Hoilett, N 1 ., J. Yang 2 , R. J. Kremer 3 , S. H. Anderson 1 , F. Eivazi 2

1. control 1%PA ALKALINE PHOSPHATASE ACID PHOSPHATASE PI SA 1400.00 1200.00 1200.00 1000.00 1000.00 800.00 800.00 CONCENTRATION PNP(ug/g soil 600.00 600.00 400.00 400.00 200.00 200.00 0.00 0.00 2004 2005 2004 2005 YEAR YEAR M 1 2 3 4 5 6 7 8 9 10 control 1%PA PI SA Microbial Properties as Affected by in Situ Phosphate Treatments in Lead Contaminated Urban Soils Introduction Results and Discussion Figure 4 illustrates the DNA banding profiles from soils treated with and without phosphate from two sampling dates. Some dissimilarity in the number of bands representing bacterial genotypes was detected, especially between sampling dates. Greater number of bands were observed for June than in October. Most dominant (dense) bands were common among all the soils regardless of phosphate treatment. This suggests that the in situ soil treatments has no detrimental effect on microbial diversity or community structure in soil. There are more than 17,000 contaminated sites in the United States (EPA), among which numerous sites contain toxic heavy metals such as lead (Pb) and Zinc (Zn) (Yang et al., 2002). Lead contamination in soil has been Identified as a threat to human health and ecosystem (Hettiarachchi et al., 2001; Kramer et al., 2003). Application of soluble phosphates is emerging as a potentially cost-effective remedial technology for treating contaminated soils (Yang, 2002; McGowan et al., 2000; Ruby et al., 1994). Phosphate treatments that transform soil Pb to insoluble species such as pyromorphites have been proven effective in reducing soil Pb bioavailability to human and the leachability to groundwater (Casteel et al., 1996; Yang et al., 2002). The impact of in situ phosphate treatment on soil microbial growth and communities that sustain the terrestrial ecosystem and soil productivity is largely unknown and has been insufficiently evaluated. Microorganisms participate in 80-90% of all processes and reactions in soil and are one of indicators of soil health and quality. Microbe-mediated processes are the most sensitive to perturbations in soil (Nannipieri et al., 2002). Thus, measurement of microbial biomass or community can be used to assess soil degradation and the management strategy designed to reverse it (Harris 2002). The adverse effects of heavy metals in soil has often been reported to result in a reduction in microbial biomass and activity (Shi et al., 2002) or cause selection of metal-resistant genotypes of soil microbes (Delmore et al., 2002). • Total organic carbon (TOC; fig.1) and Total nitrogen (TN; fig.2) gradually increased over time for both the treatments and the control, with a slightly more rapid increase in the treated soil (78%) vs. untreated soil (60%). Throughout the experiment the carbon to nitrogen ratio (C:N ratio) averaged between 15:1 and 25:1. This would indicate that the biomass was at no time stressed for either carbon or nitrogen. Differences in TOC and TN levels (figs. 1 &2) across sample dates may be attributed to either temporal variation of microbial growth or the site spatial variability by heterogeneous application of the methods. Comparisons of three application methods indicate that the control and rotilling treatments had similar trends throughout the experiment and were in general slightly higher than either the surface applied and the pressurized injection. • Microbial population analyses presented in fig 3. showed that the microbe counts were not significantly differed regardless of lead-treated mediums and application methods, suggesting that microbes have adjusted to tolerate the high level of lead within the environment. However, the reduction of the population between 2004 and 2005 for both the lead and no lead treatments may indicate the response to changes in environmental conditions. • The enzymatic activities (figs. 5 & 6) (measured as phosphatase activity) showed similar trends to the TOC/TN measurements regardless of the acid or alkaline measurements, with a 15% increase in activity for the treated soils as compared to a 13% increase for the control soils. Large variation in 2005 by application methods could result from the site variation induced by heterogeneous application of methods. Objectives TOTAL ORGANIC CARBON TOTAL ORGANIC NITROGEN Fig. 5. Acid phosphatase enzymatic activity in soils untreated or treated with 10,000 mg of P kg-1 as H3PO4 by three application methods. This study, through field treatments, was conducted to assess the alteration of soil microbial properties induced by in situ soluble phosphate treatments in lead contaminated urban soil, in order to verify ecological safety of the soil treatment. Specific tasks included: Examine alteration of microbial diversity or community in soil due to the in situ phosphate treatments as measured by the Denaturing Gradient Gel Electrophoresis (DGGE) technique; Evaluate microbial population or biomass in the phosphate-treated soils by plating/ counting techniques and the TOC/TN analyses; and Assess the impact of the soil treatment on soil enzyme activity using the acid-alkaline phosphatase enzyme assay. Fig. 6. Alkaline phosphatase enzymatic activity in soils untreated or treated with 10,000 mg of P kg-1 as H3PO4 by three application methods. 8.00 0.60 7.00 0.50 Control 6.00 SA RT PI 0.40 Hoilett, N1., J. Yang 2, R. J. Kremer3, S. H. Anderson1, F. Eivazi2 University of Missouri-Columbia; 2Lincoln University of Missouri; 3USDA-ARS 5.00 Summary and Conclusions AVE. % CARBON 4.00 0.30 AVG. % NITROGEN 3.00 0.20 Microbe-mediated processes are the most sensitive to perturbations in soil. Measurements of soil microbial biomass or diversity would provide with good indications as to treatment effects on soil health or quality. The similarity in total organic carbon (TOC), total nitrogen (TN) and phosphatase activity for both the treated and control soils suggests that the phosphate treatment does not negatively affect the original microbial population. However shift in population size between sample years indicates sensitivity of environmental conditions. The presence of common bands in DDGE patterns across treatments would suggest that phosphate treatment has limited effect on soil bacterial diversity. Combined with the decrease in lead hazard due to the phosphate treatment, the application of phosphorus can be considered beneficial. This study provides an evidence that the in situ phosphate treatment in Pb contaminated soils has no detrimental effects on soil microbial growth and diversity and would be an environmental safe remedial strategy for safeguarding human and environment from the contamination. 2.00 0.10 1.00 0.00 0.00 2005 2004 2004 2005 YEAR YEAR Materials and Methods Fig. 1. Total nitrogen from soil treated with 0, and 10,000 mg of P kg-1 as H3PO4 bythree application methods: Rototiling (RT); Surface applied (SA); Pressurized injection (PI). Fig. 2. Total organic carbon from soil treated with 0, and 10,000 mg of P kg-1 as H3PO4 by three application methods: Rototiling (RT); Surface applied (SA); Pressurized injection (PI). • Experimental Procedures: Study site was on a 2/3-acre residential lot in Joplin City within the Jasper County Superfund Site, Southwest Missouri. The soil was contaminated with lead (Pb) by a former smelter located ¼ mile away, containing an average of 2200 mg Pb kg-1 at a range of 800-60,000 mg Pb kg-1. Field experiment consisted of 2- by 4-m plots in a randomized complete block design with four replicates of each of two H3PO4 treatment levels: untreated (control) and 10,000 mg P kg-1. Each plot was bounded by installing 25-cm tall plastic edging to prevent cross-contamination between plots. Predetermined amounts of H3PO4 (85% P2O5) that treated top 15-cm soil were applied to soil by three application methods: Surface application (SA), Pressurized injection (PI); Rototilling incorporation. Soil samples at each plot were collected at three month intervals post treatment and stored in 4oC refrigerator until microbial analyses. • Microbial Analytical Procedures: • Microbial population: Total number of microbes and isolation of microbial communities were measured by plating and counting methods as described by Wollum (1982). Culture medium amended with lead nitrate based on method by Konopka et al (1999). • Microbial Diversity: The Denaturing Gradient Gel Electrophoresis (DGGE) technique as described by Hasting (1999) was performed to describe the microbial community structure and species variation in soil based on the polymerase chain reaction (PCR)-amplified gene fragments and targeted nuecleotide sequences. • Microbial Biomass: Total organic carbon (TOC) and total nitrogen (TN) that represent microbial biomass were determined by both Shimadzu SSM 5000A and the LECO, TruSpec Analyzer • Enzyme Activity: Acid- alkaline phosphatase activity was measure using the phosphomonoesterase method developed by Eivazi and Tabatabai (1997). This procedure involves colorimetric determination of p-nitrophenol released when soil is incubated with toluene and the respective buffered substrate for one hour at 370C. BACTERIAL POPULATION 16 14 12 References 10 LN OF COLONY FORMING UNITS/g SOIL 8 Deloreme, T.A., C.E. Schwartz, J.S. Angle and Rufus L. Chaney. 2001. Distribution of Bacteria with Particle Size in Soils Containing Different Levels of Metals. Agricultural Research Services. United States Department of Agriculture . Casteel, S.W.; Blanchar, R.W.; Yang, J. 1997. Effect of phosphate treatment on the bioavailability of lead from the Jasper county site-Joplin, Missouri. Draft Report to Missouri Department of Natural Resources. EPA (Environmental Protection Agency). 1986. Air Quality Criteria for Lead, June 1986. Research Triangle Park, N.C., EPA 600/8-83-018F. (Cited in Xintaras 1992). Harris, J.A. 2002. Measurements of the Microbial Community for Estimating the Success of Restoration. European Journal of Soil Science 54:801-808 (2003). McGowan, S.L., N.T. Basta and G.O. Brown. 2000. Use of Diammonium Phosphate to Reduce Heavy Metal Solubility and Transport in Smelter- Contaminated Soil. Journal of Environmental quality 30: 493-500 (2001). Nannipieri, J. Ascher, M.T. Ceccherini, L. Landi, G. Pietramellara and G. Renella 2003. Microbial Diversity and Soil Functions. European Journal of Soil Science 54: 655-670. Ruby, Micheal V., Andy Davis and Andrew Nicholson. 1994. In Situ Formation of Lead Phosphates in soils as a Method to Immobilize Lead. Environmental Scientific Technology 28: 646-654 Yang, John. 2002. Research proposal for the use of Phosphate-Based Materials As a Cost Effective Remedial Strategy in Metal-Contaminated Areas. Lincoln University. 6 4 2 0 2005 2004 2004 2005 No lead-treated Lead-treated Fig. 3. Bacterial Plating counts for soil treated with 0 and 10,000mg Of P kg-1 as function of three application methods Fig. 4. DGGE profiles of 16S rDNA gene segments from DNA extracted from soils treated with or without 1% P. M, DNA marker bands; lane 1 control soil (Oct. 2004); lane 2, soil +1%P (Oct. 2004); lanes 3,5,7,9 control soils (June 2005); lanes 4,6,8,10 soil + 1%P (June 2005). ACKNOWLEDGMENT: This research was funded by the U.S. Environmental Protection Agency(EPA), National Center of Environmental Science (NCER), Science to Achieve Results (STAR) Program. Grant Number: RD831071(Yang).