ABSTRACT. A soil perfusion system was used to measure Zn (as Zn2÷) im- mobilization by soil microorganisms in Rubicon sand (Entic Haplor- thods, pH 5.9).
1Biological
Immobilization of Zinc and Manganesein Soil 2B. ZAMANI, B. D. KNEZEK, AND F. B. DAZZO
ABSTRACT A soil perfusion system was used to measureZn(as Zn2÷) immobilizationby soil microorganisms in Rubiconsand(Entic Haplorthods, pH5.9). A 240-/~4Znsolution (320 mL)wascontinuouslyperfusedthrough25 g of gamma-irradiated (sterilized) or biologicallyactive soil. At steady-state,approximately 75.0% of the perfusateZnbecameboundto the soil columnby chemicaland physical mechanisms understerile conditions.Theintroductionof soil microorganisms and a sourceof Candenergyto the sterile soil column resultedin an additional 20.5% reductionof Znin the soil perfusate(significantdifferenceat the 99% level) to a newsteady-statelevel of 10.8p.Mafter a 72hr period. Furtherperfusiondid not removeadditionalZu. Thenutrient amendment alone did not contributeanysignificant immobilization of Zn. Thus, underconditions that are conduciveto rapid growth, zymogenoussoil microorganismscould significantly immobilize andreducethe level of the mobileZnto a level belowthat attainableby chemicalandphysicalfactorSin the soil alone.In contrast withresults in Rubicon sand,microbialimmobilization in finertexturedsoils (Brookstonclay loam--Typic Argiaquolls,pH7.5) and (Houghton mucksoil--Typic Medisaprist,pH6.0) was not detected. Biological immobilizationof Mnin Rubiconsandwas also highest whena combination of microbesand nutrients wereadded.Withthe use of the soil perfusionsystem,it is possibleto quantitatethe contribution of microorganisms to immobilization of Znand Mnin Rubiconsandreceivingutilizable organicmaterials. AdditionallndexWords:soil perfusion apparatus,~°Co-gamma irradiation,metalfixation, soil microorganisms. Zamani,B., B. D. Knezek,and F. B. Dazzo. 1984. Biological immobilization of zinc and manganesein soil. J. Environ. Qual. 13:269-273.
The role of soil microorganismsin the immobilization of micronutrients such as Zn is consideredto be insignificant whencomparedwith chemical and physical mechanisms of Znfixation in soil (16). Lindsay(15) Lucasand Knezek(16) studied Zn adsorption in calcareous soil and found that the insoluble ZnCO3 formedwas unavailable to plants. Elgabaly and Jenne (8) and Elgabaly (7) reported that a portion of the 2÷ and ÷ adsorbed on montmorillonite entered the octaZn(OH) hedral layers of clay and became nonexchangeable. Soluble soil organic matter (e.g., fulvic acid) can also influence micronutrient solubility in soil through complexation and chelation (10, 1 l, 18, 19, 21,22, 23, 25). Zinc deficiencies for crops have occurred after the topsoil was removed or following certain crop rotations. Zinc deficiencies increased because the exposed subsoil was lower in organic matter and higher in pH and carbonate than the surface soil (14). In the latter case, beans (Phaseolus vulgaris) (6) and corn (Zea mays) (14) experienced a severe Zn deficiency following the incorporation of a sugarbeet (Beta vulgaris) crop residue ~Contribution from the Dep. of Crop & Soil Sci. and Dep. of Microbiol. &Public Health, MichiganState Univ., East Lansing, MI 48824-1114.Received25 Aug.1983. 2 ResearchAssociate, Professor of Soil Sciences,Dep. of Cropand Soil Sci., and Associate Professor of Soil Microbiology,Dep. of Microbiol. and Public Health, and of Crop & Soil Sci., Michigan State Univ., respectively.
into the soil. Lindsay(14) suggested that this Zn deficiency could havebeendue to high microbialactivity, whichcompetedwith planted crops for available Zn in the soil rich in carbohydrateresidues. Although soil microorganisms are knownto immobilize heavymetals (9, 13, 25, 26, 27), the quantity immobilizedand the kinetics of this process in soil are not known.Zinc at concentrations >_ 10 mMis toxic to certain soil microorganismsand enhancesthe survival of other soil microorganismscultured in laboratory media(l). Thepresent study wasundertakento test the hypothesis that Zn could be immobilized by soil microorganisms in sufficient quantities to lower the Zn levels in the soil solution. Wewere inclined to believe prior to experimental work that this would occur when soils of low cation exchange capacity (CEC) were amended with organic wastes containing Zn and an available energy and nutrient supply, in situations similar to that in soils receiving sewage sludge. We examined several soils with a continuously perfused soil column system to test this hypothesis. MATERIALS AND METHODS Thethree different soils used in this study were Rubiconsand (sandy, mixed,frigid Entic Haplorthods),Brookstonclay loam(fineloamy, mixed, mesic Typic Argiaquolls), and Houghtonmuck(euic mesic Typic Medisaprists), with original 0.1N HCl-extractable Zn contents of 0.06, 0.17, and 0.51 mmol/kg,respectively (Table 1). Standardanalyses (5) of these soils characterizedRubiconsand as infertile, mildly acid sandysoil with a low CEC;Brookstonclay loam as a very fertile, slightly alkaline soil with a moderateCEC;and Houghtonmuckas a slightly acid, organic soil of moderatefertility with very high CEC(Tabre1). Immobilization of Zn andMnwasstudied by the use of the soil perfusion technique(24). A columncontaininga mixtureof 12.5 g sieved soil, 1 g of glass wool,and 12.5 g of whitesand waspackedinto each soil perfusionapparatus(Bellco Biological Glassware,Vineland,N.J.) ®tubingof 4, 15, (Fig. 1). This percolationunit is constructedof Pyrex 17, and 38 mmsizes, and the internal glass columnis madefrom an ordinary 15-mmPyrex®bacteriological test tube. A carefully controlled vacuumwasapplied to the apparatus to allow for continuous perfusionof 320mLof deionizeddouble-distilled waterat a rate of 25 mL/min.This circulatory action of the soil perfusion systemby controlled vacuum permittedmixingof the soil perfusatein the reservoir. In a preliminaryexperiment,the uniformityof operation for three replicate nonsterile perfusion units was examinedby measuringthe change in Zn concentration within the reservoir during continuous perfusion. A total volumeof 260 mLZn solution as ZnCl2(having an initial concentrationof 0.31rn~/) wasusedin this experiment. For sterilization, the assembled soil perfusion apparatus was wrappedin aluminumfoil and either autoclaved at 120°Cand 1.035 x 10~ Pa (15 psi) for 1 hr on 2 consecutivedays, followedby 17 hr the third day, or irradiated for 24 hr with 6°Co at a total gamma radiation dosage of 4.9 Mrads. A comparisonof both sterilization methods on subsequent Zn immobilization was done with Rubicon fine sand. A comparisonof Znimmobilizationon three different soils was then done with perfusion units sterilized by gammaradiation only. Tointroducemicrobialactivity, sterilized soil columnswereinoculated with 5 mLof a freshly preparedsoil suspensionof Rubiconsand (10-1 dilution in water) and allowedto recolonize for 31 hr at room temperature without perfusion. A similar soil suspension that had beensterilized by autoclavingwasaddedto sterile control treatments. Five milliliters of sterile reconstituted Bacto-NutrientBroth(8 g/L) (Difco Laboratories, Detroit, Mich.), whichcontained 8.40 mg J. Environ. Qual., Vol. 13, no. 2, 1984 269
Table 1-Properties of Rubicon sand, Brookston clay loam, and Houghton muck.? 0.1N HCI-extractable Particle size, % PH
Sand
Silt
Clay
Rubicon sand Brookston clay loam Houghton muck
5.9 7.5 6.0
88.5 32.5
4.4 32.4
7.1 35.1
-
Zn
%
Soil series
Organic matter
Mn
-mmoYkg 0.06 0.17 0.51
1.1 2.8 56.0
CEC cmol(p')/kg
0.09 1.2 0.11
32 175 704
t All values are the average of three replications. 214.2 183.6 i53.0
0.00
n
]I
unit 1
0 unit2 0 Unit3
I 30
0
2
6 12 18 24 Time after Zn addition to the soil column (hrs)
Fig. 2-The performance consistency of three soil perfusion units. A solution of Zn was added to each Rubicon sandy soil reservoir (not sterilized), and the Zn concentration in the reservoir during continuous operation was determined by flame atomic absorption spectrophotometry. manipulations of the perfusion apparatus were done with aseptic technique to prevent microbial contamination. Sterility tests of the perfusion apparatus were made periodically by inoculating soil from the column and perfusate samples from the reservoir into sterile tubes of Difco Fluid Thioglycollate Broth and incubating the tubes at 22OC for 1 week to test for the presence of contaminating aerobic and anaerobic microorganisms. Each perfusion experiment was replicated three times, and the average values were reported. The values of Zn concentration in the reservoir of three different soils were subjected to an analysis of variance. The experiment was arranged in a split-plot design with three replications. The main plot was a treatment consisting of four combinations of inoculation with microoganisms and amendment with nutrient broth. The subplot consisted of sampling time, which occurred at 0, 0.5, 1, 2, 4, 12, 24, 36, 48, 60, and 72 hr after Zn addition. Simple effects of treatment and time of sampling and the interaction between the two were compared.
RESULTS AND DISCUSSION Fig. 1-Assembled soil perfusion apparatus. 6.48 mg N (C/N ratio of 1.3:1), and 1.08 pmoles Zn, was added to some perfusion systems as a C and energy source at the time of microbial inoculation. Five milliliters of sterile deionized double-distilled water was added in place of nutrient broth to unsupplemented controls. After microbial recolonization of the soil, the perfusion system was started by applying a partial vacuum and 5 mL of a filter-sterilized solution of 15.4 mM ZnCI2 or MnSO. was added to the top of the column (see Fig. 1) instead of directly to the reservoir. The advancing front of the solution eluted from the column within 1 to 2 hr. Periodically during continuous perfusion, I-mL samples of perfusate in the reservoir were removed aseptically through the side-tube (see Fig. 1) and immediately replaced with 1 mL of sterile deionized double-distilled water. (The 1/320 dilution factor did not affect the outcome of the experiment.) These perfusate samples were subsequently diluted in IO-mL volumetric flasks with deionized double-distilled water and analyzed for Zn or Mn with a Perkin Elmer 303 flame atomic absorption spectophotometer. All sampling transfers and
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J. Environ. Qual., Vol. 13, no. 2,1984
Verification of consistent performance between the three nonsterile, unamended soil perfusion units was shown by similar kinetics of Zn removal from the effluent in the reservoirs during passage through replicate soil columns (Fig. 2). Maintenance of sterility of the soil column and reservoir was verified by negative results in microbial growth tests, thus eliminating the contribution of microbial immobilization of Zn to the loss of Zn2+in the perfusion solution of sterile units. A separate, nonsterile perfusion system containing quartz sand and glass wool only (no soil) did not immobilize Zn (data not shown). Data presented in Fig. 3 and 4 represent the kinetics of Zn2+removal from perfusion systems that had been previously sterilized by autoclaving and by gamma radiation, respectively. Both systems showed that sterile, nutrient-amended soil columns were less effective in removing Zn2+from the perfusion solution than
183°6f , 1 53,0 I
Sterile and amended Inoculated and unamended
0 Sterile and unamended ~ Sterile and amended T ~ Inoculated and unamended
107,1
l © Inoculated and amended
61.2
~t~eastsignificant dilference(LED)(0.01) for anytwomeans
45.9
0.00
3o.6l 0 ~ O0
0 Timeafter Zn addition to the soil column(hrs) Fig. 3--The influence of inoculation with soil microorganisms and sterile nutrient broth on Zn immobilization in a soil perfusion apparatus containing Rubiconsand that was sterilized by autoclaving. 91.8
46 60 72 2 24 36 Timeafter Znadditionto the soil column(hrs) Fig. 5--The influence of inoculation with soil microorganisms and sterile nutrient broth on Zn immobilization in a soil perfusion apparatus containing Rubicon sand that was sterilized by gammaradiation. 91.8-
76.5G Sterile and amended [] Sterile andamended; inoculatedat 45hrs © Inoculated and amended
~.~ 76.5
,-e
Sterile and unamended Sterile andamended Inoculated and unamended Inoculated and amended LSDnot significant
I
o OJ
