NC State
BioResources
Cui, L., Yan, J., Yang, Y., Li, L., Quan, G., Ding, C., Chen, T., Fu, Q., and Chang, A. (2013). "Influence of biochar on microbial activities of heavy metals contaminated paddy fields," BioRes. 8(4), 5536-5548.

Abstract

Biochar (BC) amendments might decrease the bioavailability of metals in soils that are contaminated with heavy metals. In general, soil microbial communities are sensitive to changes in soil property changes. Microbial communities were tested in a Cd- and Pb-polluted paddy field in southern China. BC was applied as a basal soil amendment before rice transplantation in 2009. The BC was applied at rates of 0, 10, 20, and 40 tons per hectare. Soil heavy metal fractions with sequential extraction procedure, soil microorganisms, and enzymes were monitored in 2011. The soil pH and soil organic carbon (SOC) were significantly increased by 2% to 5% and 16% to 51% under BC amendment, respectively. Compared to the non-BC treatment, the cadmium (Cd) and lead (Pb) acid-soluble fraction concentrations were significantly decreased by 15.3% to 26.7% and 18.2% to 30.9%. The Cd and Pb reducible fraction were decreased by 13.5% to 25.6% and 21.9% to 23.53%.The Cd and Pb oxidizable fraction by 15.4% to 69.2% and 22.7% to 29.3% with BC application, respectively. The populations of actinomycetes and fungi were increased by 19.0% to 38.5% and 3.7 to 9.3 times, respectively. Meanwhile, BC significantly increased the cellulose, urine enzyme, neutral phosphatase, and sucrase activities by 117.4% to 178.3%, 31.1% to 37.6%, 29.7% to 193.8%, and 36.5% to 328.6%, respectively. BC amendment offers a basic option to reduce Cd and Pb bioavailability and change the fractions. The BC also increases microorganism quantity and soil enzyme activity.


Download PDF

Full Article

Influence of Biochar on Microbial Activities of Heavy Metals Contaminated Paddy Fields

Liqiang Cui,a Jinlong Yan,a,* Yage Yang,a,b Lianqing Li,c Guixiang Quan,a Cheng Ding,a Tianming Chen,a Qiang Fu,a and Andrew Chang d

Biochar (BC) amendments might decrease the bioavailability of metals in soils that are contaminated with heavy metals. In general, soil microbial communities are sensitive to changes in soil property changes. Microbial communities were tested in a Cd- and Pb-polluted paddy field in southern China. BC was applied as a basal soil amendment before rice transplantation in 2009. The BC was applied at rates of 0, 10, 20, and 40 tons per hectare. Soil heavy metal fractions with sequential extraction procedure, soil microorganisms, and enzymes were monitored in 2011. The soil pH and soil organic carbon (SOC) were significantly increased by 2% to 5% and 16% to 51% under BC amendment, respectively. Compared to the non-BC treatment, the cadmium (Cd) and lead (Pb) acid-soluble fraction concentrations were significantly decreased by 15.3% to 26.7% and 18.2% to 30.9%. The Cd and Pb reducible fraction were decreased by 13.5% to 25.6% and 21.9% to 23.53%.The Cd and Pb oxidizable fraction by 15.4% to 69.2% and 22.7% to 29.3% with BC application, respectively. The populations of actinomycetes and fungi were increased by 19.0% to 38.5% and 3.7 to 9.3 times, respectively. Meanwhile, BC significantly increased the cellulose, urine enzyme, neutral phosphatase, and sucrase activities by 117.4% to 178.3%, 31.1% to 37.6%, 29.7% to 193.8%, and 36.5% to 328.6%, respectively. BC amendment offers a basic option to reduce Cd and Pb bioavailability and change the fractions. The BC also increases microorganism quantity and soil enzyme activity.

Keywords: Biochar (BC); Heavy metals; Rice paddy; Soil amendment; Soil microorganisms; Soil enzyme

Contact information: a: Key Laboratory for Ecology and Pollution Control of Coastal Wetlands (Environmental Protection Department of Jiangsu Province), Yancheng Institute of Technology, 9 Yingbin Avenue, Yancheng 224051, China; b: School of the Environment, Jiangsu University, 301 Xuefu Road, Zhenjiang 212013, China; c: Institute of Resources, Ecosystem, and Environment of Agriculture, Nanjing Agricultural University, 1 Weigang, Nanjing, 210095 China; d: Department of Environmental Sciences, University of California Riverside, CA92521, USA;

* Co-first author; Corresponding author: yjlyt4788@126.com; yjlyt@ycit.cn

INTRODUCTION

Heavy metals of anthropogenic origin degrade soil quality and detrimentally affect many environments (Recatalá et al. 2010). The introduced metals have adversely impacted the ecology of microbial communities in soil. Such contamination prompts public health concerns that humans and livestock might inadvertently be exposed to potentially harmful heavy metal elements via staples harvested from contaminated soils (Chaney et al. 2004). In China, combatting cadmium contamination of rice harvested from industrial waste-tainted paddy fields has become a national priority.

Biochar, a by-product of biomass pyrolysis to generate energy, has shown promise in remediating heavy metal-contaminated soils and might reduce the potential of cadmium transfer up the food chain (Lehmann et al. 2006; Lee et al. 2010). Direct benefits of biochar amendment may include: increasing pH, organic matter content, and moisture retention of soils; enhancing the soils’ ability to adsorb heavy metals; improving soil structure and nutrient retention; and reducing N2O and CH4 emissions (Lehmann and Joseph 2009; Cao et al. 2009; Atkinson et al. 2010; Sohi et al. 2010). Cui et al. (2011, 2012) reported that crops responded to biochar application, and heavy metals contents of harvested plant tissues and grains were significantly reduced. Gomez-Eyles et al. (2011) observed significant reductions in available Cd and Cu and increases in pH two months after soils received the biochar treatments. Following amendment at rates of 4% and 8%, the biochar reduced plant-available Cu, Ni, Zn, and Pb of the affected soils. When the mobile fractions of soil Cu, Ni, Zn, Cd, and Pb were lowered, the risk of metals leaching in agricultural soils was retarded (Méndez et al. 2012). Through biochar (producing by straw, husk) was applied at rate of 5%, Cd, Zn, and Pb concentrations of rice shoots harvested at the affected soils decreased by up to 98%, 83%, and 72%, respectively, compared to that of the control in a pot experiment (Méndez et al. 2012).

Microbes (bacteria, actinomycetes, and fungi) are ubiquitous and integral parts of soils; they play significant roles in the recycling of soil-borne C, N, P, S, and metallic elements (Yang et al. 2007), making them available to plants (Rajkumar et al. 2012). Heavy metals in bioavailable forms adversely affect soil microbes by reducing their populations and changing the community structure and diversity (Renella et al. 2005). The adverse impacts are reflected in terms of decreased soil enzyme activities (Belyaeva et al. 2005) and interferences in plant-soil-metals association (Wang et al. 2008). Kandeler et al. (1996) noted that heavy metals reduce functional diversity of the affected soil microbial communities. Soil microbes by extension would reflect the overall health of the heavy metal-contaminated soils (Kennedy and Smith 1995). Activities of soil enzymes such as invertase, urease, and acid phosphatase become metrics of the soil quality (Chen et al. 2012). Ormsby et al. (2012) found that soil enzyme activity was reduced significantly in all fractions subjected to heavy metal pollution in the following order: arylsulfatase > phosphatase > urease > xylanase. The finding is corroborated by comparable effects on cellulose, invertase, sucrose, etc. (Lee et al. 2003). Soil enzyme activities are realistic, sensitive measurements of changes in the structure and diversity of the microbial community in metal-polluted soils (Kandeler et al. 2000).

If biochar is able to reduce metal uptake of plants, will the biochar amendments protect the microbial communities in heavy metal-polluted soils from harm (Wang et al. 2008)? We hypothesized that the biochar amendments in soil would enhance the health of soil microbial communities in Cd and Pb-polluted rice paddies. Our objectives were to illustrate changes of bioavailable Cd and Pb of the contaminated soils and show the corresponding changes in activities of cellulase, neutral phosphatase, urase, and arylsulfatase in relation to the biochar amendments.

EXPERIMENTAL

Site Description

The experiment was set up at a production field (31º24.434’N and 119º41.605’E) where atmospheric fallouts and effluent discharges of an iron smelter had contaminated the soils in the 1970s. Cadmium and lead were the primary pollutants. The local climate was humid subtropical with a mean annual temperature of 22 ºC and annual precipitation of 1,100 mm.

The paddy soil was characterized as ferric-accumulic stagnic anthrosols. The summer rice (Oryza sativa L.) and winter wheat (Triticum aestivum) rotation has been practiced at this location for a long time, and the heavy metals pollutants were well acclimated in the soils.

Experiment Design

The experiment followed a randomized complete block layout with three replicates for each treatment, and each plot measured 4 m x 5 m. The treatment included application of biochar at levels of 0(Control), 10, 20, and 40 metric tons per hectare in May, 2009. These biochar amendments were applied only at the beginning of the three-year study.

The biochar stock was a local product made from wheat straw pyrolyzed at 450 °C and then ground to pass through a 2-mm sieve. Upon application, the surface-deposited biochar was plowed-in and mixed thoroughly with the upper 20 cm soil. Before sowing, basal fertilizers, N, P2O5, and K2O were applied at 125, 120, and 125 kg per hectare in the forms of urea, calcium biphosphate, and KCl, respectively. Wheat (Zhenmai-5) was planted by direct seeding, and rice (Wugeng-13) seedlings were transplanted. Properties of the biochar and receiving soil were noticeably different, especially in terms of pH, carbon content, and Cd and Pb levels (Table 1).

Table 1. Chemical Properties of the 0-15 cm Depth Topsoil of the Paddy Soil and Biochar Stock

Soil Sampling and Analysis

The 0 to 15 cm depth soils were sampled after rice harvested in October, 2010. Three undisturbed cores were obtained from each plot to make one composite sample with soil collector tool of Eijkelkamp (Netherlands). Each soil sample was cleared of plant debris, air-dried at room temperature, and ground to pass through a sieve of 2 mm openings. A subsample of the soils was ground to pass through a sieve of 0.15 mm openings for Cd and Pb determinations. Soils were analyzed according to procedures described in Lu (2000).

Sequential extraction was performed using the modified four-stage procedure recommended by the European Community Bureau of Reference (BCR) (Žemberyová et al. 2006).

The total numbers of culturable heterotrophic bacteria and Colony Forming Units (CFU) of fungi and actinomycetes were determined by serial dilution and plating on selective media (Olsen and Bakken 1987; Davis et al. 2005).

Soil neutral phosphatase activity was measured spectrophotometrically by the disodium phenyl phosphate method of Wang et al. (2007). The assay for soil cellulose was adapted from Kandeler and Gerber (1988). The assay for soil urine enzyme was adapted from Guan (1986). The assay for soil sucrase was adapted from Schinner et al. (1996).

Infrared radiation spectra of biochar was detected by Fourier Transform Infrared Spectrometer (Nicolet 670, USA).

Data Processing and Statistics

All data were reported as mean standard deviation. Differences between the treatments were examined using a two-way analysis of variance. All analyses were carried out using SPSS, version 18.0.

RESULTS

Soil pH and Organic Carbon

Due to the alkaline nature of biochar, amendments significantly increased the pH of soils amended with 10, 20, and 40 ton per hectare by 0.11, 0.27, and 0.29 pH units, respectively, over that of the control treatment (Table 1). The biochar-induced pH changes were significant, roughly equivalent to 0.01 pH unit rises for 1 ton per hectare biochar up to 20 ton per hectares, beyond which the effect of biochar became less noticeable. Meanwhile, soil organic carbon content (SOC) increased by 16.2%, 33.1%, and 51.0%, respectively, over that of the experimental control.

Table 2. pH and Organic Carbon Content of Biochar Amended Paddy Soil (n=3, mean±SD)

Different lower case letters represent significant difference between the treatments.

Cd and Pb in Soils

Cd and Pb in soils were sequentially fractionated into acid soluble, reducible, oxidizable, and residual fractions. The overall mass recovery of the fractionation procedure was >90%.

Outcomes of the experimental control revealed that Cd was distributed primarily in the acid-soluble and reducible fractions (Table 3). In other words, the Cd derived from the pollution had primarily acclimated into the readily soluble mineral forms and into the oxidized state. Less than 10% of the soil-borne Cd were minerals in reduced forms such as sulfites or were bonded with the soil matrices. Lead was deposited overwhelmingly in the reducible fraction of the soil, and the acid-soluble Pb was the second largest fraction. The acid-soluble fraction was characterized by Cd and Pb minerals in carbonate forms, and these accounted for 50% and 10% of the total metals in polluted soils, respectively. The reducible fraction was characterized by Cd and Pb in association with the Fe-Mn minerals and accounted for 40% and 75% of the total metals in the polluted soils, respectively.

The biochar treatments resulted in distinctively different Cd distribution patterns. In proportion to application rates, Cd in acid-soluble and reducible fractions shifted to the residual fraction (Table 3). The shifts were clearly noticeable and were significantly higher than that of the experimental control. The Cd in acid-soluble and reducible fraction was reduced by 15.3%, 17.1%, and 26.7% and 18.8%, 13.5%, and 25.6%, respectively, over their respective experimental control with 10, 20, and 40 tons per hectares biochar treatments. The Cd contents of the oxidizable fraction were not significantly changed by the biochar treatments. Accordingly, the amount of Cd in the residual fraction proportionally increased. As the biochar amendment did not involve any reductive or oxidative reaction, it appeared that biochar possessed properties to shift a portion of the Cd from the acid soluble and reducible chemical forms in soils to be tightly bonded to the chemical matrices of biochar. Despite the biochar-induced reductions, the acid soluble and reducible forms remained the primary chemical species of Cd in the soil.

Biochar treatments again reduced the Pb in acid soluble and reducible fractions, while Pb in the oxidizible fraction remained unchanged, and the residual fraction proportionally increased (Table 3). Apparently, the same chemical mechanisms shifted the distributions of Cd and Pb in the biochar amended soils. Pb in the acid-soluble fraction was significantly decreased by 24.7%, 18.2%, and 30.9% over that of the experimental control for biochar amendments of 10, 20, and 40 tons per hectare, respectively. Pb of reducible fractions was reduced by 21.9%, 23.53%, and 22.9%, respectively. The changes had consequences on the ability of plants to absorb Cd and Pb (Cui et al. 2011).

Table 3. Cd and Pb in Fractions of Biochar Amended Paddy Soil (n=3, mean±SD, mg kg-1)

Different lower case letters represent significant differences between the treatments.

Soil Microorganism and Enzyme Activity

The biochar amendments up to 40 tons per hectare did not significantly affect the bacterial population of the Cd and Pb-polluted soils (Table 4).

Table 4. Populations of Microorganisms with BC Application in 2011(n=3, mean±SD)

Different lower case letters represent significant differences between the treatments.

The fungi and actinomycete populations of the polluted soils were significantly affected. Populations of actinomycetes in treated soils increased by 19.9%, 19.0%, and 38.5%, respectively, and populations of fungi increased by 370%, 460%, and 930%, respectively over the respective experimental controls under 10, 20, and 40 tons per hectare. It appeared that heavy metals were not harmful to the microorganisms in the polluted soils. The soil enzyme activities were assessed to determine if metals inhibit microbial functions.

Compared to the non-BC treatment, cellulose significantly increased by 117.4%, 123.2%, and 178.3%, urine enzyme by -2.6%, 31.1%, and 37.6%, neutral phosphatase by 29.7%, 73.2%, and 193.8%, and sucrase by 36.5%, 254.0%, and 328.6% in 2011 at rates of 10, 20, and 40 tons per hectare under BC application, respectively (Fig. 1).

Fig. 1. Enzyme Activity with BC Application in 2011 (n=3, mean ± SD)

DISCUSSION

The Cd and Pb-polluted soils were ideal for testing how biochar amendments mitigated the harmful effects of heavy metals on soil microbial populations and their metabolism. The experiment was carried out in a production-scale paddy field to test the hypothesis that biochar amendments would enhance microbial populations and metabo-lisms in Cd and Pb-polluted soils. The soil was contaminated by effluent discharges in the 1970s and has been cultivated without interruptions. In over 30 years, the anthropogenic metals and the inherent microbial ecosystems were all acclimated to the new status quo in the soils. The conditions of this soil were ideal for testing the hypothesis.

Biochar treatments shifted Cd and Pb in polluted soils from readily bioavailable forms to less active forms and added organic carbon into receiving soils. For biochar to enhance microbial populations and metabolisms in the Cd and Pb-polluted soils, the treatments needed to reduce the bioavailability of Cd and Pb in the receiving soils. Cui et al. (2011) reported that concentrations of CaCl2-extracted Cd and Pb in the polluted soils could vary by a wide margin, indicative of transformations that have taken place upon biochar treatments. In practice, soil-borne biochar reduced Cd phytoavailability (Fellet et al. 2011). In a more precise manner, our results showed that the biochar amendments caused the Cd and Pb in the acid-soluble sulfate and carbonate precipitates and on surfaces of the reducible Fe-Mn oxides and hydroxide forms to migrate toward the lattices of clay minerals and biochar (Table 2). Biochar accelerated the processes that the metals’ bioavailability reduced. The amendments would also be beneficial to microorgan-isms and microbial metabolisms.

The most direct impacts of biochar treatment in the receiving soils were in pH modulation and increasing stable organic matter levels (Sauve et al. 2000). Evidently, the concentrations of acid soluble Cd and Pb of the polluted soils decreased in proportion to the decreases in the receiving soils’ pH and SOC contents (Fig. 3). In this regard, biochar resembled other soil amendments, such as active carbon (Pyrzyńska and Bystrzejewski 2010), red mud (Gray et al. 2006), cyclonic ashes (Ruttens et al. 2010), and calcium magnesium phosphate (Zhang et al. 2009), that were capable of reducing metals’ activities through increased soil pH and improved metal ion occlusion. Biochars contain alkaline and macro-organic carbon-based materials (Fig. 2) possessing -NH (at 1628.25 cm-1), -OH (at 3443.78 cm-1), -PO(at 1089.20 cm-1), and -C-Cl (at 769.23 cm-1) surface functional groups that are able to form complexes with Cd and Pb (Yuan et al. 2011).

Fig. 2. Infrared radiation spectra of biochar

Fig. 3. Correlation of acid-soluble Cd and Pb fractions with soil pH and SOC

Principal Component Analysis (PCA) has been applied to indicate the effects of biochar amendment on heavy metals pollution, and the results of PCA are listed in Table 5 and Table 6. According to the results, the elements (Table 6) could be grouped into a two-component model, which accounted for 95.9% of all the data variation. Nearly all the elements were associated and showed high values in the first component (PC1), while the different fraction of the Oxidizable Cd, Residual Cd, and Oxidizable Pb were grouped into the second component (PC2). The PCA results imply that biochar amendment led to a change in soil properties, heavy metals fractions, soil enzymes, and microbial community of the slightly acid paddy soil and they affected each other in soil (Chen et al. 2013).

Table 5. Initial Eigenvalues and Extraction Sums of Squared Loadings of Principal Component (PCs) for Paddy Soil with Biochar Application

Table 6. Component Matrices of Principal Component (PCs)

With reductions of Cd and Pb bioavailability, the biochar-amended soils supported greater populations of fungi and actinomycetes. Soil microorganisms and their metabolisms are sensitive indicators of soil fertility and environmental changes (Wang et al. 2007). Bacterial, fungal, and actinomycetes populations of soils are functions of the soils’ organic carbon contents (Entry et al. 2008); a pH at the upper end of the normal range often has been observed to encourage microbial growth (Silva and Nahas 2002). However, heavy metals might alter the population size, diversity, and activities of microbes in the receiving soils (Rajapaksha et al. 2004). In this experiment, bacteria appeared to be less susceptible to heavy metals in the soils (Table 3; Plassart et al. 2008). The fungal and actinomycetes populations, however, responded to the biochar treatments and significantly increased over those of the experimental control. Again, it was the soil pH modulation and the addition of organic carbons that encouraged growth. The fungal and actinomycetes populations of the polluted soils were inversely correlated to concentrations of acid soluble Cd and Pb, which suggests this was a function of the biochar treatments (Fig. 4.).

Fig. 4. Correlation of acid-soluble Cd and Pb fractions with fungal and actinomycetes populations

Soil enzyme activities of the Cd and Pb-polluted soils were enhanced by biochar. Modulating the pH and adding organic carbon into the receiving soils were primarily responsible for the enhancement. Enzyme activities can be considered as measurements of soil health because they respond to environmental stresses such as pollution (Lee et al. 2009). Heavy metals, when present in the soils, reduce the enzyme activities (Renella et al. 2005; Kizilkaya et al. 2004) and retard soil fertility (Sivakumar et al. 2012). As the biochar amendments increased, the acid-soluble and reducible Cd and Pb of the polluted soils decreased proportionally, and cellulase activities of the soils increased accordingly. The activities of neutral phosphatase, urease, and sucrase in the polluted soils also showed considerable improvements according to the biochar treatments.

CONCLUSIONS

Biochar amendments improved the health of microbial communities and metabo-lisms of heavy metal-polluted paddy soils via modulating pH and adding organic carbon into the treated soils. Biochar has a great potential to ameliorate soils contaminated with the heavy metals and improve the soil ecosystem. Long-term effects on soil health and potential offsetting effects deserve further field monitoring studies.

ACKNOWLEDGMENTS

This study was partially supported by the Talent Introduction Plan under grants kjc2012022, Jiangsu Six Talents Peak Project (2011 Agricultural Fields) grants, Ministry of Science and Technology of China under grants 2006BAD17B06, and Open Project of Key Laboratory for Ecology and Pollution Control of Coastal Wetlands (Environmental Protection Department of Jiangsu Province) KLCW1208.

REFERENCES CITED

Atkinson, C. J., Fitzgerald, J. D., and Hipps, N. A. (2010). “Potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: A review,” Plant Soil 337, 1-18.

Cao, X., Ma, L., Gao, B., and Harris, W. (2009). “Dairy-manure derived biochar effectively sorbs lead and atrazine,” Environ. Sci. Technol. 43, 3285-3291.

Chaney, R. L., Reeves, P. G., Ryan, J. A., Simmons, R. W., Welch, R. M., and Angle, J. S. (2004). “An improved understanding of soil Cd risk to humans and low cost methods to phytoextract Cd from contaminated soils to prevent soil Cd risks,” BioMetals 17, 549-553.

Chen, J., Liu, X., Zheng, J., Zhang, B., Lu, H., Chi, Z., Pan, G., Li, L., Zheng, J., Zhang, X., Wang, J., Yu X. (2013). “Biochar soil amendment increased bacterial but decreased fungal gene abundance with shifts in community structure in a slightly acid rice paddy from Southwest China,” Appl. Soil Ecol. 71, 33-44.

Chen, R., Blagodatskaya, E., Senbayram, M., Blagodatsky, S., Myachina, O., Dittert, K., and Kuzyakov, Y. (2012). “Decomposition of biogas residues in soil and their effects on microbial growth kinetics and enzyme activities,” Biomass Bioenergy, 45, 221-229.

Cui, L., Li, L., Zhang, A., Pan, G., Bao, D., and Chang, A. (2011). “Biochar amendment greatly reduces rice Cd uptake in a contaminated paddy soil: A two-year field experiment,” BioResources 6, 2605-2618.

Cui, L., Pan, G., Li, L., Yan, J., Zhang, A., Bian, R., and Chang, A. (2012). “The reduction of wheat Cd uptake in contaminated soil via biochar amendment: A two-year field experiment,” BioResources 7, 5666-5676.

Davis, K. E. R., Joseph, S. J., and Janssen, P. H. (2005). “Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria,” Appl. Environ. Microbiol. 71, 826-834.

Entry, J. A., Mills, D., Mathee, K., Jayachandran, K., Sojka, R. E., Narasimhan, G. (2008). “Influence of irrigated agriculture on soil microbial diversity,” Appl. Soil Ecol. 40, 146-154.

Fellet, G., Marchiol, L., Delle Vedove, G., and Peressotti, A. (2011). “Application of biochar on mine tailings: Effects and perspectives for land reclamation,” Chemosphere 83, 1262-1267.

Gomez-Eyles, J. L., Sizmur, T., Collins, C. D., and Hodson, M. E. (2011). “Effects of biochar and the earthworm Eisenia fetida on the bioavailability of polycyclic aromatic hydrocarbons and potentially toxic elements,” Environ. Pollut. 159, 616-622.

Gray, C. W., Dunham, S. J., Dennis, P. G., Zhao, F. J., and McGrath, S. P. (2006). “Field evaluation of in situ remediation of a heavy metal contaminated soil using lime and red-mud,” Environ. Pollut. 142, 530-539.

Guan, S. (1986). “Soil enzyme research method,” Agriculture Press, 274-340.

Kandeler, E., Tsherko, D., Bruce, K. D., Stemmer, M., Hobbs, P. J., Bardgett, R. D., and Amelung, W. (2000). “Structure and function of the soil microbial community in microhabitats of a heavy metal polluted soil,” Biol. Fertil. Soils 32, 390-400.

Kandeler, F., Kampichler, C., and Horak, O. (1996). “Influence of heavy metals on the functional diversity of soil microbial communities,” Biol. Fertil. Soils 23, 299-306.

Kandeler, E., and Gerber, H., (1988). “Short-term assay of soil urease activity using colorimetric determination of ammonium,” Biol. Fertil. Soils 6, 68-72.

Kennedy, A. C., and Smith, K. L. (1995). “Soil microbial diversity and the sustainability of agricultural soils,” Plant Soil 170, 75-86.

Kizilkaya, R., Askin, T., Bayrakli, B., and Sağlam, M. (2004). “Microbiological characteristics of soils contaminated with heavy metals,” Eur. J. Soil Biol. 40, 95-102.

Lee, J., Bae H., Jeong J., Le, J., Yang Y., Hwang I., Martinoia, E., and Lee, Y. (2003). “Functional expression of a bacterial heavy metal transporter in Arabidopsis enhances resistance to and decreases uptake of heavy metals,” Plant Physiol. 133, 589-596.

Lee, J. W., Hawkins, B., Day, D. M., and Reicosky, D. C. (2010). “Sustainability: The capacity of smokeless biomass pyrolysis for energy production, global carbon capture and sequestration,” Energy Environ. Sci. 3, 1695-1705.

Lee, S. H., Kim, E. Y., Hyun, S., and Kim, J. G. (2009). “Metal availability in heavy metal-contaminated open burning and open detonation soil: Assessment using soil enzymes, earthworms, and chemical extractions,” J. Hazard. Mater. 170, 382-388.

Lehmann, J., Gaunt, J., and Rondon, M. (2006). “Bio-char sequestration in terrestrial ecosystems – A review,” Mitig. Adapt. Strateg. Global Change 11, 403-427.

Lehmann, J., and Joseph, S. (2009). “Biochar for environmental management: An introduction,” in: Lehmann, J., and Joseph, S. (eds.), Biochar for Environmental Management: Science and Technology, Earthscan, London, 1-12.

Lu, R. K. (2000). “Methods of inorganic pollutants analysis,” In: Soil and Agro-chemical Analysis Methods, Agricultural Science and Technology Press, Beijing, 205-266.

Méndez, A., Gómez, A., Paz-Ferreiro, J., and Gascó, G. (2012). “Effects of sewage sludge biochar on plant metal availability after application to a Mediterranean soil,” Chemosphere 89, 1354-1359.

Olsen, R. A., and Bakken, L. R. (1987). “Viability of soil bacteria: Optimization of plate counting technique and comparison between total counts and plate counts within different size groups,” Microbiol. Ecol. 42, 59-74.

Ormsby, R., Kastner, J. R., and Miller, J. (2012). “Hemicellulose hydrolysis using solid acid catalysts generated from biochar,” Catal. Today 190, 89-97.

Plassart, P., Akpa Vinceslas, M., Gangneux, C., Mercier, A., Barray, S., and Laval, K. (2008). “Molecular and functional responses of soil microbial communities under grassland restoration,” Agric. Ecosyst. Environ. 127, 286-293.

Pyrzyńska, K., and Bystrzejewski, M. (2010). “Comparative study of heavy metal ions sorption onto activated carbon, carbon nanotubes, and carbon-encapsulated magnetic nanoparticles,” Colloids Surf. A 362, 102-109.

Rajapaksha, R. M. C. P., Tobor-Kaplon, M. A., and Bååth, E. (2004). “Metal toxicity affects fungal and bacterial activities in soil differently,” Appl. Environ. Microbiol. 70, 2966-2973.

Rajkumar, M., Sandhya, S., Prasad, M. N. V., and Freitas, H. (2012). “Perspectives of plant-associated microbes in heavy metal phytoremediation,” Biotechnol. Adv. 30, 1562-1574.

Recatalá, L., Sánchez, J., Arbelo, C., and Sacristán, D. (2010). “Testing the validity of a Cd soil quality standard in representative Mediterranean agricultural soils under an accumulator crop,” Sci. Total Environ. 409, 9-18.

Renella, G., Mench, M., Gelsomin, A., Landi, L., and Nannipieri, P. (2005). “Functional activity and microbial community structure in soils amended with bimetallic sludges,” Soil Biol. Biochem. 37, 1498-1506.

Ruttens, A., Adriaensen, K., Meers, E., De Vocht, A., Geebelen, W., Carleer, R., Mench, M., and Vangronsveld, J. (2010). “Long-term sustainability of metal immobilization by soil amendments: Cyclonic ashes versus lime addition,” Environ. Pollut. 158, 1428-1434.

Sauve, S., Hendershot, W., and Allen, H. E. (2000). “Solid solution partitioning of metals in contaminated soils: Dependence on pH, total metal burden, and organic matter (TOC),” Environ. Sci. Technol. 34, 1125-1131.

Schinner, F., Öhlinger, R., Kandeler, E., Margesin, R. (1996). “Methods in soil biology,” Springer, Berlin, Heidelberg, New York.

Silva, P., and Nahas, E. (2002). “Bacterial diversity in soil in response to different plants, phosphate fertilizers and liming,” Braz. J. Microbiol. 33, 304-310.

Sivakumar, S., Nityanandi, D., Barathi, S., Prabha, D., Rajeshwari, S., Son, H. K., and Subbhuraam, C. V. (2012). “Selected enzyme activities of urban heavy metal-polluted soils in the presence and absence of an oligochaete, Lampito mauritii (Kinberg),” J. Hazard. Mater. 227–228, 179-84.

Sohi, S. P., Krull, E., Lopez-Capel, E., and Bol, R. (2010). “A review of biochar and its use and function in soil,” Adv. Agron. 105, 47-82.

Wang, Y., Shi, J., Lin, Q., Chen, X., and Chen, Y. (2007). “Heavy metal availability and impact on activity of soil microorganisms along a Cu/Zn contamination gradient,” Global J. Environ. Sci. 19, 848-853.

Wang, Y. P., Li, Q. B., Shi, J. Y., Lin, Q., Chen, X. C., Cai, X., Wu, W. X., and Chen, Y. X. (2008). “Assessment of microbial activity and bacterial community composition in the rhizosphere of a copper accumulator and a non-accumulator,” Soil Biol. Biochem. 40, 1167-1177.

Yang, R., Tang, J., Chen, X., Hu, S. (2007). “Effects of coexisting plant species on soil microbes and soil enzymes in metal lead contaminated soils,” Appl. Soil Ecol. 37, 240-246.

Yuan, J. H., Xu, R. K., and Zhang, H. (2011). “The forms of alkalis in the biochar produced from crop residues at different temperatures,” Bioresour. Technol. 102, 3488-3497.

Žemberyová, M., Barteková, J., and Hagarová, I. (2006). “The utilization of modified BCR three-step sequential extraction procedure for the fractionation of Cd, Cr, Cu, Ni, Pb and Zn in soil reference materials of different origins,” Talanta 70, 973-978.

Zhang, L., Li, L., Pan, G., Cui, L., and Hu, Z. (2009). “Effects of phosphorus and foliar zinc fertilizers on reducing grain Cd concentration of rice grown in a polluted paddy,” Ecol. Environ. Sci. 18, 909-913 (In Chinese).

Article submitted: Dec. 16, 2012; Peer review completed: July 2, 2013; Revised version received: Sept. 2, 2013; Accepted: Sept. 10, 2013; Published: September 13, 2013.