Methodological interference of biochar in the determination of extracellular enzyme activities in composting samples

Introduction Conclusions References


Introduction
Agricultural use of biochar has been paid attention as an alternative strategy for mitigation of greenhouse gas (GHG) emission as well as improvement of soil properties,.In addition, high sorption character of biochar, similarly to activated carbon, makes it possible to contribute to reduction of several hazards (heavy metals, pesticide, and hydrocarbon) in soil (Yang et al 2009).Furthermore, it has been reported the suitability of biochar as an additional component for enhancing the composting quality by reducing the nitrogen volatilization due to sorption on surface of biochar (Steiner et al 2010), mitigating CH 4 emission due to the higher aeration in composting pile (Sonoki et al., 2012) and improving compost quality such as an intense humification process and more recalcitrant character (Dias et al. 2010;Jindo et al 2012).
Lately, the application of compostbiochar blended biocharcompost to soil can promote a synergistic effect on enhancing plant the nutrition content and water holding capacity (Lieu et al.,2012) as well as contributing the reduction of organic pollutants and heavy metal (Beesley et al., 2010).
In terms of the decomposition of organic matter during composting, enzymatic activities such as -glucosidase and phosphatase are useful tool to reflect dynamics of biodegradation process and provide valuable information about stability and maturity of the compost (Vuorinen 2000;Mondini et al., 2004).
These hydrolytic enzymes are measured by colorimetric determination of p-nitrophenol (PNP) which is formed as the reaction-product of hydrolysis of different p-nitrophenylnitrophenol derivatives used as a substrate: nitorophenyl--d-glucopiranoside (PNG) for -glucosidase activity, and p-nitrophenyl phosphatase (PNPP) for alkaline and acid phosphatase activities..By contrast, p-nitrophenol is a well-known toxic compound in industrial sector, and is treated by absorption in on activated carbon (Tang et al 2007;Ivančev-Tumbas al 2008).Furthermore, the biochar, which has similar absorption character to activated carbon (Hale et al., 2013), interferes with the extraction of soluble organic compounds, leading to underestimation of soil microbial activities (Chan et al., 2007).Even though several works on the relation between microbial measurements and biochar exposure has been reported (Durenkamp et al 2010;Bailey et al 2011;Luo et al., 2013), further research are required for understanding the biochar interaction from the chemical, physical and biochemical point of view.Thies and Rillig (2009) proposed the utilization of spiking assays with specific molecules as internal standard to overcome potential interferences in the estimation of the microbial parameters.
The aim in present work was to study the influence of biochar as a composting component on the retention of the PNP generated from three colorimetric-based enzymatic assays (alkaline and acid phosphatases and glucosidaseglocosidase).The retention of PNP was tested in two different composting mixtures (poultry manure (PM) and cow manure (CM)) and other two similar composting mixtures containing biochar as additional component (PM+B, CM+B).

Biochar preparation
The production of biochar, made from broad-leaved tree (Quercus serrate serrata Murray), was carried out using a Japanese traditional kiln at atmospheric pressure and a temperature range of 400-600 °C with final temperature of 550 °C .To analyze the physical properties of biochar, we grounded and sieved the biochar less than 0.5 mm in diameter.The elemental content was measured with an elemental analyzer (Thermo Finnigan EA1112, Thermo Fisher Scientific, Inc., MA, USA).The pH of each mixture pH at 1:20 (w/v) ratio was measured with a compact pH meter B-212 (HORIBA Ltd., Kyoto, Japan) Microporosity was evaluated by the iodine (I 2 ) number method and methylene blue (MB) adsorption capacity, respectively, were measured, following the methodology used by Gaspard et al., 2007.Surface area was measured with a BELSORP18PLUS (BEL Japan, Inc., Osaka, Japan).The main characteristics of the obtained biochar are shown in Table 1.: pH (H 2 O)= 7.23; C = 791.5 g kg −1 ; O = 91.5 g kg −1 ; H = 18.9 g kg −1 ; ash = 78.7 g kg −1 ; N = 37.6 g kg −1 ; P = 2.3 g kg −1 ; K = 14.1 g kg −1 ; Surface area = 255.0m 2 g −1 ; methylene blue (MB) absorption capacity: 8.3 mg g -1 ; iodine adsorption capacity: 100 mg g -1 .

Raw materials and composting process
Composting was carried out at Kanagi experimental farm of Hirosaki University.Two composting mixtures were prepared following initial proportion of organic waste: CM -cattle manure (100.9 kg) mixed with apple pomace (76.8 kg), rice straw (9.7 kg) and rice bran (12.7 kg);, PM -poultry manure (35.2 kg) mixed with apple pomace (141.8 kg), rice straw (9.9 kg) and rice bran (13.0 kg).
Another two composting mixtures (CM+B and PM+B) were prepared by enriching the initial composting mixtures CM and PM with 20kg of biochar.The organic waste mixtures were composted in cone shaped windrows with regular turnings and continuous monitoring of pile temperature and moisture.The principal physicochemical properties of the composting mixtures are described in Table 1, and further information on the composting process and characteristics of the composting mixtures can be found elsewhere (Sonoki et al, 2012).The composting process lasted approximately 3 months for all piles.A representative sample of each organic material was taken at the initial stage (I) and after maturation stage (M).These samples were collected from different spots of piles, mixed together, air dried and grounded to 0.5mm.

Thermogravimetric analysis (TGA)
Thermal analysis of the organic material was measured using a SDT-2960 simultaneous DSC-TGA thermal analyzer (TA instruments) under static air atmosphere as follows: a temperature equilibrating at 30 ºC followed by a linear heating rate of 5 ºC min -1 from 30 to 105 ºC, an isotherm for 10 min and then continued ramping of 5 ºC min -1 from 105 to 680 ºC.An index of thermal lability of the organic matter (W 2 /W 1 ), shown in Table 1, was calculated from the ratio: Mass loss at 350-550 ºC (W 2 ) / Mass loss at 110-350 ºC (W 1 ) (Plante et al., 2009).

Enzymatic analysis
Alkaline and acid phosphatase and -glucosidase activities were determined following the methods reported by Tabatabai and Bremmer (1971), and Eivazi and Tabatabai (1988) respectively using 0.5 g of organic material, and 2 ml of modified universal buffer (MUB) containing the following substrate: Alkaline phosphatase activity assay was performed at pH 11 using p-nitrophenyl phosphatase (PNPP) as substrate, meanwhile acid phosphatase activity assay was performed with the same substrate at pH 5.5; -glucosidase activity was assayed at pH 6 using p-nitrophenyl -D-glucopiranoside (PNG) as substrate.In the three cases, the suspensions were incubated at 37ºC for 1 hour.Enzymatic reactions were stopped by cooling in ice for 15 min.Then, 0.5 ml of CaCl 2 0.5 M and 2 ml of NaOH 0.5 M (for phosphatases) or 2 ml of Tris (hydroxymethyl) aminomethane-sodium hydroxide (THAM-NaOH) 0.1 M pH 12 (for glucosidase) were added.The p-nitrophenol (PNP), formed as product reaction from the three enzymatic assays, was determined at 398 nm of spectrophotometer.

PNP retention assay during the enzymatic activity analysis
To study the retention of PNP during the analysis of the different enzymatic activities, following spiking assay was performed: Instead of adding the substrates (PNG and PNPP) at the beginning of the procedure, reactionproduct (PNP) was added with difference different concentration (0, 50, 100 and 150 mg L -1 ) to buffer solution (pH=5, 6,5 and 11, corresponding to the glucosidase, acid and alkaline phosphatase activity assays, respectively).After the incubation, the same procedure as for enzymatic assay, described in previous section was taken place for PNP determination..This procedure allows evaluating the retention of PNP by the biochar during the analysis.Controls were performed similarly by adding the same amounts of PNP after the incubation period and before the measurement of the absorbance in the calibrated spectrophotometer (with an external PNP standard solution).These results were shown in Fig. 2. (CM and CM+B) and Fig. 3. (PM and PM+B).Lately, the PNP retention was calculated by fitting the amount of PNP measured after the enzymatic determination (PNP exp ) and the amount of PNP added in the control (PNP add ) to a linear equation (PNP exp = k x PNP add ), where k was the slope of the linear fitting.The percentage of PNP recovered in the enzymatic determination was calculated as 100•k, whereas the percentage of PNP retention was calculated as 100• (1-k).PNP retention assays were performed in duplicate for all treatments and shown in Table 2.

Characteristics of the composting mixtures
Different composting mixtures were selected at different stages of the composting process to cover the range of organic matter stabilization degree.
The different nature of the organic matter at different stages of the biodegradable process and the property of the recalcitrant biochar was assessed by thermogravimetry.(Lyons et al., 2006;Tsui and Juan, 2010;Manya et al., 2013).Basically, the TGS-DSC diagrams are characterized by two main mass losses, showing two exothermic peaks, and these are respectively corresponded which correspond to volatilization of light compounds such as aliphatic molecules or carbohydrates and another to oxidation of high molecular weight components (Fig. 1.).Comparing the graph shapes between the samples from initial stage (Fig. 1.A and C) and from maturity stage (Fig. 1.B and D), the second wave of peak, generated by mass loss at 350-550 ºC, was pronouncedly shown at maturity stage, due to the selective degradation of labile organic materials during the composting process.As a consequence, the index of lability of W 2 /W 1 in all samples of maturity stage is higher than those of initial stage (Table 1).
The influence of additional biochar into the composting mixture at the initial phase (Fig. 1.A and C) is observed by higher peak of second wave in biochar blended composts (PM+B, CM+B), which are described in dotted lines.This has resulted from that biochar originated from hard-wood mostly consists of recalcitrant compounds, which are combusted at W 2 range (350-550 0 C) in an oxidant atmosphere of air.Consequently, W 2 /W 1 ratio at initial time (Table 1) increased in biochar blended piles (PM+B, CM+B) from the piles of non-biochar addition (PM, CM).After maturation stage (Table 1), W 2 /W 1 ratio markedly increased in the biochar blended composts (PM+B, CM+B, 2.3, and 1.6, respectively), reflecting the high relative proportion of recalcitrant biochar.

Study of the PNP retention on biochar blended compost.
The colorimetric determination of PNP was influenced by the degree of stability of the composting mixtures, which affected the relative proportion of biochar in the mixture.The biochar blended composts showed more retention of PNP, especially in the case of maturity stage (Figure 2 and 3).The amount of PNP retained by the biochar blended composting mixtures (CM+B and PM+B) varied from 41% in the starting composting mixtures up to 74% in mature composts.This result might have attributed to gained dominance of biochar amount inside composting mixtures which was gradually increased during the composting process.The recalcitrance of biochar character was remained retained until the maturation stage, while labile organic materials in the composting piles were lost due to the selective degradation, as already shown by TGS measurement (Fig. 1.).Therefore the effect of the physico-chemical properties of biochar on the compost structure is expected to be more dominant in the mature stage than at the initial stage.
The PNP retention by biochar also depends on pH status of the buffer solution, used by each specific enzymatic activity.At high pH condition (pH 11), representing alkaline phosphatase essay, the PNP retention is observed in the range between 15 and 30% of the added PNP (Table 2).However, the same spiking assays performed a low pH (pH 6.5 and 5 from acid phosphatase and βglucosidase activities, respectively) exhibited high PNP retention from 30% (acid phosphatase determination in PM+B) up to 70% which is the case of the β-glucosidase determination in CM+B.These results are in agreement with the pH dependence of phenol adsorption efficiency by activated carbon reported by several authors (Ayranci and Duman 2005;Tang et al 2007), concluding that the absorption efficiency of activated carbon is lower in alkaline solution than neutral or acid solution.An increase in the amount of OH ions in alkaline solution reduces the diffusion of phenol ions due to an electrostatic repulsion of negatively charged site of the sorbent and phenolic ions.As the pH increases, the surface charge of pyrogenic materials became negative and decreases its sorption capacity (Beker et al 2010).Furthermore, other authors (Zhang et al 2010) reported that, regarding the mobility of biochar particle, the lower the pH solution, the lesser transport of the biochar particle.
Sorption affinity of pyrogenic material is also influenced by physical properties such as microporosity and surface area, as well as chemical properties such as hydrophobicity in relation with O/C content (Al-Asheh et al., 2004;Ko et al., 2007;Tsui and Juang, 2010).Micropore and mesopore structure, estimated respectively by the iodine number and the methylene blue adsorption, are usually enlarged at high pyrolysis temperature together with surface area.. Overall, aAll these biochar properties are dominantly defined by feedstock and the pyrolysis conditions used for the preparation of the biochar (Uchimiya et al., 2010).
The PNP retention by the organic matter of the composting mixtures prepared without biochar (PM and CM) was also affected by the pH gradient.Table 2 shows that at low pH solution (pH 5) the initial stage of composting, CM has 69% of PNP recovery, meaning 31% of PNP was retained.This methodological problem in the determination of the enzymatic activities is well-known in clay mineral soils or soils enriched with organic matter (Tabatabai and Bremer 1971;Trasar-Cepeda et al 1988).The organic material containing large amount of humic substances are known to easily absorb PNP molecules (Chen et al 2009).
In order to tackle this obstacle, several authors have recently recommended to test the soil enzymatic assays in samples blended with biochar to ensure the assumption of saturating substrate concentrations, and if necessary to amend the protocols before initiating the assays (Swine (et al., 2013).In practice, and in order to overcome the underestimation by absorption on biochar, Paz-Fierro et al (2012 and2014) used different calibration curves for each different type of amendment to acquire an accurate measure of soil enzymatic activities.This problem is even more complex in composting samples, where the degradation of labile organic matter causes a progressive enrichment in the proportion of biochar in the mixture.The different proportion of biochar in the starting mixtures and the mature compost also requires the adaptation and optimization of the enzymatic assay to the different composting stage.
In conclusion, the presence of biochar limited the validity of enzymatic essays for the colorimetric determination of PNP since PNP was strongly retained in biochar blended compost.It is challengeable to improve the colorimetric methods of PNP determination for biochar interaction, and clear-cut solution has not been found until present day.Further research is necessary in order to correctly quantify enzymatic activity in presence of biochar.More other factors are necessary to be considerable for understanding the biochar interaction with enzymatic activity assay.