Use of biomass ash to reduce toxicity affecting soil bacterial community growth due to tetracycline antibiotics

1 Use of biomass ash to reduce toxicity affecting soil bacterial community growth 1 due to tetracycline antibiotics 2 3 V. Santás-Miguel1, M.J. Fernández-Sanjurjo2, A. Núñez-Delgado2, E. Álvarez-4 Rodríguez2, M. Díaz-Raviña3, M. Arias-Estevez 1, D. Fernandez-Calviño1* 5 6 1Área de Edafoloxía e Química Agrícola. Facultade de Ciencias. Universidade de Vigo. 7 As Lagoas 1, 32004 Ourense. Spain 8 2Departamento de Edafoloxía e Química Agrícola, Escola Politécnica Superior de 9 Enxeñaría, Universidade de Santiago de Compostela. Campus univ. 27002 Lugo. Spain 10 3Departamento de Bioquímica del Suelo, Instituto de Investigaciones Agrobiológicas de 11 Galicia (IIAG/CSIC). Santiago de Compostela. Spain 12 13 * Corresponding author E-mail: [email protected] (V. Santás Miguel) 14 15 Declarations of interest: none. 16 17 18 2 Abstract 19 Tetracycline antibiotics (TA) used in veterinary medicine reach terrestrial ecosystems 20 mostly via the repeated applications of animal manures and slurries on agricultural soils, 21 where they may cause toxic effects on bacterial communities. In the current work, we 22 studied the efficacy of adding doses of 0, 6, 24 and 48 g kg-1 of biomass ash (BA) to 23 four different soils to reduce potential negative effects of tetracycline antibiotics. 24 Specifically, soil samples were polluted with different concentrations of tetracycline, 25 oxytetracycline or chlortetracycline, and the bacterial community growth was estimated 26 using the 3H leucine incorporation technique. Soil amendment with BA increased soil 27 pH (1.3-4.8 units), total carbon (0.7-5.8 g kg-1) and Fe and Al oxides concentrations 28 (0.25-3.98 g kg-1), as well as bacterial activity (1-9 times compared to the control). In 29 addition, BA amendment at high doses (24 or 48 g kg-1) resulted in a similar toxicity 30 decrease for the three antibiotics, but with variations among soils. The reductions in 31 antibiotics toxicity were very variable, ranging between 5% and 100% (total recovery). 32 In view of that, the spreading of BA could be interesting as management practice to 33 reduce risks of soil pollution and subsequent toxicity on bacterial communities due to 34 tetracycline antibiotics. 35 36 Keywords: Biomass ash; chlortetracycline; leucine incorporation; oxytetracycline; 37 tetracycline 38 39 1. Introduction 40 Veterinary antibiotics are widely used for animal health, and even as animal 41 growth promoters in countries where this parallel use is not banned (Anderson et al., 42 2005). These antibiotics reach terrestrial ecosystems via the repeated applications of 43 3 animal manures and slurries as amendments on agricultural soils (Pan and Chu, 2017). 44 Once in the soil, veterinary antibiotics may cause harmful effects on non-target 45 organisms, such as soil bacterial communities (Warman, 1980). The presence of 46 antibiotics in the soil may cause a reduction in microbial biodiversity (Thiele-Bruhn and 47 Beck, 2005; Hammesfahr et al., 2008), influence the growth and enzymatic activities of 48 bacterial communities (Liu et al., 2009; Ma et al., 2016; Song et al., 2017; Santás-49 Miguel et al., 2020), and hence, ecological functions and functional stability (Zielezny 50 et al., 2006; Demoling and Bååth, 2008; Pallecchi et al., 2008; Toth et al., 2011). 51 Tetracyclines are the antibiotics most used in the European Union, especially 52 tetracycline, oxytetracycline and chlortetracycline (ESVAC, 2016), which motivated 53 that previous studies have focused on the effects of tetracycline residues on soil 54 microbial functions (Thiele-Bruhn, 2005; Yang et al., 2010; Liu et al., 2012; Ma et al., 55 2016; Song et al., 2017). It has been established that the main agents responsible for the 56 sustainability of soils are microorganisms (Pulleman et al., 2012), since they are 57 involved in nutrient cycles and renewal of organic matter (Thiele-Bruhn et al., 2012). 58 Therefore, the correct growth of soil bacterial communities will help to maintain an 59 optimal soil fertility and organic matter turnover. However, there are only few studies 60 estimating the effects of tetracycline antibiotics on soil bacterial community growth 61 (Rousk et al., 2008; Rousk et al., 2009a; Santás-Miguel et al., 2020). These studies have 62 shown a marked toxicity due to tetracycline antibiotics, which was highly dependent on 63 soil characteristics. 64 In order to prevent harmful effects of antibiotics accumulated into soils, the use 65 of appropriate waste and/or by-products as soil amendments may be a low cost 66 alternative aiding to decrease undesirable toxicity affecting soil bacterial communities. 67 It has been previously demonstrated that soil amendment with certain by-products, such 68 4 as mussel-shell (Fernández-Pazos et al., 2013; Osorio-López et al., 2014; Otero et al., 69 2015; Romar-Gasalla et al., 2018), pyritic material (Fernández-Pazos et al., 2013; 70 Osorio-López et al., 2014; Otero et al., 2015), pine bark (Romar-Gasalla et al., 2018), 71 oak ash (Romar-Gasalla et al., 2018) and hemp waste (Romar-Gasalla et al., 2018) may 72 reduce the availability of different types of pollutants, both anionic and cationic. 73 However, even if is considered that the amendment of soils with by-products may 74 stimulate or inhibit microbial communities, the potential effects of by-products on soil 75 bacterial community growth has been poorly studied. Within the few researches 76 previously carried out in this regard, Fernández-Calviño et al. (2015a) assessed the 77 effect of different doses of crushed mussel shell applied in two Cu-polluted acid mine 78 soils, showing long-term positive effects. In a subsequent study, Fernández-Calviño et 79 al. (2018) measured bacterial and fungal growth in acid mine soils amended with pine 80 bark, crushed mussel shell and a mixture of both, showing general positive effects on 81 microbial growth. However, no previous studies have dealt with the effects of biomass 82 ash amendment on bacterial community growth in soils. 83 Biomass ash (mainly derived from biomass energy plants and combustion 84 boilers of variate sizes) is a very abundant waste material, which increases soil pH, as 85 well as the contents of major nutrients, and decreases the availability of Al and some 86 minor elements (Demeyer et al., 2001). Also, biomass ash may reduce the availability of 87 different pollutants in soils (Núñez-Delgado et al., 2015; Núñez-Delgado et al., 2017; 88 Romar-Gasalla et al., 2018). Specifically regarding effects on microbiota, Zimmermann 89 and Frey (2002) studied the effect of wood ash amendment on soil microbial process, 90 showing an increase in pH and a subsequent stimulation of microbial biomass, basal 91 respiration and enzymatic activities. 92 5 In view of this background, in the current work we hypothesize that adding 93 appropriate doses of biomass ash to soils, the bacterial community growth would be 94 stimulated, and toxicity effects due to tetracycline antibiotics would decrease. 95 Therefore, the main objective of this work is to assess the effect of biomass ash 96 amendments on bacterial community growth in soils polluted with the tetracycline 97 antibiotics tetracycline, oxytetracycline and chlortetracycline. 98 99 2. Material and Methods 100 2.1. Chemicals 101 Tetracycline hydrochloride (CAS. 64-75-5; ≥95% in purity), Oxytetracycline 102 hydrochloride (CAS 2058-46-0; ≥95% in purity) and Chlortetracycline hydrochloride 103 (CAS 64-72-2; ≥97% in purity), all three supplied by Sigma–Aldrich (Steinheim, 104 Germany), were used for soil spiking. 105 106 2.2. Procurement of soil and biomass ash samples 107 Four soils devoted to cultivation of potatoes in rotation with cereal, located in 108 Galicia (NW of Spain), previously analyzed by Conde-Cid et al. (2019a), were selected 109 for the current study. On each sampling site 10-20 sub-samples were taken in the soil 110 surface horizon (0-20 cm) using an Edelman probe, and were subsequently mixed in 111 one composite sample. At the laboratory, the soil samples were air dried, sieved by a 2 112 mm mesh light screen, homogenized and stored in polyethylene bottles. Soil pH was 113 determined in water (soil ratio: 1:2.5), using a combined glass electrode. Total carbon 114 and total nitrogen were determined by elemental analysis in a LECO CHN-1000 (LECO 115 Corporation, St. Joseph, MI, USA). Exchangeable basic cations (Ca, Mg, Na and K) 116 were extracted with 0.2 M NH4Cl (Sumner and Miller, 1996), while exchangeable Al 117 6 was extracted with 1 M KCl (Bertsch and Bloom, 1996), and then determined by flame 118 atomic absorption (Ca, Mg and Al) or emission spectroscopy (Na and K). The effective 119 cation exchange capacity (eCEC) was estimated as the sum of the exchangeable basic 120 cations and Al. Fe and Al oxides (Feo and Alo) were extracted with 0.2 M ammonium 121 oxalate-oxalic acid (Blakemore, 1978). 122 The main characteristics of these soils are shown in Table 1. Briefly, these soils 123 present pH values (measured in water) between 4.6 and 5.1, organic carbon contents 124 between 1.1 and 3.8%, and effective cation exchange capacity between 4.1 and 6.8 125 cmolc kg-1. Al and Fe oxides have values ranging between 855-5040 mg kg-1, and 126 between 1150-2585 mg kg-1, respectively. 127 The biomass ash (BA) used was from a combustion boiler in Lugo (Spain). 128 Previously, this by-product was analyzed by Romar-Gasalla et al. (2018). Briefly, it 129 showed a pH in water of 11.3, a total carbon content of 11.7%, a total nitrogen content 130 of 0.21%, and a C/N ratio of 55.5. It presented high contents of amorphous Al and Fe 131 oxides (8323 and 4233 mg kg-1 respectively), and also relatively high levels of oxides of 132 Ca and other elements (Table 1), due the combustion process. 133 134 2.3. Experimental Design 135 Four soils were amended with four doses of biomass ash (0, 6, 24 and 48 g kg-1), 136 resulting in 16 soil mixtures. In brief, 288 dry grams of each soil sample were weighed 137 in four 500 mL polyethylene bottles (72 g per bottle) and mixed with different amounts 138 of biomass ash (0, 0.43, 1.73, 3.46 g) in order to obtain the final concentrations of 0, 6, 139 24 and 48 g of biomass ash per kg-1 of soil. These concentrations were satisfactorily 140 used in previous works testing the effects of different by-products on soils (Ramírez-141 Pérez et al., 2013; Fernández-Calviño et al., 2015a; Conde-Cid et al., 2020a; Conde-Cid 142 7 et al., 2020b). Later on, the resulting mixtures of dry soil and different concentrations of 143 biomass ash were rewetted up to 60-80% of water-holding capacity, and the bottles 144 sealed with lab-film (Parafilm) to avoid soil drying. Then, these mixtures were 145 incubated during 30 days at 22 ºC in the dark. 146 After this incubation time, different amounts of tetracycline, oxytetracycline and 147 chlortetracycline were dry added (via talc) separately (by triplicate) to the 16 soil 148 mixtures in 50 mL polypropylene tubes, reaching 8 concentrations for each antibiotic 149 and each mixture (0, 0.5, 2, 7.8, 31.3, 125, 500 and 2000 mg kg-1). The bottles were 150 sealed with Parafilm to avoid soil drying (thus maintaining water-holding capacity). The 151 resulting microcosms (totalizing a number of 960, 1 g each one) were initially studied 152 for 1 day, focusing on the assessment of the subsequent bacterial community growth, 153 performed after extraction from soil samples, and estimated using the 3H Leucine 154 incorporation method (Bååth 1994; Bååth et al., 2001). Briefly, 1 g of soil (fresh 155 weight) was mixed with 10 mL distilled water using a multivortex shaker for 3 min at 156 maximum intensity, followed by low-speed centrifugation at 1000 x g for 10 min, to 157 create a bacterial suspension in the supernatant. An aliquot (1 mL) of this suspension 158 was transferred to 2 mL micro-centrifugation tubes. Then, 2 μL [3H]Leu (3.7 MBq mL−1 159 and 0.574 TBq mmol−1; Perkin Elmer, USA) were added with non-labeled Leu to each 160 tube, resulting in 275 nM Leu in the bacterial suspensions. After incubation for 2 h at 22 161 °C, bacterial growth was stopped with 75 μL 100% trichloroacetic acid. Washing was 162 performed as described by Bååth et al. (2001), and the amount of leucine incorporated 163 was determined using scintillation liquid counting (Tri-Carb 2810 TR, Perkin Elmer, 164 USA). 165 166 2.4. Data analysis 167 8 Data on bacterial growth obtained for each soil and each concentration of 168 tetracycline, oxytetracycline or chlortetracycline were normalized (Santás-Miguel et al., 169 2020) respect to the control (samples without antibiotic) to be able to compare the 170 inhibition curves of different soils and concentrations of biomass ash. The normalized 171 data were represented as inhibition curves of relative bacterial growth as a function of 172 antibiotic doses. From the inhibition curves, a toxicity index (log IC50) was calculated to 173 compare the effects of different doses of BA regarding the toxicity exerted by 174 antibiotics on soil bacterial communities. The logarithm of the concentration that 175 inhibits 50% of bacterial growth (log IC50) was calculated using the following logistic 176 model: Y= c/[1+eb(a-x)], where Y is Leu incorporation for each antibiotic concentration, 177 x is the logarithm of the concentration of antibiotic added, a is the value of log IC50, b is 178 a parameter related to the slope of inhibition curves, and c is the bacterial growth 179 without antibiotic addition. 180 181 3. Results and discussion 182 3.1. Biomass ash effect on general soil characteristics and bacterial community growth 183 In soils without BA (dose 0 g kg-1) the values of soil pH varied between 4.6 and 184 5.1 (Table 1). However, soil pH changed significantly after biomass ash addition, 185 following dose-response curves (Fig. 1). Thus, in soil samples amendment with 6 g kg-1 186 of biomass ash, pH increased between 1.3 and 2.4 units. Regarding soils amended with 187 24 g kg-1 and 48 g kg-1 of BA, pH values increases between 2.9 and 4.2 units, and 188 between 3.6 and 4.8 units, respectively. Total carbon in soils without BA varied 189 between 11 and 38 g kg-1 (Table 1), values in the range of those usually found in Galicia 190 (Calvo de Anta et al., 2015). Carrying out a theoretical calculation, the addition of BA 191 (which contains 11.7% of C, Table 1) to agricultural soils contributed to an increase of 192 9 total carbon amounting: 0.7 g kg-1 for a dose of 6 g kg-1 of BA; 2.9 g kg-1 for a dose of 193 24 g kg-1 of BA; and 5.8 g kg-1 for a dose of 48 g kg-1 of BA. The contents of Fe and Al 194 oxides in the soils studied varied between 1.15 and 2.59 g kg-1, and between 0.86 and 195 5.04 g kg-1, respectively (Feo and Alo contents in Table 1). Carrying out the theoretical 196 calculation of Fe and Al oxides added to each soil by BA amendment, the results 197 indicated that the addition of 6 g kg-1 of BA to soils contributed to an increase of 0.25 198 and 0.50 g kg-1 in the contents of Fe and Al oxides; while the increase ranged between 199 1.01 and 1.99 g kg-1 for a BA dose of 24 g kg-1; and reached up to between 2.02 and 200 3.98 g kg-1 for a dose of 48 g kg-1 of BA (Table 2). 201 The behavior regarding mean bacterial growth in response to soil amendment with 202 biomass ash (Fig. 2) was different for each soil. In soil 1 and 2 it was observed that the 203 effect on bacterial growth due to BA addition was low (increases between 1-1.9 times as 204 compared to control), while it was high for soils 3 and 4 (increases of up to 9.8 and 7.0 205 times as compared to the control). Previous studies also found increases in bacterial 206 community growth in response to soil amendment with various by-products, such as 207 crushed mussel shells (Fernández-Caviño et al., 2015a), pine bark (Fernandez-Calviño 208 et al., 2018), and marble wastes or organic wastes (Zornoza et al., 2016). These 209 increases were generally attributed to both increases in soil pH and in total carbon, two 210 characteristics also increased by biomass ash amendments. In relation to soil pH, Rousk 211 et al. (2009b) found increases of 5-fold in bacterial community growth for a gradient of 212 soil pH going from 4 up to 8. Organic matter also shows high importance for bacterial 213 growth, and different authors found important increased in bacterial community growth 214 in mine soils amended with organic materials (Zornoza et al., 2016; Fernández-Calviño 215 et al., 2018). 216 217 16 Conde-Cid, M., Fernández-Calviño, D., Nóvoa-Muñoz, J. C., Núñez-Delgado, A., 332 Fernández-Sanjurjo, M. 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Soil respiration and microbial properties in an acid 476 forest soil: effects of wood ash. Soil Biol. Biochem. 34, 1727-1737. 477 Zornoza, R., Acosta, J.A., Faz, A., Bååth, E., 2016. Microbial growth and community 478 structure in acid mine soils after addition of different amendments for soil 479 reclamation. Geoderma 272, 64-72. 480 481 482 23 Table 1. General characteristic of the soils and biomass ash studied. Average values 483 (n=3) with coefficients of variation always <5% 484 485 pHW is pH measured in water; C is total organic carbon; NT is total nitrogen; eCEC is the effective cation exchange capacity (cmolckg- 486 1). Xe is exchangeable concentration of the element (cmol(+) kg-1); Alo, Feo: extracted with ammonium oxalate (mg kg-1). 487 488 489 Soil 1 Soil 2 Soil 3 Soil 4 Biomass ash C (%) 1.07 3.75 3.10 3.39 11.65 N T (%) 0.09 0.34 0.25 0.31 0.21 pH w 4.80 5.05 4.63 4.74 11.31 Ca e (cmol (+) kg-1) 1.53 3.67 1.51 2.24 95.03 Mg e (cmol (+) kg-1) 0.41 0.71 0.52 0.64 3.26 Na e (cmol (+) kg-1) 0.25 0.12 0.21 0.35 12.17 K e (cmol (+) kg-1) 1.27 1.22 0.93 1.00 250.7 Al e (cmol (+) kg-1) 0.61 1.10 2.16 1.68 0.07 eCEC (cmol (+) kg-1) 4.08 6.82 5.33 5.92 361.2 Al o (mg kg-1) 855 4335 4950 5040 8323 Fe o (mg kg-1) 1150 2430 1390 2585 4233 24 490 Table 2. Final concentrations of Fe and Al oxides in soils for each dose of biomass ash 491 added to the soils studied. Average values (n=3) with coefficients of variation always 492 <5% 493 Biomass ash dose 0 g kg-1 6 g kg-1 24 g kg-1 48 g kg-1 Soil Alo Feo Alo Feo Alo Feo Alo Feo Soil 1 0.86 1.15 1.35 1.40 2.85 2.16 4.84 3.17 Soil 2 4.34 2.43 4.83 2.68 6.33 3.44 8.32 4.45 Soil 3 4.95 1.39 5.45 1.64 6.94 2.40 8.93 3.41 Soil 4 5.04 2.59 5.54 2.84 7.03 3.60 9.02 4.61 Alo, Feo: extracted with ammonium oxalate (mg kg-1) 494 495 25 Table 3. Values of logarithm IC50 (estimated values ± error) and R2 from fits to logistic 496 model for the 3 antibiotics and 4 soils studied as a function of different biomass ash 497 (BA) doses added. 498 Soil 1 Soil 2 Soil 3 Soil 4 Antibiotic BA dose Log IC50 R2 Log IC50 R2 Log IC50 R2 Log IC50 R2 TC 0 g kg-1 2.26±0.09 0.975 2.71±0.06 0.985 2.51±0.05 0.989 2.68±0.08 0.973 6 g kg-1 1.84±0.09 0.992 3.14±0.01 0.998 2.49±0.05 0.991 2.83±0.12 0.957 24 g kg-1 - - 2.80±0.1 0.963 3.35±0.03 0.992 2.85±0.18 0.941 48 g kg-1 - - 5.84±2.91 0.541 3.58±0.62 0.814 4.64±1.12 0.735 OTC 0 g kg-1 2.11±0.13 0.975 1.90±0.07 0.995 2.70±0.10 0.948 2.46±0.16 0.934 6 g kg-1 2.77±0.10 0.971 2.96±0.21 0.906 1.80±0.05 0.997 2.49±0.07 0.983 24 g kg-1 - - 2.87±0.10 0.970 2.84±0.11 0.921 3.09±0.10 0.921 48 g kg-1 - - 4.75±1.53 0.695 3.64±0.13 0.929 3.35±0.46 0.818 CTC 0 g kg-1 1.82±0.12 0.985 2.00±0.08 0.982 2.15±0.08 0.965 2.42±0.03 0.996 6 g kg-1 1.79±0.09 0.985 1.88±0.18 0.953 1.63±0.10 0.990 1.81±0.16 0.973 24 g kg-1 - - 2.17±0.12 0.987 3.08±0.07 0.953 3.99±0.26 0.943 48 g kg-1 - - 3.36±0.18 0.280 - - 2.43±0.45 0.926 TC: tetracycline; OTC: oxytetracycline; CTC: chlortetracycline 499 500 501