Chemosphere 119 (2015) 209–216

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Combining phytoextraction and biochar addition improves soilbiochemical properties in a soil contaminated with Cd

http://dx.doi.org/10.1016/j.chemosphere.2014.06.0240045-6535/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Address: South China Botanical Garden, ChineseAcademy of Sciences, #723 Xingke Road, Tianhe District, Guangzhou 510650,China. Tel.: +86 20 37252631; fax: +86 20 37252905 (Z. Li).

E-mail addresses: lizan@scbg.ac.cn (Z. Li), jpaz@udc.es (J. Paz-Ferreiro).

Huanping Lu a,b, Zhian Li a,⇑, Shenglei Fu a, Ana Méndez c, Gabriel Gascó d, Jorge Paz-Ferreiro a,d,⇑a Key Laboratory of Vegetation Restoration and Management of Degraded Ecosystems, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, Chinab University of Chinese Academy of Sciences, Beijing 100049, Chinac Departamento de Ingeniería de Materiales, E.T.S.I. Minas, Universidad Politécnica de Madrid, C/Ríos Rosas n�21, Madrid 28003, Spaind Departamento de Edafologia, ETSI Agrónomos, Universidad Politécnica de Madrid, Avenida Complutense 3, Madrid 28040, Spain

h i g h l i g h t s

� Biochar increases overall enzyme activity in a soil contaminated with Cd.� Results were enzyme specific.� Changes in enzyme activity are not exclusively driven by alterations in soil pH.� Synergistic effects between plant and biochar on soil biological activity are plausible.

a r t i c l e i n f o

Article history:Received 14 December 2013Received in revised form 17 April 2014Accepted 9 June 2014

Handling Editor: I. Cousins

Keywords:BiocharCadmiumAmaranthus tricolor L.PhytoremediationSoil qualitySoil enzymes

a b s t r a c t

The main goal of phytoremediation is to improve ecosystem functioning. Soil biochemical properties areconsidered as effective indicators of soil quality and are sensitive to various environmental stresses,including heavy metal contamination. The biochemical response in a soil contaminated with cadmiumwas tested after several treatments aimed to reduce heavy metal availability including liming, biocharaddition and phytoextraction using Amaranthus tricolor L. Two biochars were added to the soil: eucalyp-tus pyrolysed at 600 �C (EB) and poultry litter at 400 �C (PLB). Two liming treatments were chosen withthe aim of bringing soil pH to the same values as in the treatments EB and PLB. The properties studiedincluded soil microbial biomass C, soil respiration and the activities of invertase, b-glucosidase, b-gluco-saminidase, urease and phosphomonoesterase. Both phytoremediation and biochar addition improvedsoil biochemical properties, although results were enzyme specific. For biochar addition these changeswere partly, but not exclusively, mediated by alterations in soil pH. A careful choice of biochar mustbe undertaken to optimize the remediation process from the point of view of metal phytoextractionand soil biological activity.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Since the beginning of the industrial revolution there has beenan escalating trend in the use of heavy metals, which has resultedin increased contamination levels. Unlike organic contaminants,heavy metals are not degraded in the environment and can accu-mulate in soils and sediments. As a consequence, there is anupsurge in studies concerning soil heavy metal contamination(Khan et al., 2010; Vig et al., 2003), which constitutes a burden

for the environment (Kabata-Pendias, 2010) and for human health(Järup et al., 1998). This includes complications (Laskowski, 1991)derived from increases in metal concentration as the elementpasses from lower to higher trophic levels a process known asbiomagnification.

The background level of Cd in soils is less than 1 mg kg�1

(Adriano, 2001), however its presence in the environment hasincreased steadily in the last years as a consequence of man-madeactivities. The main anthropogenic sources of cadmium in theenvironment are coal combustion, municipal waste incineration,zinc, lead or copper smelter, electroplating, pigments productionand nickel–cadmium batteries (World Health Organization,http://www.euro.who.int/en/home). Thus, the use of sewagesludges and phosphatic fertiliser in soils could increase their

210 H. Lu et al. / Chemosphere 119 (2015) 209–216

cadmium content (Kabata-Pendias, 2010). Cadmium is a perniciousheavy metal whose presence in the environment can result in animpact on soil ecosystem functioning. Cadmium also induces prob-lems in human health ranging from cancer to Itai–Itai disease(Järup et al., 1998).

Starting from the 1990s there has been an increasing interest inusing phytoremediation to improve soil quality in contaminatedareas. Phytoremediation techniques are the most cost-efficientprocesses and enjoy a better public perception compared to ex situdecontamination techniques (Ali et al., 2013). Phytoextractors areplant species that can grow in areas heavily contaminated withmetals and that can concentrate these metals in the harvestableparts. In the case of Cd, a plant species is believed to have thepotential to phytoextract this element when its presence in theplant shoots exceeds concentrations of 100 mg Cd kg�1 shoot dryweight (Baker et al., 2000). The number of Cd hyperaccumulatorsis scarce compared to elements like Ni that can be accumulatedby more than 300 plant species (Ali et al., 2013).

In the last years there has been an increasing interest on theeffect of biochar on metalliferous plants, in particular in those spe-cies capable of accumulating Cd, but also other elements such asZn, Pb and Tl (Houben et al., 2013; Fellet et al., 2014). With thisaim in mind, these authors have explored the possibility to com-bine biochar and phytoremediation for environmental remedia-tion, focusing on the fate of heavy metals. Those studies havedemonstrated that biochar strongly immobilizes soil heavy metals,at least at the doses used in these experiments, and thus, plantuptake of the contaminant was to some extent impeded by the bio-char. This resulted in an unsatisfactory recovery of heavy metal inthe plant tissue.

A holistic approach to soil phytoremediation must be per-formed as the ultimate goals of soil remediation processes areboth, to immobilize or reduce the amount of pollutant from thecontaminated site, and to restore the capacity of the soil to performits normal functions. In this sense, planting a vegetative cover canhave a number of beneficial effects on soil, including reduced ero-sion and an improvement of soil quality and ecosystem function-ing, while adding biochar to soil can result in an enhancement incarbon sequestration (Lehmann et al., 2006) and soil biologicalproperties (Paz-Ferreiro et al., 2012) or in a reduction in soil ero-sion (Jien and Wang, 2013). These co-benefits of biochar applica-tion have not been evaluated in previous studies dealing withthe interaction between biochar and metalliferous plants.

Indicators of soil quality that can properly assess the efficiencyof a phytoremediation process must be chosen accordingly. Thereis a considerable and ever-increasing bibliography regarding theuse of soil enzymes as indicators of soil quality due to their rapidresponse after land use changes or alterations in soil management(Paz-Ferreiro et al., 2010, 2011). In fact, several mathematicalexpressions using soil enzymes as indicators of soil quality, eitherby themselves or combined with other biological, physical orchemical soil properties have been proposed in the last years(Paz-Ferreiro and Fu, in press). In spite of this, there are few studiesthat tried to assess the effect of phytoremediation (Epelde et al.,2009; Moreno-Jiménez et al., 2012) or biochar (Paz-Ferreiroet al., 2012; Wu et al., 2013) separately on soil enzyme activities.

In general, heavy metals have a negative impact on soil bio-chemical properties and, in particular, towards soil enzymes(Hinojosa et al., 2004; Khan et al., 2010), although this effect canbe different from an enzyme to another and depend also on thepollutant (Shen et al., 2005; Khan et al., 2010). Most soil enzymeshave negative relationships with increasing amount of extractableheavy metal (Hinojosa et al., 2004) and it is a well-known fact thatsoil restoration can improve the biochemical activity of a contam-inated area (Hinojosa et al., 2004).

Thus, the aim of our work is to study the soil biochemicalresponse after the use of biochar and phytoextraction for remedi-ation purposes. In addition we employed liming treatments toassess the contribution of pH changes to alterations in soil biolog-ical properties mediated by biochar, as due to the frequentlyreported proximity between organisms and biochar surfaces, asreviewed by Lehmann et al. (2011), biochar pH could have a keyinfluence on total microbial abundance. We hypothesized that, inspite of the effectiveness of phytoextraction being diminished inthis soil by biochar use (Lu et al., 2014), soil biochemical qualitycould benefit from a combination of phytoextraction and biocharaddition. We also hypothesized that biochars prepared from differ-ent feedstocks and at different temperatures would affect differ-ently enzyme activity patterns.

2. Materials and methods

The soil and experimental design used in this study have beendescribed previously (Lu et al., 2014). Basically, soil was collectedfrom the surface layer (0–20 cm) of a cropland area. Sampling tookplace in 20–25 points over a 0.5 hectare area, totaling an amount of50 kg of soil. The sampling area was located near a waste landfillsite in the suburb of Guangzhou, China. Guangzhou is located inthe subtropical humid area having an average annual temperatureof 12.7 �C and annual average precipitation of 1700 mm. Accordingto FAO, the soil is classified as a Fimic Anthrosol.

Part of the soil was air dried and sieved to 2 mm to conduct gen-eral analyses, while the rest was moist sieved to 10 mm to conductthe experiment. Before the starting of the pot experiment, the soilhad a total organic carbon content of 1.98%, total nitrogen contentof 0.142%, pH of 6.00, total phosphorus of 687 mg kg�1, availablephosphorus of 126 mg kg�1. Soil had a sandy-loam texture, with17% clay, 7% of silt and 76% of sand. Its total Cd content was6.1 mg kg�1, a figure more than 20 times higher than the ChineseSoil Environmental Quality Standard Guide value of 0.3 mg kg�1

(China GB 15618-1995, 1995). At this level of Cd contaminationAnthrosols in South China show a reduction of soil microbialproperties, including soil enzymes (unpublished data from theauthors).

2.1. Preparation and characterisation of biochar

The two biochars used in the present experiment were obtainedfrom poultry litter and eucalyptus as feedstocks (PLB and EB,respectively). The preparation of these materials, the methods tocharacterise the biochars and the reasons to select these materialsare described in more detail in Lu et al. (2014), while the mainproperties of the biochars are shown in Table 1.

The concentration of total polycyclic aromatic hydrocarbons(PAH) was determined as in Tammeorg et al. (in press). Briefly,Soxhlet extractions (0.5 g of biochar, 90 mL of toluene, 6 h,160 �C) were spiked with 1,1-binaphthyl as an internal standardbefore extraction. The toluene was reduced to 1 mL and thecontent of 18 PAHs (naphthalene, acenaphthylene, acenaphthene,fluorene, phenanthrene, anthracene, fluoranthene, pyrene,benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[j]fluoranthene benzo[k]fluoranthene benzo[a]pyrene, indeno[1,2,3-cd]pyrene, dibenz[a,h]anthracene, benzo[g,h,i]perylene,benzo[e]pyrene) were determined by gas chromatography massspectroscopy analysis.

For heavy metal analysis, samples (0.2 g) were digested with6 mL HNO3 and 2 mL H2O2 using a microwave closed system (Mul-tiwave3000, Anton Paar, Austria). Heavy metal concentrationswere analysed using an ICP-MS spectrometer (Agilent 7700x, USA).

Table 1General characteristics of the biochars used in the experiment.

Poultry litter biochar Eucalyptus biochar

pH 10.02 10.40Carbon (%) 16.77 81.03Nitrogen (%) 1.37 1.07C/N ratio 8.9 78.7O (%) 35.7 7.4H (%) 1.50 1.99O/C ratio 2.13 0.09H/C ratio 0.09 0.02Cd (mg kg�1) <0.001 <0.001Cu (mg kg�1) 96.0 16.7Zn (mg kg�1) 614 52As (mg kg�1) 2.1 0.4Pb (mg kg�1) 8.2 10.3Surface area (m2 g�1) 7.418 334.56Average pore width (nm) 15.406 1.928Naphthalene (mg kg�1) 0.4 0.2Sum of 18 PAH (mg kg�1) 0.4 0.2Particle size (mm) <1 <0.5Pore volume (cm3 g�1) 0.0286 0.1612Ash (%) 74.95 1.74Volatile matter (%) 7.66 2.20Fixed carbon (%) 17.39 96.06

H. Lu et al. / Chemosphere 119 (2015) 209–216 211

2.2. Experimental design

On the 13th of April of 2013, a mesocosm using a fully repli-cated randomised experiment was set up in a greenhouse in SouthChina Botanical Garden. Contaminated soil (1.5 kg for the treat-ments with plants and 500 g for the treatments without plants)at an initial water holding capacity (WHC) of 50% was mixed withbiochar or with lime. The mixtures were homogeneous through the10 cm depth pot. In all cases the biochar was dried when mixedwith the soil. After this procedure, the soils were watered to 60%of water holding capacity. Every day the pots were weighted anddistilled water was added to the soils in order to account for mois-ture losses. The experiment studied two factors, namely the type ofamendment and the presence/absence of phytoremediator, having4 replicates per treatment. The treatments in the amendment fac-tor were: contaminated soil, hereafter referred to as control (C),poultry litter biochar (3% w/w) (PLB), eucalyptus biochar (3% w/w) (EB), poultry litter biochar (1.5% w/w) + eucalyptus biochar(1.5% w/w) (BB), liming with CaO as necessary to increase soil pHas much as after EB addition (CaO1; pH value of 7.01) and limingwith CaO as necessary to increase soil pH as much as after PLBaddition (CaO2; pH value of 7.62).

With respect to the factor involving presence/absence of phyto-remediator, half of the mesocosm were planted with 3 seeds of redamaranth (Amaranth tricolor L.), while in the remainder half thevegetative cover was absent.

After 60 d soils were collected. Moist soils were sieved to 4 mmand pre-incubated at 25 �C and 60% WHC during seven days for theanalysis of biochemical properties.

2.3. Soil respiration and soil microbial biomass

Soil basal respiration was determined at the end of the experi-ment by static incubation. The CO2 produced during a 2 h period bythe soil (25 g) incubated at 60% WHC inside 1 L jars and at 25 �Cwas collected in 10 mL of 0.01 M NaOH solution, which was thentitrated with HCl.

Active microbial biomass was measured at the end of the exper-iment using the substrate-induced respiration (SIR) method(Anderson and Domsch, 1978). In short, SIR was determined afteradding a 1:4 glucose/talcum mixture to the samples, which wereincubated in the same conditions as for the respiration study, at

a concentration of 12.0 g glucose kg�1 soil (Anderson andDomsch, 1978). The period of static incubation, as in the case ofrespiration measurements, was 2 h.

The metabolic quotient (qCO2) was calculated as the ratiobetween microbial respiration and soil microbial biomass as ameasure representative of the efficiency of the soil (Andersonand Domsch, 1978). It was expressed as microgram of CO2–Creleased per milligrams of biomass carbon per hour.

2.4. Soil enzyme activities

Acid phosphomonoesterase and b-glucosidase activities weredetermined following modifications of the original methods(Eivazi and Tabatabai, 1988; Saá et al., 1993) as described byPaz-Ferreiro et al. (2007). Briefly, these enzyme activities weredetermined after incubating soils at 37 �C and then measuring, byspectrophotometry, the amount of p-nitrophenol released duringenzymatic hydrolysis. Acid phosphomonoesterase was estimatedfollowing the method of Saá et al. (1993), using 16 mMp-nitrophenyl phosphate as substrate. b-glucosidase activity wasdetermined similarly as described for phosphomonoesteraseactivity, but using 25 mM p-nitrophenyl-b-D-glucopyranoside assubstrate (Eivazi and Tabatabai, 1988). The p-nitrophenol releasedduring enzymatic hydrolysis was determined using a spectropho-tometer at a wavelength of 400 nm. The activity of each of thesethree enzymes was expressed as lmol p-nitrophenol g�1 h�1.

b-glucosaminidase activity was assayed as described by Parhamand Deng (2000), but stopping the reaction with 2 M CaCl2 instead of0.5 M CaCl2. Essentially, soil (1 g) was incubated in the presence of4 mL of 0.1 M acetate buffer (pH 5.5) and 1 mL of 10 mMp-nitrophenyl-N-acetyl-b-D-glucosaminide solution at 37 �C. Afterstopping the reaction with CaCl2 the activity was measured at awavelength of 400 nm and expressed as lmol p-nitrophenolg�1 h�1.

Invertase activity was determined after incubating the sampleswith 35.06 mM saccharose in 2 M acetate buffer (pH 5.5) at 50 �Cduring 3 h and assessing the released reducing sugars followingthe method of Schinner and von Mersi (1990). Invertase activitywas measured at 690 nm and expressed as lmol glucose g�1 h�1.

Urease activity was determined as in Kandeler and Gerber(1988). A modified Berthelot reaction is used in this essay to obtainan NH4

+ coloured complex that is measured at 610 nm. The activitywas expressed as lmol NH4

+ g�1 dry soil h�1.All enzyme activities were determined in triplicate. Different

standard curves were prepared for every treatment in order toaccount for both, the adsorption that some of the biochars hadon the product of the reaction (Paz-Ferreiro et al., 2012) andadsorption of the product of the enzymatic reaction by soil organicmatter. In all cases we ensured that the substrate was not limitingthe reaction.

For each soil sample, the geometric mean of the assayedenzyme activities (GMea) was calculated as:

GMea ¼ ðPm� Glu� Inv� Ure� GsmÞ1=5

where Pm, Glu, Inv, Ure and Gsm are phosphomonoesterase, b-glu-cosidase, invertase, urease and b-glucosaminidase respectively. Thisalgorithm is sensitive to soil quality shifts in plots under differenttype of management and is responsive to heavy metal contamina-tion (Paz-Ferreiro et al., 2012; Paz-Ferreiro and Fu, in press).

2.5. Statistical analysis

Statistical analyses (calculation of means and standard devia-tions, differences between treatments) were performed using SPSS15.0 package. Differences of means were tested using a two-way

212 H. Lu et al. / Chemosphere 119 (2015) 209–216

ANOVA with the presence/absence of plant and the type of amend-ment (eucalyptus biochar, poultry litter biochar, CaO1, CaO2, mix-ture of biochar or none) as factors. Mean values were considered tobe different when P < 0.05 using the Tukey’s test.

A principal component analysis (PCA) was carried out to estab-lish the overall effect of the treatments on soil biochemical proper-ties. The component extraction was made by means of the Kaisercriterion which assumes that the eigen-value should not be lessthan 1. After extraction, the variance was maximized usingorthogonal factor rotation (varimax).

Soil microbial biomass C

Soil respiration

qCO2

Fig. 1. Soil microbial biomass, respiration and metabolic coefficient (qCO2) fordifferent treatments with and without plant cover.

3. Results

The biochars used in this experiment had a low amount of PAH,in both cases under the limit of 12 mg kg�1 or 4 mg kg�1 set by theEuropean Biochar Certificate (2013) for basic and premiumbiochar, respectively. Only naphthalene was above the detectionlimit (see Table 1). The heavy metal content complied with theUS and Canadian guidelines for compost preparation. In addition,they complied with the heavy metal regulation of most EU coun-tries. As only exceptions, PLB had a Cu content over that acceptedby the Dutch standard and a Zn content over the Belgian, German,Irish, Dutch, Swedish and British standard. The contents of heavymetals also agreed with the stricter European Biochar Certificate(2013) for premium biochar, except for the Zn content in PLB.

The soil microbial biomass, determined after the SIR methodwas significantly greater in soils planted with Amaranthus com-pared to unplanted soils (F = 17.57, P < 0.001). The treatment BBhad a value 35% higher than that of the control, while the treat-ment PLB had 28% more microbial biomass C than the control soil(see Fig. 1).

Basal respiration values were higher in soils with the presenceof plant species (F = 10.61, <0.010, Table 2). Respiration was 52%,48% and 90% higher in the treatments amended with biochar(PLB, EB and BB respectively) than in the control soil.

The metabolic coefficient (qCO2) was not affected by the pres-ence or absence of a plant species (F = 0.10, P = 0.75). The treat-ments EB and BB had significantly higher metabolic coefficientthan the control soil.

Similar to the changes in microbial biomass and respiration, soilenzyme activities were generally larger in the soil planted withAmaranthus. The activities of invertase, b-glucosidase,b-glucosaminidase and phosphomonoesterase were modified bybiochar addition (see Figs. 2 and 3).

Invertase activity increased in the treatments with plants(F = 6.10, P < 0.05). Invertase activity values were 25% larger inthe treatment PLB compared to the control soil. However, valuesfor BB were only 55% of those in the control. b-glucosidase activitywas 57%, 25% and 33% higher in the soils treated with PLB, EB or BBcompared to the control soil, but was not affected by the presenceof a phytoremediator species (F = 1.10). b-glucosaminidase activi-ties was enhanced by the presence of the phytoremediator(F = 43.73, P < 0.001) but the addition of biochar or liming had noeffect on this enzyme activity. Urease activity was enhanced bythe presence of Amaranthus (F = 4.51, P < 0.05). The value of ureasewas double in the BB treatment in comparison to the control phos-phomonoesterase activity increased with plant presence (F = 32.88,P < 0.001) and was enhanced by the treatments PLB and EB in com-parison to the control soil. Values in those samples were 12% and16% higher than in the control soil.

Finally, GMea increased in the pots with Amaranthus (F = 57.97,P < 0.001). On the other hand, GMea had a value 27%, 14% and 7%higher in the PLB, EB and BB treatments (average for the treat-ments with and without Amaranthus), respectively, compared tothe control soil.

The PCA extracted three principal components, which contrib-uted 78% of the total variance. The first axis (PC1) described around35%, PC2 nearly 22% and PC3 around 21% of the variance. Soilmicrobial biomass, soil respiration and urease activities were bestdescribed by PC1, while PC2 had a large loading (eigenvector val-ues) for b-glucosaminidase and phosphomonoesterase activities(see Table 3). Finally, PC3 was dominated by invertase activity.The component scores of the PCA are presented in a two-dimen-sional graph for the case of PC1 and PC2 (Fig. 4). The differencesbetween the treatments were mainly controlled by PC1, whilePC2 described the differences between samples with or withoutplant species. The treatments with biochar (PLB, EB and BB) werefound more displaced towards the right along PC1 compared to

Table 2Results of the ANOVA showing the differences between treatments for the properties analysed.

Source of variation Variable Sum of squares Degrees of freedom F P value

Amendment Soil microbial biomass C 25.38 5 7.69 0.000Respiration 33.33 5 12.67 0.000qCO2 0.31 5 5.33 0.001Invertase 1017.40 5 16.84 0.000b-glucosidase 0.24 5 24.30 0.000b-glucosaminidase 0.16 5 2.71 0.036Urease 1026.16 5 7.29 0.000Phosphomonoesterase 2.32 5 9.43 0.000GMea 5.33 5 23.59 0.000

Plant Soil microbial biomass C 11.61 1 17.57 0.000Respiration 5.59 1 10.61 0.002qCO2 0.001 1 0.10 0.750Invertase 73.73 1 6.10 0.018b-glucosidase 0.003 1 1.37 0.249b-glucosaminidase 0.46 1 38.52 0.000Urease 127.08 1 4.51 0.041Phosphomonoesterase 1.65 1 33.61 0.000GMea 0.52 1 11.45 0.002

H. Lu et al. / Chemosphere 119 (2015) 209–216 213

the liming treatments (CaO1 and CaO2). This would indicate thatbiochar addition had greatest impact on soil biochemicalproperties.

4. Discussion

When using materials to amend heavy metal contamination insoil it is fundamental to avoid the introduction of new contami-nants. In this sense, the biochars that we used did not possess anunsuitable amount of heavy metals or PAHs. As an example, bioch-ars prepared from sewage sludge can contain higher amounts ofheavy metals (Méndez et al., 2012) than the ones reported in thisstudy (Table 1). The improvement of soil biological and biochemi-cal properties is fundamental to define the success of the remedi-ation process. For this reason these properties are increasinglyused in relation to soil remediation (Yang et al., 2013).

The results obtained in this study showed that the respirationincreased relatively more than soil microbial biomass after euca-lyptus biochar addition (EB) and combined biochar addition (BB),leading to increases in metabolic quotient values. Studies in thebibliography show contrasting results concerning the effect of soilremediation on respiration. This could be due to the fact that sev-eral processes, directly or indirectly concerned with the size of themicrobial biomass and its activity and metabolism can be alteredby contaminants and subsequent remediation processes. Thehigher values of respiration found here for some of the biocharamended soil could have been mediated by an improved soilstructure, leading to enhanced aeration (Busscher et al., 2010) orlessened complexation between the substrate available for respira-tion and the heavy metal (Landi et al., 2000) or due to the presenceof labile C compounds in the biochar (Smith et al., 2010). Thesynergistic effect in BB could be due to some improvement in thebiochar properties, including higher ash content and a better C/Nratio, in comparison to EB.

Changes in microbial biomass due to biochar addition aremostly positive and are believed to be mainly due to biocharproviding a habitat for soil microbial communities and substrateavailability (Kolb et al., 2009). This process would be more impor-tant for EB due to its higher surface area. Other processes such assorption of bacteria to the biochar surface and processes mediatedby pH changes might also have played an important role. In fact,Steiner et al. (2004) found that microbial biomass correlatedpositively with pH after biochar application.

Similarly to that found in our work, Kolb et al. (2009) alsoreported increases in soil metabolic quotient following biocharaddition. This was not the case for PLB which had the same qCO2

values than the control soil. The value of qCO2 has been suggestedto be indicative of the bioenergetic status of microbial biomass. Itshould also be noted that soils that have been exposed for longtime to elevated heavy metal concentrations, and in particularCd, have shown consistently lower values of soil metabolicquotient that unpolluted soils (Landi et al., 2000).

It was observed that almost all of the biochemical parametersmeasured showed higher values in planted compared to unplantedpots. Soils with a vegetative cover usually present higher rates ofmicrobial activity and biomass as compared to bare soil (Ross,1976). This has been usually attributed to the presence of surfacesfor microbial colonization and easily metabolizable root exudates(e.g., fatty acids, amino compounds, sugars, organic acids andnucleotides). Similarly, higher values of soil biological andbiochemical properties have been reported in rhizospheric soilcompared to bare soil in other phytoextraction experiments(Wang et al., 2006; Epelde et al., 2009). The present study supportsthese findings, as we found the activity of four out of the fivehydrolases studied increasing with the presence of plant cover.

In general, soil enzymes showed lower values in the controlcontaminated soil than in the soils amended with lime or, spe-cially, with biochar. Heavy metals have frequently been reportedto have a negative effect on soil quality and soil biochemical prop-erties, inhibiting soil enzymes. These contaminants are known toaffect and the metabolism and morphology of soil microorganisms,mainly as a consequence of protein denaturation, due to interac-tion with the protein-active functional groups and via destructionof cell membrane integrity (Leita et al., 1995). Changes in enzymeactivity as an indirect consequence via community structuremodification are also possible (Nannipieri 1994). This can lead tothe alteration of the conformation of enzymes or in essential func-tional groups being blocked. The effects of soil contamination onsoil enzymes are variable, depending on soil type, contact timeand the particular enzyme activity. In particular, high Cd concen-trations have been reported to have a harmful effect on soil micro-bial biomass, microbial respiration and enzymatic activities (Viget al., 2003; Cardelli et al., 2009; Masto et al., 2011). With theapproach used in our work we cannot explain why some enzymeshave been more sensitive than others to the different treatments.

In fact, the bioavailability of Cd, which is particularly importantto understand its effects on soil microbial organisms, depends onseveral factors, including soil type, Cd speciation, aging, form ofCd applied, and the soil microbial community (Vig et al., 2003).Thus, it can be expected that treatments with less plant availableCd, as it was the case of the biochar treatments (Lu et al., 2014),showed an improvement in soil functioning which is indicative

Fig. 2. Invertase, b-glucosidase and b-glucosaminidase for different treatmentswith and without plant cover.

Urease

Phosphomonoesterase

Geometric mean of enzyme activities

Fig. 3. Urease, phosphomonoesterase and GMea for different treatments with andwithout plant cover.

214 H. Lu et al. / Chemosphere 119 (2015) 209–216

of a certain metabolic recovery. Other works have also reported animprovement in soil functioning as a consequence of the reductionin soil heavy metal contamination (Jiang et al., 2010). However, notall the microbial effect can be attributed to changes in the level ofsoil contamination. It is known that biochars with very contrastingcharacteristics (elemental ratios, specific surface area, etc.) canmediate interactions with soil microorganisms, resulting inenhanced soil enzymatic activities in different soil types

(Paz-Ferreiro et al., 2014). Our work also adds up to other studiesthat have evidenced that biochar addition can have positive effectsin the enzyme activity and size of the microbial community in soilscontaminated with different heavy metals (Cui et al., 2013).

The effect of liming on soil enzyme activities was less pro-nounced in this study than that of biochar addition. While thereare some reports of liming mostly, although with exceptions,enhancing enzymatic activities as a consequence of improved pHvalues (Perez de Mora et al., 2005; Garau et al., 2007), others(Paz-Ferreiro et al., 2012) do not report any improvement in soilbiological activity after liming. Discrepancies in these studies can

Table 3Eigenvector values for the rotated component matrix of the principal componentanalysis.

Principal component

PC1 PC2 PC3

Soil microbial biomass C 0.830 0.279 0.096Respiration 0.842 0.150 0.131Invertase �0.147 0.075 0.915b-glucosidase 0.573 0.059 0.681b-glucosaminidase �0.078 0.895 �0.032Urease 0.756 �0.205 �0.266Phosphomonoesterase 0.330 0.786 0.205

H. Lu et al. / Chemosphere 119 (2015) 209–216 215

be attributed to the difference in soil types and rates ofamendments.

As revealed by the PCA, soils with presence and absence ofplants and with different treatments were clearly separated fromeach other. The shifts between the soil treatment occurred mostlyalong PC1, with the biochar treatments are located at the rightmargins of the PC1 axis. It is interesting to notice that the changesnoticed in PC1 were more intense for the soils treated with biocharthan for the limed soils. On the contrary, the changes due to plantcover were more pronounced along PC2. It would have been inter-esting to have an unpolluted soil to compare it with our samples.Thus, we could have confirmed whether or not the treated samplesapproached to the reference soil and therefore strengthen theconclusions obtained in the present work.

It should also be stressed that in some of the determined prop-erties (soil respiration and urease) we have found additive effectsof different biochar types. This could be postulated that this isdue to a lesser bioavailability of the contaminant, but this is aneffect that has not been reported (Lu et al., 2014). Alternativeexplanations could include an improvement in the C/N ratio or alarger range in pore sizes when more than one biochar is used asamendment. However, the reasons cannot be inferred from ourexperimental design. Therefore, it will be necessary to furtherinvestigate the mechanisms implicated.

Finally, it should be mentioned that the biochar that resultedinto more immobilization of heavy metal, thus inhibiting Cd phy-toextraction (PLB, see Lu et al., 2014) brought about more positiveeffects on microbial activity compared to the other biochars used.Therefore, a complicated balance seems to be involved in theelection of a suitable biochar for remediation purposes in contam-inated lands, which should depend on an equilibrium betweenimproving soil biological properties, immobilizing the contaminantand, if possible, also contribute to soil carbon sequestration. Thiselection must be also constrained by how perdurable is the effect

Fig. 4. Score plot of the principal component analysis (PCA) based on soilbiochemical parameters. PC = principal component. Filled squares represent thesoil with amaranth, while open circles represent the unplanted soil.

of biochar addition. Thus, the experiment reported in this articleis a medium term experiment and further research should be doneto determine the effects of biochar aging on both, heavy metalimmobilization and soil biochemical properties.

5. Conclusions

This study has shown that phytoremediation combined withbiochar addition to soil can enhance soil biology. The biochemicaleffects of biochars in soils were more strongly influenced bybiochar addition than by other practices to elevate soil pH suchas liming. The mechanisms behind the stimulatory effect of biocharon soil biochemical parameters are not yet fully understood, butseem to be more pH dependent for microbial biomass and soil res-piration than for soil enzymes. Our results indicate that a carefulchoice of biochar must be undertaken in order to optimize theremediation process from the point of view of metal phytoextrac-tion and soil biological activity.

Acknowledgements

J. Paz-Ferreiro was sponsored by the Chinese Academy ofSciences (fellowship for young international scientists number2012Y1SA0002). We acknowledge support from the NationalNatural Science Foundation of China (Nos. 40871221 and41301571), and the Research Fund Program of GuangdongProvincial Key Laboratory of Environmental Pollution Control andRemediation Technology (2013K0008).

We thank three anonymous reviewers for the many insightsprovided in a previous version of the manuscript.

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