Company; Eppendorf RS-311 micropipette by Shanghai Kenqiang Instrument Co., Ltd. DOI: 10.3303/CET1655065 Please cite this article as: Yang Z., 2016, Selection of heavy metal zinc adsorption strains in contaminated soil, Chemical Engineering Transactions, 55, 385-390 DOI:10.3303/CET1655065 385 2.2 The collection of soil samples The soil samples are collected by the five-spot method. The author gathers the contaminated soil around the sewage treatment plant, and in front of the Miaogou Iron Mine, and the bottom mud at the estuary of Xiaotang River. After that, the author mixes the soil samples, shovels off the surface soil, puts the soil at the depth of 5- 10cm into aseptic bags, records the sampling location and time, returns to the lab, and stores the bags in the 4°C refrigerator for future use. 2.3 Experiment methods 2.3.1 Strain enrichment and separation The soil suspension is prepared by mixing 5g of soil sample in Zn2+-containing fluid medium. After shaking culture for 3 days, it is transferred to fresh fluid medium until Zn2+ concentration reaches 500 mg/L. This continuous enrichment results in a highly pure, stable, enrichment culture. The acclimated strain is then spread to a solid medium into which 500mg/L of zinc chloride has been added. Single colony is repeatedly streaked on the heavy metal-containing solid medium until the pure culture is obtained. 2.3.2 Determination of strain adsorption capacity (1) Cell Preparation The activated cells are picked from the slant, shaken for 20 hours, collected, and washed with deionized water and centrifuged. Repeat the washing and centrifuging process three times, and the cells are ready for use. (2) Adsorption Test Weigh a certain amount of dry cells, add them into 50mL of Zn2+-containing aqueous solution, conduct oscillation adsorption according to different experimental requirements, and carry out centrifuging at 13000r/min for 5min. (3) Calculation Heavy metal adsorption rate is calculated with the following formula: Adsorption rate = (C0-Ct)/C0×100% Where, C0 is the initial concentration of heavy metal ions (mg/L); Ct is the equilibrium concentration of heavy metal ions (mg/L). 3. Experimental results 3.1 Stain enrichment and purification The obtained strains were numbered and streaked and purified. See Table 1 for the colony morphology. Table 1: Description of colony morphology Stain Characteristic of Colony Shape Surface Humidity Color Size Edge ZnA-1 Circular Flat Dry White Large Regular ZnA-2 Circular Convex Sticky Ivory Extra-large Regular ZnA-3 Sub-circular Flat Dry Ivory Extra-large Regular ZnA-4 Sub-circular Flat Dry White Large Jagged ZnA-5 Sub-circular Flat Dry White Large Jagged ZnB-1 Circular Convex Sticky Red Medium Regular ZnB-2 Circular Flat Wet Ivory Medium Regular ZnB-3 Circular Flat Dry White Medium Regular ZnB-4 Oval Convex Sticky White Extra-large Regular ZnB-5 Circular Flat Wet Ivory Large Regular ZnC-1 Sub-circular Flat Dry White Large Snowflake ZnC-2 Circular Flat Wet Ivory Large Regular ZnC-3 Circular Flat Sticky Ivory Large Regular ZnC-4 Circular Flat Dry White Large Jagged ZnC-5 Circular Flat Dry White Large Irregular ZnD-1 Circular Flat Dry White Extra-large Regular ZnD-2 Circular Convex Sticky Ivory Medium Regular ZnD-3 Circular Convex Wet Ivory Medium Regular ZnE-1 Irregular Convex Dry White Large Snowflake ZnE-2 Circular Flat Wet Ivory Large Regular ZnE-3 Circular Convex Wet Ivory Medium Regular 386 Stain Characteristic of Colony Shape Surface Humidity Color Size Edge ZnF-1 Circular Flat Dry Light red Extra-large Irregular ZnF-2 Circular Flat Sticky Light red Large Regular ZnF-3 Circular Flat Dry White Large Regular ZnF-4 Circular Flat Sticky Light red Medium Irregular ZnF-5 Circular Convex Sticky White Extra-large Jagged ZnF-6 Irregular Convex Sticky Light red Large Jagged ZnF-7 Circular Flat Dry Ivory Medium Regular ZnF-8 Circular Flat Dry White Extra-large Jagged ZnH-1 Irregular Convex Dry White Large Snowflake ZnH-2 Sub-circular Flat Wet Ivory Large Jagged ZnH-3 Sub-circular Flat Dry Ivory Large Regular ZnH-4 Sub-circular Flat Dry White Large Jagged ZnH-5 Circular Convex Sticky White Small Regular ZnI-1 Circular Flat Wet Ivory Extra-large Regular ZnI-2 Circular Flat Sticky White Extra-large Regular ZnI-3 Circular Flat Sticky White Medium Jagged ZnJ-1 Circular Flat Relatively Wet Ivory Large Regular ZnJ-2 Sub-circular Flat Dry White Extra-large Jagged ZnJ-3 Sub-circular Convex Sticky White Extra-large Jagged ZnK-1 Circular Convex Sticky White Medium Regular ZnK-2 Circular Flat Sticky Ivory Medium Regular ZnK-3 Circular Flat Sticky Ivory Extra-large Jagged ZnL-1 Oval Flat Wet Ivory Medium Regular ZnL-2 Circular Flat Sticky Ivory Large Regular ZnN-1 Circular Flat Wet Ivory Extra-large Regular ZnN-2 Circular Convex Wet Brown Small Regular ZnM-1 Circular Flat Sticky Ivory Large Regular ZnM-2 Irregular Convex Sticky Ivory Extra-large Irregular ZnM-3 Circular Flat Sticky White Medium Regular ZnO-1 Sub-circular Flat Sticky White Extra-large Irregular ZnO-2 Circular Flat Wet Light red Extra-large Regular ZnO-3 Circular Flat Wet Ivory Large Regular ZnO-4 Circular Flat Dry White Extra-large Regular ZnP-1 Sub-circular Flat Sticky White Large Jagged ZnP-2 Circular Flat Wet Ivory Medium Regular ZnP-3 Circular Flat Sticky White Extra-large Regular ZnP-4 Circular Convex Sticky Grey in the middle and transparent on the edge) Small Regular 3.2 Zinc adsorption test 3.2.1 Drawing of Zn2+ standard curve (1) Preparation of the developer solution Take a 4mL transparent tube, add 3mL of 4g/mL NaOH solution with a micropipette, and add 300μL of 5 g/L dithizone stock solution, stir evenly and allow the mixture to stand until two layers are formed. Then, take the upper layer as the developer solution. The developer solution should be prepared right before use. (2) Absorption Spectrum Scanning and Standard Curve Drawing Take a series of 15mL microcentrifuge tubes, add 0 μL, 10μL, 20μL, 30μL, 40μL, 50μL, 60μL, 70μL, 80μL, 90μL and 100μL of 100mg/L Zn2+ standard solution to the tubes respectively. Then, add deionized water into the tubes till the level reaches 700μL. Next, add 100μL of Triton X-100 solution, 100μL of developer solution, and 100μL of acetic acid-sodium acetate buffer solution into each tube, shake the tubes up to prepare a 1mL reaction system. With a UV-1700 spectrophotometer, measure the absorption spectrum of the reaction system at 520 nm with Zn2+ content of 0μg/mL and 1 μg/mL, respectively, and draw the standard curve. (See Figure 387 1) Use this method to determine content of metal ions in the solution after adsorption and calculate the adsorption rate (Yu et al., 2004; Li et al., 2007). Figure 1: Zn2+ Standard Curve (2) Results of Zn2+ Adsorption by Strains See Table 2 for the adsorption rate of Zn2+ by strains. Out of the 58 zinc resistant strains that have been screened out, 34 are capable of absorbing zinc. ZnH-2 strain boasts a zinc adsorption rate of 48.30%, higher than that of any other strain, followed by ZnL-2 with an adsorption rate of 42.95%., ZnE-3, and ZnA-4. As these four strains are more valuable for the remediation of zinc-contaminated soil, the author studies the biological characteristics of them. Table 2: Results of Zn2+ Adsorption by Strains Strain Adsorption Rate% Strain Adsorption Rate% ZnB-2 6.005 ZnB-1 3.133 ZnD-3 7.050 ZnM-3 3.303 ZnP-4 3.394 ZnK-2 1.697 ZnD-1 1.305 ZnB-5 1.567 ZnE-3 11.88 ZnL-2 42.95 ZnA-5 5.222 ZnD-2 3.916 ZnH-3 4.83 ZnH-4 1.697 ZnH-5 3.916 ZnA-4 10.84 ZnB-6 4.569 ZnF-4 6.658 ZnC-1 5.614 ZnC-3 4.439 ZnH-2 48.30 ZnL-1 3.916 ZnP-1 9.008 ZnK-1 3.133 ZnF-7 5.352 ZnO-3 4.439 ZnE-2 5.483 ZnP-3 6.658 ZnH-1 4.178 ZnF-5 3.524 ZnJ-1 5.22 ZnF-6 1.305 ZnI-1 3.394 ZnM-2 1.305 3.3 Strain identification 3.3.1 Colony characteristics Table 3: Description of Colony Morphology Stain Colony Characteristics Shape Surface Humidity Color Size Edge ZnA-4 Sub-circular Flat Dry White Large Jagged ZnE-3 Circular Convex Wet Ivory Medium Regular ZnH-2 Sub-circular Flat Wet Ivory Large Jagged ZnL-2 Circular Flat Sticky Ivory Large Regular 388 Observing the colony morphology of the four strains of ZnA-4, Zn-3, Zn-2 and Zn-2, the author draws the preliminary conclusion that these strains are bacterial colonies which are circular/sub-circular, and white. See Table 3 and Figure 2 for detailed description. ZnA-4 ZnE-3 ZnH-2 ZnL-2 Figure 2: Colony morphology and characteristics 3.3.2 Gram staining After Gram staining, the four strains are observed under the microscope. The author concludes that all of them arte bacillus. ZnE-3 is Gram-positive bacteria and all the other three strains are Gram-negative bacteria. See Figure 3. ZnA-4 ZnE-3 ZnH-2 ZnL-2 Figure 3: Photos of gram stained strains 3.2 The results of physiological and biochemical experiments See Table 4 for detailed results. Table 4: The results of physiological and biochemical experiments Item Strain ZnA-4 ZnE-3 ZnH-2 ZnL-2 V.P test - - - - Nitrate reduction test + + + + Starch hydrolysis test + - + + Contact enzyme test + + + + Sugar fermentation test + + + + Citrate test - - - - Methyl red test + + + - Ammonia production test + - + - Phenylalanine deaminase test - - - - Malonate test + + + + Denitrification test - + + + Dextrin production test + + - + Glucose fermentation test + - + + + - + + Urease test - + + - 389 Item Strain ZnA-4 ZnE-3 ZnH-2 ZnL-2 Lipase test - + - - 3-ketolactose determination - - - - Production of pyocyanine - - - - Nitrite reduction + + + + Carbon source utilization + - + + Note: “+”≥90% indicates that the strain is positive; “–”≥90% indicates that the strain is negative. Comparing the above results to the Common Bacteria Identification Manual, the author draws the preliminary conclusion that: ZnA-4, ZnE-3, ZnH-2 and ZnL-2 are Bacillus spp. 4. Conclusion In light of the adsorption capacity of heavy metal zinc in contaminated soil, this test obtains four strains with high zinc adsorption capacity, namely ZnA-4, ZnE-3, ZnH-2 and ZnL-2 respectively. ZnH-2 strain boasts a zinc adsorption rate of 48.30%, higher than that of any other strain, followed by ZnL-2 with an adsorption rate of 42.95%. As these four strains are more valuable for the remediation of zinc-contaminated soil, the author studies the biological characteristics of them, and preliminarily identifies them as Bacillus. In the future, researchers can make further studies on how to optimize the adsorption capacity of the strains. Reference He Q.X. 2012. Review of the Remediation Technology of Soils Pollution by Heavy Metals. Guangzhou Chemical Industry, 40(22): 44-46. Li Y.P., Liu X.D., Gong H.L. 2007. Adsorption Characteristics of Soils for Lead, Copper, Zinc and Cadmium. Rock And Miner A analysis, 26(6):455-459. 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