BIOLOGÍA DEL SUELO

Effect of biochar on enzymatic activity in a brazilian oxisol

 

Efeito of biochar sobre a atividade enzimática em latossolo brasileiro

 

Fabiano André Petter1, Luiz Ferando Carvalho Leite2, Diogo Milhomem Machado3, Larissa Borges de Lima1, Leandro Pereira Pacheco4, Ademir Sergio Ferreira de Araujo5*

1. Universidade Federal do Mato Grosso (UFMT), Instituto de Ciências Agrárias e Ambientais, CEP: 78557-267, Sinop, MT.
2. Embrapa Meio Norte, Caixa Postal 01, CEP 64006-220 Teresina, PI.
3. Universidade Federal do Piaui (UFPI), CEP: 64900-000, Bom Jesus, PI.
4. Universidade Federal do Mato Grosso – UFMT, Departamento de Engenharia Agrícola e Ambiental, CEP 78735-910. Rondonópolis, MT.
5. Universidade Federal do Piauí, Centro de Ciencias Agrarias, CEP: 64049-550, Teresina, PI.
*Autor de contacto: asfaruaj@yahoo.com.br

 

Recibido: 19/04/2018
Recibido con revisiones: 20/09/2018
Aceptado: 20/09/2018

 

ABSTRACT
The objective of this work was to assess the long-term effect of biochar application on enzyme activity in anOxisol of Brazilian Cerrado. The experiment was conducted with a randomized block design consisting of thecombination of two levels of fertilization (0 and 200 kg ha-1, NPK 00-20-20 formulation) and five levels ofbiochar (0, 2, 4, 8 and 16 Mg ha-1). The activity of urease, β-glucosidase, acid phosphatase, and total soilenzyme (hydrolysis of fluorescein diacetate - FDA) were evaluated in soil samples. The activity of urease andβ-glucosidase were significantly increased by the application of biochar with and without chemical fertilization,respectively. Intermediate doses of biochar increased total soil enzyme activity. There was no effect of biocharon acid phosphatase activity. The application of biochar associated with chemical fertilization promotes urease,β-glucosidase and total soil enzyme activity.

Key words: Soil quality; Charcoal; Microbiology.

RESUMO
O objetivo do trabalho foi avaliar o efeito de biochar sobre a atividade das enzimas em um Latossolo sob Cerradono Brasil. O experimento foi conduzido em blocos casualizados consistindo de dois níveis de fertilização (0 e200 kg ha-1 NPK 00-20-20) e cinco níveis de biochar (0, 2, 4, 8 e 16 Mg ha-1). A atividade das enzimas urease,β-glucosidase, fosfatase acida e hidrolise de diacetato de fluoresceína – FDA, foi avaliada. As atividades daurease e β-glucosidase foram aumentadas pela aplicação do biochar com e sem fertilização química. As dosesintermediarias do biochar aumentaram a atividade do FDA no solo. Não houve efeito do biochar sobre a fosfataseacida. A aplicação do biochar promove aumentada atividade das enzimas urease, β-glucosidase e a atividadeenzimática total.

Palavras-chave: Qualidade do solo, carvão; Microbiologia

 

INTRODUCTION

Growth of the cultivated area in the BrazilianCerrado has placed this region as one of the mostimportant agricultural regions in Brazil. Some factorshave contributed to the rapid developmentof agriculture in the Cerrado, such as wide availabilityof land, predominance of favorable topography,and use of adapted crop varieties (Queiroz,2009). Nevertheless, inadequate land managementhas brought about land degradation throughsoil erosion, organic matter losses, and compaction(Carvalho et al., 2010), therefore reducingthe productive potential of these areas.

On the other hand, the adoption of conservationistpractices for increasing soil organicmatter (SOM) content and soil microbial activity,through the introduction of cover crops and applicationof organic residues, are essential for thesustainability of production systems (Azadi et al.,2011). Recently, the use of carbonized biomass,known as biochar, formed by means of a pyrolysisprocess in which there is incomplete combustion(in a reduced oxygen environment) of plantbiomass, has increased worldwide (Du et al.,2014; Mierzwa-Hersztek et al., 2016). Amongthe main benefits of biochar on soil properties arethat this residue provides a suitable habitat formicroorganisms, promoting their activity in thesoil and affecting different microbial processesinvolved in nutrient cycling and dynamics of SOM(Jindo et al., 2012; Petter & Madari, 2012).

The activity of microorganisms can be measuredthrough of the activity of enzymes that occursin all intact and viable microbial cells (Gianfredaet al., 2005). Also, the activity of soilenzymes indicates biochemical functions and itmay provide quantitative changes on SOM (Silvaet al., 2012). Some important soil enzymes, suchas dehydrogenase, hydrolysis of fluorescein diacetateand arylsulphatase, are involved in the biogeochemicalcycles (C, N and S) and consequently,may reflect changes in the soil nutrient cycling(Karaca et al., 2011). Therefore, the activity ofenzymes could be an useful indicator for evaluatingthe effect of biochar on soils under Cerrado.

The effect of different types of biochar on enzymeactivity has been assessed in some previousstudies (Mierzwa-Hersztek et al., 2016; Du etal., 2014). Mierzwa-Hersztek et al. (2016) haveevaluated the application of poultry litter biocharin temperate soils and did not find effect on theactivity of enzymes. Previously, Du et al. (2014)assessed the consecutive application of crushedcorncob biochar, during 4 years in Nothern Chinaand found variable responses of soil enzymes.However, information about the influence of biocharon biological properties in tropical soils arescarce. Also, it is unclear the effect of biocharproduced with plant species of Cerrado on the activityof soil enzymes. Here we hypothesized thatapplication of biochar produced with species ofCerrado could affect differently the soil enzymaticactivity in the Brazilian Cerrado. Therefore, theaim of this study was to evaluate the effect ofrates of biochar produced with species of Cerradoon the activity of urease, β-glucosidase, acidphosphatase and hydrolysis of fluorescein diacetatein a Cerrado Oxisol.

MATERIAL AND METHODS

The study was conducted in Nova Xavantina,MT (14°35'36" S and 52°24'04" W, altitude of310m). The soil was classified as Oxisol of sandyclay texture. Before implementation of the experiment,we determined the following chemicaland physical characteristics of the soil (0-0.20m), using the methodology of Embrapa (2006):pH (H2O) 5.6, P (Mehlich method): 67 mg dm-3,K+: 61.5 mg dm-3, Ca2+: 1.4 cmolc dm-3, Mg2+:0.4 cmolc dm-3; Al3+: 0.13 cmolc dm-3, H+ +Al3+: 4.98 cmolc dm-3, V%: 27%; CEC: 6.93cmolc dm-3, OM: 12.6 g kg-1, Fe: 75.0 mg dm-3,Mn: 49.0 mg dm-3, Zn: 45.0 mg dm-3, Cu: 1.7mg dm-3; Clay: 307 g kg-1; Silt: 73 g kg-1; Sand:620 g kg-1.

The study area was native Cerrado forest until1985. After removal of the forest, soybean wascultivated under a no-tillage system using milletas a cover crop until 2006. Then the present experimentwas initiated, using a randomized blockdesign, composed of two levels of fertilizer application(0 and 200 kg ha-1 of 00-20-20 formulaof NPK chemical fertilizer, the latter representingthe presence of 40 kg ha-1 P2O5 and 40 kg ha-1K2O applied to the soil), and five doses of biochar (0, 2, 4, 8 and 16 Mg ha-1) randomly distributed,with four replications. Each plot was 10 m longand 4 m wide, totaling 40.00 m2; the useful areafor evaluations was 25.00 m2 since the edgeswere discarded.The biochar was made via slow pyrolysis in acylindrical metal kiln using temperatures around400–500 ºC. The biochar from Cerrado specieswas finely ground to a particle size of ≤ 2 mmand applied only once in September 2006, beingmanually distributed and incorporated by arotary hoe to a depth 0-0.15 m. A single pointsurface area of biochar was determined by theBrunauer, Emmelet and Teller (BET) nitrogen absorptionmethod (Brunauer et al., 1938), usingnitrogen gas sorption analysis at 77.3 K (-195.9ºC). The specfic surface area (SSA) of the biocharapplied, with a bulk density of 0.3 g cm-3, was2.9±0.4m2g-1.The elemental composition of it is presentedin Table 1. After the incorporation of biochar, millet(Pennisetum sp.) was sown as a cover crop,which was desiccated two months later, followedby planting of soybeans (Table 2).

Table 1. Elemental composition (total values ) of vegetablebiochar used in this experiment.
Tabela 1. Composicao elementar (valores totais) do biocharutilizado neste experimento.

Table 2. Timeline of crop sequence, fertilization and cultivation in the field experiment in Nova Xavantina, Mato Grosso State,Brazil.
Tabela 2. Cronologia das culturas, fetilizacao e cultivo no campo experimental em Nova Xavantina, Mato Grosso do Sul, Brasil.

ZT: zero tillage, the main crops were sown directly through the mulch of the cover crop, the synthetic fertilizer was applied in the same operation assowing, into the furrow. *Glyphosate acid (1080 g ha-1) and 2,4-D acid (242 g ha-1); ** Trifloxystrobin + cyproconazole (66 + 28 g ha-1), tebuconazole(100 g ha-1) and methamidophos (420 g ha-1); *** Biochar doses were applied once: 0, 2, 4, 8, 16 Mg ha-1; **** P-K (20-20) synthetic fertilizer doses: 0e 200 kg ha-1; ***** In 2010 and 2011, due to insufficient precipitation in March and April the U. ruziziensis was sown only in September, when therainy season began.

For the present study, soybean planting wasdone on 5 December 2012. Twelve seeds weredistributed per meter, with the spacing of 0.45m between rows and a depth of 0.02-0.03 m. In this same operation, the fertilization was done.The soybean cultivar used was TMG 108 RR®.

The evaluations for this study were performedin the sixth year following implementation(2012/2013 season), i.e. the effect on mediumto long-term application of biochar. Soil samplingwas conducted in February 2013, at the time ofsoybean flowering, at a depth of 0-0.10 m. Ineach plot, three simple samples were taken toform a composite, with the help of an auger, respectingthe useful area of the plot. After collection,the samples were homogenized, milled andsieved (0.177 mm). For enzymatic activity, thesamples were placed in plastic bags and transportedto the laboratory where they were homogenized,sieved (2 mm) and refrigerated at 4°C, atfield humidity, until the time of analysis.

We determined the potential activities of threesoil enzymes: urease, β -glucosidase, acid phosphatase,and total enzymatic activity (based on hydrolysisof fluorescein diacetate - FDA), accordingto the methods described by Tabatabai (1994).These methods are based on the colorimetric determinationof p-nitrophenol (yellow color) formedafter the addition of colorless substrate specificfor each enzyme measured. For each soil sampleanalytical replicates were performed in the laboratory.The soil enzymatic activity is expressed in μgp-nitrophenol h-1 g-1 dry soil. For the determinationof urease, β -glucosidase, acid phosphatase,and the total enzymatic activity (hydrolysis of fluoresceindiacetate) the substrates used were 0.2M urea solution, 0.05M p-nitrophenol-β-D-Glucopyranoside(0.05 M PNG), 0.05 M p-nitrophenolphosphate (0.05 M PNP) and 100 μl 2.4 M –diacetylfluorescein, respectively. For total enzymeactivity absorbance was read at 490 nm. The resultswere analyzed using the statistical programsSisvar 5.1 (Ferreira, 2011) and SigmaPlot 10.0(SPSS, 2001) using multiple regression in whichthe independent variables were the chemical fertilizer(NPK) and biochar and dependent variableswere the enzyme activity.

RESULTS AND DISCUSSION

The application of 16 Mg ha-1 of biochar associatedwith the application of chemical fertilizersinduced higher urease activity (Figure 1A).This result is due to the highest input of N andalso biomass that stimulated urease activity withhigher availability of easily decomposed organicN (Saya-Cork et al., 2002). In addition, biocharcan serve as substrate (N-biochar) and microhabitatfor soil microorganisms (Jindo et al., 2012),protecting urease against the action of other compoundsnaturally present in the soil and maintainingthe activity for a longer period of time.

Figure 1. Values of theenzymes urease (A),β-glucosidase (B), acidphosphatase (C), and totalenzyme activity (D) at adepth of 0-0.10 m in Oxisol,for different doses of biochar,with NPK fertilization of0 and 200 kg ha-1, in NovaXavantina, MT, 2012.*Significant at 5% probabilityby "t" test for biochar. ns notsignificant.
Figura 1. Valores das enzimasurease (A), β-glucosidase (B),fosfatase acida (C) e atividadeenzimática total (D) nasprofundidades 0-0.10 m emum oxisol, para diferentesdoses de biochar, comfertilização NPK de 0 e 200kg ha-1, em Nova Xavantina,MT, 2012. *Significante a5% de probabilidade peloteste "t" para biochar. ns nãosignificante.

For β-glucosidase, the significant effect of biocharwas found in the treatments without chemicalfertilization (Figure 1B). There was a significantdifference when comparing the means as afunction of fertilization. In the treatments withoutchemical fertilization, the response curve behavedin a linear way, demonstrating an increasein β-glucosidase activity with increasing doses ofbiochar. These results differ from those obtainedby Bailey et al. (2011), who found no significantvariability in the activity of β-glucosidase with theapplication of biochar. The main reason for thisdifferent result may be related to the difference inthe time between application of biochar and determinationof β-glucosidase activity. In our study,the activity of β - glucosidase was determined inlong-term (7 years), while Bailey et al. (2011)evaluated β - glucosidase activity in short-term(less than 1 year). This fact determines the magnitudeof response of β-glucosidase to the applicationof biochar, since the release of phenoliccompounds derived from pyrolysis of the biocharreduces over years. According to Warnock et al.(2010), phenolic compounds have the power toinhibit the enzyme β-glucosidase. Hence the importanceof evaluating the dynamics of biochar inthe long term.

The greater availability of plant residues as afunction of chemical fertilization may have minimizedthe direct effect of biochar on the β-glucosidaseactivity, since these residues are readilyavailable for the decomposition process. Non-pyrolysedplant residues can afford higher β-glucosidaseactivity compared to those in which therewas burning of vegetation, due to the aromaticstructures present in the pyrolyzed materials.These results indicate that in soils with higherlevels of organic matter, the values of β-glucosidaseactivity are higher, as evidenc e d in this study, thereby showing a positive corr e lation ofTOC with the activity of β-glucosidase (Turner etal., 2002).

There was no significant effect of application ofbiochar on acid phosphatase activity, which wasinfluenced only by chemical fertilization (Figure1C). The fertilization reduced the activity of acidphosphatase compared to treatments without fertilization.According to Gatiboni et al. (2008), thegreater the availability of soluble P in the soil, afterchemical fertilization, decreases the activity ofacid phosphatase, since this enzyme is releasedby plants and microorganisms when there is a lowavailability of soluble P in soil.

Regardless of the presence or absence ofchemical fertilization, biochar significantly affectedtotal enzymatic activity (Figure 1D). In bothtreatments, the response curve showed a quadraticresponse, with the highest average recordedin the presence of chemical fertilization. Theseresults agree with Lammirato et al. (2011) whoalso found quadratic response of total enzymaticactivity due to the application of biochar. This behaviorwith elevated quantities of biochar may beassociated with increased release, even if slowly,of soluble C-compounds which can be aromatic,aliphatic or carboxylic, and which have the potentialability to inhibit the enzyme activity in the soilsolution (Smith et al., 2010).

CONCLUSION

In conclusion, the application of biocharproduced with species of Cerrado promoted increasedactivity of urease and β-glucosidase andtotal enzyme activity. As these enzymes are involvedin important nutrient cycles and are indicatorsof soil microbial activity, biochar may be asuitable option to ameliorate the land degradationand improve the soil quality.

ACKNOWLEDGEMENTS

We thank the Brazilian council of scienceand technology (Conselho Nacional de DesenvolvimentoCientífico e Tecnológico - CNPq) for financial support for the Projeto Biochar (CNPq471205220133), coordinated by Fabiano A.Petter. Ademir S. F. Araujo is supported by CNPqby Research Productivity.

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