key: cord-0793482-5f15l9tv
authors: Zhi, Lin; Zhipeng, Rui; Minglong, Liu; Rongjun, Bian; Xiaoyu, Liu; Haifei, Lu; Kun, Cheng; Xuhui, Zhang; Jufeng, Zheng; Lianqing, Li; Marios, Drosos; Stephen, Joseph; Natarjan, Ishwaran; Genxing, Pan
title: Pyrolyzed biowastes deactivated potentially toxic metals and eliminated antibiotic resistant genes for healthy vegetable production
date: 2020-09-19
journal: J Clean Prod
DOI: 10.1016/j.jclepro.2020.124208
sha: 723816afedb17531269af52b38ac5c5ab4937dc8
doc_id: 793482
cord_uid: 5f15l9tv

Potentially toxic metals (PTEs) and antibiotic resistance genes (ARGs) present in bio-wastes were the major environmental and health risks for soil use. If pyrolyzing bio-wastes into biochar could minimize such risks had not been elucidated. This study evaluated PTE pools, microbial and ARGs abundances of wheat straw (WS), swine manure (SM) and sewage sludge (SS) before and after pyrolysis, which were again tested for soil amendment at a 2% dosage in a pot experiment with a vegetable crop of pak choi (Brassica campestris L.). Pyrolysis led to PTEs concentration in biochars but reduced greatly their mobility, availability and migration potential, as revealed respectively by leaching, CaCl(2) extraction and risk assessment coding. In SM and SS after pyrolysis, gene abundance was removed by 4 to 5 orders for bacterial, by 2-3 orders for fungi and by 3-5 orders for total ARGs. With these material amended, PTEs available pool decreased by 25% to 85% while all ARGs eliminated to background in the pot soil. Unlike a >50% yield decrease and a >30% quality decline with unpyrolyzed SM and SS, their biochars significantly (p>0.05) increased biomass production and overall quality of pak choi grown in the amended soil. Comparatively, amendment of the biochars decreased plant PTEs content by 23-57% and greatly reduced health risk of pak choi, with total target hazard quotient values well below the guideline limit for subsistence diet by adult. Furthermore, biochar soil amendment enabled a synergic improvement on soil fertility, product quality, and biomass production as well as metal stabilization in the soil-plant system. Thus, biowastes pyrolysis and reuse in vegetable production could help build up a closed loop of production-waste-biochar-production, addressing not only circular economy but healthy food and climate nexus also and contributing to achieving the United Nations sustainable development goals.

dried in a water bath before another reaction with 25 mL ammonium acetate (1 M, pH 283 2.0) by shaking for 1 h at 25 °C. Finally, the residue was extracted with 10 mL 284 HNO 3 -HClO 4 (4:1, v/v) for UWS, WSN, USM and SMB, or with 15 mL HF-HNO 3 -285 HClO 4 (4:1:1, v/v) for USS and SSB, to obtain the residual fraction (F4) of metals 286 existed in mineral phase. 287

The solubility products of PTEs were measured of the biowastes before and after 288 pyrolysis, with the modified toxicity characteristic leaching procedure (TCLP) based 289 on USEPA1311 method (EPA, 1992) . Briefly, a portion of a sample (1.00 g of dry 290

weight equivalent) was soaked in 20 mL unbuffered glacial acetic acid solution (pH 291 2.88) for 18 h, and centrifuged at 4000 rpm for 10 min. After filtering through a 0.45 292 μm membrane, the solution was collected for metal content determination. 293

For metal contents of the digestions/extractions obtained above, Cu and Zn, and Pb 294 and Cd were determined respectively with flame and graphite furnace atomic 295 absorption spectrometry (FAAS /GFAAS, A3, Persee, Analytical Instruments, China). 296

Analytical quality was guaranteed using certified reference materials GSS-1 for USS, 297 SSB and soil samples, and GBW(E) 080684 for other biowastes and plant samples. 298

These reference materials were provided by the Chinese National Research Centre for 299

Standards. Recovery of the analyzed metals was in a range of 89% -109%. For 300 samples of plant collected at harvest in the pot experiment, content of soluble sugar, 301 total protein and nitrate was determined with colorimetry respectively using the 302 anthrone method, coomassie brilliant blue method and salicylic acid method. Content 303 of vitamin C (Vc) was determined using the titration method. 304

In addition, the biowaste samples were examined for surface morphology with 305 scanning electron microscopy (SEM) (S3400, Hitachi, Japan) equipped with Brunauer-Emmett-Teller (BET) method with Flow Sorb II 2300 (Micromeritics, 309 Aachen, Germany). Meanwhile, cation exchange capacity was measured with a 310 modified ammonium-acetate compulsory displacement method (Gaskin et al., 2008) . 311

Samples of SM and SS before and after pyrolysis, and the amended pot soils were 313 analyzed for abundance of microbial genes and ARGs. For this, 0.25 g of a sample 314 was extracted with a FastDNA SPIN Kit for Soil (MP Biomedicals, USA) according 315 to the manufacturer's instructions. After qualitative detection using standard PCR 316 described in the Supporting Information (Table S1) , bacterial 16s rDNA, fungal 18s 317 rDNA,five tetracycline genes (tetC, tetG, tetM, tetO, tetW), two sulfonamide genes 318 (sul1 and sul2) and one class 1 integron-integrase gene (intI1) with bright target bands 319 were analyzed by quantitative PCR (qPCR) using QuantStudio ® 5 Real-Time PCR 320 systems (Thermo Fisher) (Panda et al., 2017) . The reaction mixture consisted of 0.4 321 μL of 20 pM of each primer, 10 μL of SYBR Green II master mix and 2μL of template 322 DNA in a total volume of 20 μL. And the conditions as following: denaturation at 323 94 °C for 5 min and 40 cycles of 94 °C for 45 s, annealing for 30 s, extension at 72 °C 324 for 1 min, and extension at 72 °C for 7 min. Absolute copy numbers of the genes in 325 the samples were calculated using the external standard curve method, which was 326 generated by a 10-fold serial dilution of plasmid DNA (R 2 ≥ 0.990). The amplification 327 efficiency of each pair of primer was between 87 -110%. 328

For assessing the environmental and health risks of soil and plant treated with the 330 biowastes, a number of dimensionless indices were estimated as follows.

x 100

(1) 334

Where, C F1 and C t is respectively the content of extractable fraction with acetic acid 335 and the total content of a metal in a material, obtained as per BCR procedure in 336 Section 2.2. A PTE in a material is concerned as no, low, medium, high and very 337 high risk with a RAC value of <1, 1-10, 11-30, 31-50 and >50, respectively. 338 Lastly, a dimensionless parameter of target hazard quotient (THQ) ( up to give an overall potential health risk (THQ total ) via food exposure to pak choi. 370

All the data were expressed as mean ± standard deviation and processed with 371 Excel 2016. Statistical analysis was performed using SPSS Statistics 22. Data means 372 of biowaste between before and after pyrolysis, and among the amendment treatments 373 were tested for difference with one-way ANOVA. Multiple comparisons were 374 conducted using Tukey's test to determine the significance of difference between the 375 biowaste types or among amendment treatments. Pearson's correlation was also 376 performed for relationships between/among the analyzed parameters. A difference or a 377 correlation was considered significant at p <0.05, as per the relevant statistical 378 protocols. 379

The basic properties are presented in Table 1 and the SEM images in Fig. 2 , of the 382 biowastes before and after pyrolysis. UWS and USM seemed soft and more or less 383 porous. After pyrolysis, their biochars had markedly higher porosity of nano-pores, 384 with a surface area in a range of 6-20 m 2 g -1 . In comparison to before pyrolysis, the 385 pH of biochars was all significantly (p<0.05) higher by 2.6-3.9 units while EC was 386 significantly (p<0.05) higher by almost 80% for WSB but lower by 30% and 90% for 387 SMB and SSB, respectively. Total OC was significantly (p<0.05) increased by 27% 388 for WSB but decreased for SMB by 14% and SSB by 12%. Being different, total N 389 was insignificantly (p>0.05) increased for WSB but significantly (p<0.05) decreased 390 by almost 50% for SMB and SSB, respectively compared to their feedstock. Whereas, 391 total K after pyrolysis was almost doubled (p<0.01) as before pyrolysis. Moreover, 392 total P was unchanged (p>0.05) in WSB but significantly (p<0.05) increased by 393 almost 40% in SMB and by 20% in SSB over their feedstock. In contrast, ash content 394 was seen very significantly (p<0.01) increased by 2-3 folds in WSB and SMB and by 395 80% in SSB, respectively compared to the feedstocks. As per the EDS spectra coupled 396 with SEM provided in Fig.S1 , more elements were detectable in high intensity in the 397 biowastes after pyrolysis than before, including Mg, Cu and Zn from wheat residue, 398 Compared to before pyrolysis, the PTEs were all highly (p<0.01) concentrated in 425 the biochars after biowaste pyrolysis, except for Cd reduction in SSB. In contrast, in a 426 small portion (<6%) to total, CaCl 2 -extractable pool was small (<1 mg kg -1 ) for the CaCl 2 portion to total) was very significantly (p<0.01) but greatly reduced by over 97% 440

for Cu and Zn, by 74-86% for Pb but by 21% (in SMB) -75% for Cd. Coincidently, 441 the leaching potential, the portion of TCLP pool to total, was very significantly 442 (p<0.01) but greatly reduced by 80-98 % for Cu, Zn and Pb in all biochars except by 443 50% for Pb in SSB. That of Cd, however, was significantly (p<0.01) lowered by 59% 444 in WSB and 80% in SMB despite of a significant (p<0.05) 25% increase in SSB. 445

The proportions of PTEs chemical fractions for the biowastes before and after 446 pyrolysis are plotted in Fig. 3 , with the original measurement data presented in Table  447 S2. The speciation procedure yielded a recovery between 95.55-108. 7% 

For the biowastes of SM and SS, data of gene abundance of microbial 491 communities and total ARGs are shown in Fig. 4 . Before pyrolysis, gene abundance 492 (copies g -1 ) of bacterial was 2.1×10 12 and 7.0×10 9 and that of fungi 7.4 ×10 7 and 1.6 493 ×10 7 , respectively in SM and SS. With individual genes varied in a wide range of 10 3 494 -10 9 copies g -1 (Table S3) , total ARGs was 8.5 × 10 9 in SM and 1.6 ×10 7 in SS. 495

Overall, bacterial and ARGs gene abundance as well as bacterial to fungal ratio was 496 higher by almost 2 orders in SM than in SS. 497

After pyrolysis, whereas, gene abundance (copies g -1 ) of bacterial was 9.0 ×10 5 in 498 SMB and 1.8×10 6 in SSB while that of fungal was 2.0 ×10 4 in SMB and 1.3 ×10 5 in 499 SSB. Both in SSB and SMB, gene abundance (copies g -1 ) of individual ARGs was all 500 very low (Table S3) , at levels of 2.3 ×10 2 to 6.8 ×10 3 while sul1 gene undetected. 501

Unlike the biowastes before pyrolysis, there was no difference in microbial and total 502 ARG abundance between SMB and SSB though bacterial to fungal ratio was 503 relatively higher in SSB than in SMB. After pyrolysis, abundances of microbial and 504 Data of soil fertility changes following amendment of the biowastes before and 509 after pyrolysis are organized in Table 3 . Under biowaste amendment compared to no 510 amendment (CK), significantly (p<0.05) positive changes were found for soil pH, 511 SOC, and C/N ratio, at varying extents with different parameters and the biowaste 512 types. Whereas, total N and available P were insignificantly (p>0.05) or slightly 513 increased under WS and SS regardless of pyrolysis. Differently, total N, available K 514 and P were all moderately to greatly enhanced (p<0.05) under SM regardless of 515 pyrolysis. Comparing the biowastes before and after pyrolysis, soil pH was increased 516 by 0.2-0.3 units under all the biochars than the biowastes unpyrolyzed. But increases 517 in SOC, available K and P were significantly (p<0.05) higher by 20-40% under SWB 518 and SMB respectively compared to UWS and USM though similar between USS and 519 SSB. No differences (p>0.05) in these parameters were observed between USS and 520 SSB. Also, total N was 12% lower (p<0.05) under SMB and SSB compared to USM 521 and USS, respectively. 522

In Table 4 are presented the data of total and extractable heavy metals in the pot 524 soil following amendment with the biowastes before or after pyrolysis. Compared to 525 CK, soil total content of the PTEs was significantly (p<0.05) increased at varying 526 extents under all amendments despite unchanged (p>0.05) under UWS. The increases 527 of Cu, Zn and Pb contents under the biochars were significantly (p<0.05) higher by 528 16-53% than under unpyrolyzed biowastes, corresponding generally to the metal 529 USS, relevant to their high available pool (Table 2) . However, these available pools 541 were all significantly (p<0.05) lower under the biochars than under unpyrolyzed 542 biowastes. Moreover, the portion of available pool of these metals was found either 543 unchanged (p>0.05) or reduced significantly (p<0.05) under the biochar amendments 544 despite of a significant increase for Zn under SMB and for Cd under SSB, 545 respectively over amendment of the biowastes before pyrolysis. 546

Abundances of the microbial genes and total ARGs in pot soil following 548 amendment are shown in Fig. 5 , with original data of the individual ARGs given in 549 Table S4 . Gene abundances (copies g -1 ) of both bacteria and fungi were significantly 550 (p<0.05) increased by 3-10 folds under UWS (3.3×10 12 and 6.6 ×10 9 ), and under 551 USM (3.70×10 12 and 4.20×10 9 ) but insignificantly (p>0.05) changed under other 552 amendments, compared to CK without amendment (7.6 × 10 11 and 2.6 × 10 8 ). While 553 no difference between USS and SSB, a great reduction (p<0.05) of gene abundance 554 Fig. 5 , total abundance (copies g -1 ) of ARGs in the pot soil after 563 amendment was in a range of 2.9 ×10 7 to 4.9 ×10 7 . Over CK, the total ARGs gene 564 abundance was unchanged (p>0.05) with all the amendments other than USM. 565

Notably, soil total abundance of ARGs was found as low as 3.8 ×10 7 under SMB 566 compared to 4.9 ×10 8 under USM with high abundance (up to 10 9 copies g -1 ) in the 567 material. Specifically, the intI1 gene abundance (copies g -1 ) was significantly (p<0.05) 568 reduced by 80% under SMB (7.1 ×10 6 ) over USM (3.5 ×10 7 ) (Table S4) . 569

The biomass yield and overall quality of pak choi grown in the soil amended with 571 the unpyrolyzed and pyrolyzed biowastes are organized in Fig. 6 , with the original 572 nutrition quality in Table 5 Table 5 (Table S5) . 597

The target hazard quotients (THQ) of the PTEs in pak choi estimated for adults is 598 plotted in Fig. 7 the biochar products after pyrolysis though total Cd declined in SSB, relative to 619 before pyrolysis (Table 2) . This was resulted from thermal decomposition of organic 620 tissues in the feedstock containing metals and volatilization of carbon molecules 621 during pyrolysis at high temperature (Kistler et al., 1987) , particularly when sewage 622 sludge pyrolyzed (He et al., 2010) . In this study, there were no significant (p<0.05) 623 correlations of extractable and leachable pool to total contents of the studied PTEs, for 624 their divergent variation with the different biowaste types. In terms of the pool portion 625 to total burden, chemical mobility with CaCl 2 extraction and leaching potential with 626 TCLP of the PTES in the biowates, including swine manure and sewage sludge, were 627 shown mostly largely or even completely reduced following pyrolysis (Table 2,  628 Section 3.1). Although leaching potential of Cd was unexpectedly increased in 629 pyrolyzed sewage sludge, the study could confirm a general but sharp deactivation of 630 available or leachable PTEs present in the biowaste through pyrolysis at temperatures Such deactivation of PTEs in the pyrolyzed biowastes could be further evidenced 633 with the BCR fractionation analysis ( Fig. 3 ; Table S2 ). Thereby, lower proportion of 634 PTEs were largely immobilized after pyrolysis as OM bound F3 fraction and mineral 648 bound F4 fraction, with a net 17% increase relative to before pyrolysis (Table S2; pool to the total was found significantly (p<0.05) reduced, so was often the pool itself 696 (Table 4 ), in the pot soil originally low in pH and SOC. This was particularly 697 important for Cu and Zn in swine manure, and Cd in sewage sludge originally highly 698 toxic (Guo et al., 2018) . As a result, plant concentration (Table 6) and 699 bio-accumulation (Table S5) of Cu, Zn and Cd were significantly (p<0.05) lower with 700 pyrolzyed swine manure and sewage sludge than the unpyrolyzed ones. There was a 701 very significant (p<0.01) but strong correlation (Fig. S4) 

In this study, pyrolysis almost completely (>99.99%) removed the microbes in 719 swine manure and sewage sludge originally in high abundance (Fig.4) . This, of course, 720 was caused by the direct sterilization with high temperature pyrolysis (Lehmann and 721 Joseph, 2015) . Resultantly, all the biochars (and USS high in Cd) unchanged 722 microbial abundance of the amended soil (Fig. 5 a,b) . Of course, the significant 723 increase compared to CK in bacterial and fungal abundance under UWS and USM 724 could be attributed to a sharp microbial response to addition of fresh organic matter 725 with nutrients (Tables 1). Whereas, the relatively lower microbial abundance under 726 WSB than UWS and under SMB than USM could be explained with the addition 727 (Table 1) with sul1 even undetected in SMB and SSB and other ARGs eliminated by an order 743 of 10 2 to 10 5 though by one order for tetW in SSB (Table S3) . When amended at 2%, 744 abundance of total ARGs in pot soil was at the background level of CK under all the 745 biowaste materials other than under USM, in which total abundance of ARGs was 746 increased by 10-fold over CK (Fig. 4c) . Also, the total ARGs abundance in amended 747 pot soil tended lower under the biochars than their unpyrolyzed counterparts. As 748 generally carried on Gram-negative bacteria (Wellington et al., 2013) , the ARGs in the 749 biowastes, particularly in swine manure with highly concentrated ARGs (Fig. 4) other ARGs in municipal waste-water biosolids following pyrolysis at temperatures counterparts (Table S3) . Further in the amended soil, the abundance of this gene was 759 reduced by 80% and 40% in SMB and SSB respectively over USM and USS, though 760 already close to the background level in the control. 

Compared to the unpyrolyzed biowaste, pak choi biomass production in the 774 amended pot soil was higher by folds with SMB and SSB but unchanged with WSB 775 (Table 5) . Over control, pak choi biomass yield greatly (p<0.01) declined (by almost 776 60%) under USM and USS, probably owing to the constraints by high salinity (high 777 EC, Table 1) (Table 4 ). This could be true also for that the 787 yield change between USW and SWB, and between USS and SSB was relevant to 788 difference in RAC (mobility) values of Cu and Zn between them (Fig.S2) . Toxicity of 789 Cu and Zn for vegetables were often reported in manure and sewage sludge applied 790 soils (Påhlsson, 1989; Tandi et al., 2004) . While variable changes with biochar in 791 biomass production had been noted (Liu et al., 2013) , this study confirmed that 792 pyrolyzed sewage sludge maintained but pyrolyzed swine manure promoted vegetable 793 production, as a recycling solution for the biowastes with concentration of total PTEs. 794

As for the pak choi nutrition quality, the contents of individual nutrition 795 components were not changed markedly with the amendment treatments including 796 nitrate content, which was high but below the guideline limit of 2500 mg kg -1 (EC, 797 2006 (Table S6) . Notably, a very significant 810 (p<0.01) correlation (Fig. S5) (Table 5) , 823 being not ascribed for yield change (Fig. 6) . Pak choi was a leafy crop with high 824 affinity for Zn and Cd, its plant concentration and total uptake of Cd and Zn as well as 825

Cu was significantly (p<0.05) but positively correlated to the soil CaCl 2 extractable 826 pool (Fig. S4) . Like in the work by Chen et al. (2018) , values of BCF as a plant 827 accumulation factor (Table S5) synergetic improvement of crop production and overall quality (Fig. S5 a,b) , 839 minimizing PTE accumulation in produced food in amended soil. 840

More interestingly, soil quality improvement with carbon increase was prominent 841 in this study. With the microbial abundance preserved or even enhanced, soil reaction 842 and SOC storage in the acid OC poor soil was much improved together with nutrient 843 enrichment of N, P and K, following amendment of the biowaste biochars (Table 3) . 844

Following one season of pak choi production, SOC loss was much smaller under the 845 biochars than under the unpyrlyzed biowastes. Likewise, there were higher increases 846 in soil P and K storage under pyrolyzed than unpyrolyzed biowastes. It should be 847 noted that pak choi biomass production was very well (p<0.01) correlated to soil 848 organic carbon (Fig. S5 c) . This could add value to soil organic carbon sequestration, 849 concerned mainly for climate change mitigation (Rumpel et al., 2018) . The higher 850 storage of available P and K through the amendment input (Tables 1 and 3) , as 851 potential contributor of biochar-assisted soil fertility improvement (Jeffery et al., 852 2011) , could indicate better circularity of nutrients. As these were originally provided 853 with agrochemical input in primary crop production, using pyrolyzed biowastes could 854 potentially contribute to a more sustainable world in the context of bioeconomy 855 smaller and thus the availability greatly lower under biochar than under pyrolyzed 858 biowastes (Table 4 ). Enhancement of SOC and soil available nutrient pool together 859 with declined metal availability could likely bring about improvement of soil fertility 860 quality (Table 3 ). This could benefit not only plant growth for less metal uptake were well below the above mentioned safety thresholds. Whereas, pak choi Zn under 877 USM was over the guideline limit of 20 mg kg -1 but was fortunately cut down by 10 878 mg kg -1 with safety under the biochar SMB. Nonetheless, the calculated individual largely due to Cd (Fig. 7) . However, this value was reduced to 0.97 under its biochar 882 SSB, meeting the guideline limit. Convincingly, pyrolysis minimized health risk 883 through diet consumption, assuming pak choi as the sole source for adult vegetable 884 intake, even though pak choi was a crop with high Cd affinity (Wang and Stuanes, 885 2003) . letters above all the bars, represents a significant difference at p<0.05 (One-way 1454 ANOVA followed by Turkey test) between before and after pyrolysis, and 1455 among all the treatments, respectively. 1456 Table 1 Physico-chemical properties of the biowastes before and after pyrolysis used 1483 in this study. 1484 Table 2 Contents (mg kg -1 ) of total, CaCl 2 extractable and TCLP leachable heavy 1485 metals of the biowastes before and after pyrolysis. 1486 Table 3 Fertility properties of pot soil collected at pak choi harvest under amendment 1487 treatments of the bio-wastes before and after pyrolysis in pot experiment. 1488 Table 4 Total and CaCl 2 -xetractable heavy metals of pot soil collected at pak choi 1489 harvest under amendment of biowastes before and after pyrolysis. 1490 Table 5 Properties of nutrition quality and metal contents of fresh pak choi at harvest 1491 under amendment of the biowastes before and after pyrolysis. Data as mean ± standard deviation of four replicates of a treatment. CK, control; UWS and WSB, USM and SMB, and USS and SSB, wheat 1551 residue, swine manure and sewage sludge respectively before and after pyrolysis. In a single column, different uppercase letters following a 1552 single pair of amendment and different lowercase letters across the column represents a significant difference at p<0.05 between before and after 1553 pyrolysis, and among all the treatments, respectively(One-way ANOVA followed by Turkey test Table 5 Properties of nutrition quality and metal contents of fresh pak choi at harvest under amendment of the biowastes before and after 1556 pyrolysis 1557

Data as mean ± standard deviation of four replicates of a treatment. Sugar and protein both tested in soluble form. CK, control; UWS and WSB, 1558 USM and SMB, and USS and SSB, wheat residue, swine manure and sewage sludge respectively before and after pyrolysis. In a single column, 1559 different uppercase letters following a single pair of amendment and different lowercase letters across the column represent a significant Table S3 Abundance of antibiotic resistance genes (ARGs) and class 1 integron gene 1604 of swine manure and sewage sludge before and after pyrolysis. 1605 Table S4 Abundance (copies g -1 ) of antibiotic resistance genes (ARGs) and class 1 1606 integron gene of pot soil collected at pak choi harvest under amendment of the 1607 biowastes before and after pyrolysis 1608 

Chemistry and phytotoxicity of soil trace elements 1007 from repeated sewage sudge applications

Pyrolysis of crop residues in a mobile bench-scale pyrolyser: Product 1011 characterization and environmental performance

Pyrolysis of contaminated wheat straw to stabilize 1015 toxic metals in biochar but recycle the extract for agricultural use

A 1019 three-year experiment confirms continuous immobilization of cadmium and lead 1020 in contaminated paddy field with biochar amendment

Contamination of organic nutrient 1024 sources with potentially toxic elements, antibiotics and pathogen microorganisms 1025 in relation to P fertilizer potential and treatment options for the production of 1026 sustainable fertilizers: A review

Exploring global changes in 1033 nitrogen and phosphorus cycles in agriculture induced by livestock production 1034 over the 1900-2050 period

Livestock waste-to-bioenergy 1037 generation opportunities

Impact of 1040 pyrolysis temperature and manure source on physicochemical characteristics of 1041 biochar

Biochar's role in mitigating soil nitrous oxide emissions: A review 1045 and meta-analysis

Consistent increase in abundance and diversity but variable change 1049 in community composition of bacteria in topsoil of rice paddy under short term 1050 biochar treatment across three sites from South China

Long-term field application

Effect of biochar 1056 amendment on the alleviation of antibiotic resistance in soil

Crop residue 1061 harvest for bioenergy production and its implications on soil functioning and 1062 plant growth: A review

Urban Drainage Statistical Yearbook 1065 of China

Potential of aeration flow 1067 rate and bio-char addition to reduce greenhouse gas and ammonia emissions 1068 during manure composting

Competing uses for china's straw: the economic and carbon abatement potential 1072 of biochar

Setting maximum levels for certain 1074 contaminants in foodstuffs

uri=CONSLEG:2006R1881: Environ. 141

Synergisms between compost and biochar for 1107 sustainable soil amelioration

Pyrolysis treatment of sewage sludge: A 1110 promising way to produce phosphorus fertilizer

Simple and sequential extractions of heavy metals from different 1114 sewage sludges

Effect of 1117 low-temperature pyrolysis conditions on biochar for agricultural use

Impacts of 1121 anthropogenic activity on the ecology of class 1 integrons and 1122 integron-associated genes in the environment

Toxicity of heavy metals to 1125 microorganisms and microbial processes in agricultural soils: a review

Heavy metal leaching and plant uptake in mudflat soils 1131 amended with sewage sludge

Sector 1134 perception of circular economy driver interrelationships

Physico-chemical properties and microbial responses in biochar-amended soils: 1138 Mechanisms and future directions

Increased occurrence of heavy metals, antibiotics and 1142 resistance genes in surface soil after long-term application of manure

The fate of Cu, Zn, Pb and Cd during the pyrolysis of sewage sludge at 1146 different temperatures

A review of biochar as a low-cost adsorbent for 1150 aqueous heavy metal removal

A quantitative 1157 review of the effects of biochar application to soils on crop productivity using 1158 meta-analysis

Comparison 1161 of soil organic carbon, microbial diversity and enzyme activity of wetlands and 1162 rice paddies in Jingjiang area of Hubei

Effect of Biochar Amendment on Bioavailability and Accumulation of Cadmium 1166 and Trace Elements in Brassica chinensis L. (Chinese cabbage)

Impact of 1169 different feedstocks derived biochar amendment with cadmium low uptake 1170 affinity cultivar of pakchoi (Brassica rapa ssb

of Cd to reduce potential dietary toxicity

Global potential bioethanol production from wasted crops 1174 and crop residues

Microbial load of drinking water reservoir tributaries during 1182 extreme rainfall and runoff

Behavior of chromium, nickel, copper, 1185 zinc, cadmium, mercury, and lead during the pyrolysis of sewage sludge

Circular economy as an 1189 essentially contested concept

Compressed biogas-diesel dual-fuel engine 1192 optimization study for ultralow emission

Usage of biogas in a diesel engine: Compressed bio gas (CBG)

Antibiotic 1197 uptake by plants from soil fertilized with animal manure

Biochar from biomass and waste. Waste biofuel

A review on waste to energy processes using 1207 microwave pyrolysis. Energies 5

Biochar for Environmental Management: Science, 1209 Technology and Implementation

Understanding and enhancing soil biological health: 1214 the solution for reversing soil degradation

Effects 1217 of different biochar components on the yield and quality of non-heading Chinese 1218 cabbage (Brassica campestris ssp. chinesis)

Biochar's effect on crop productivity and the dependence on experimental 1223 conditions-a meta-analysis of literature data

Treatment 1226 technologies for urban solid biowaste to create value products: a review with and Technology Press

Legacy of soil health improvement with carbon increase 1233 following one time amendment of biochar in a paddy soil -A rice farm trial

Characteristic of heavy metals 1236 in biochar derived from sewage sludge

Soil health gap: A concept to establish a 1239 benchmark for soil health management

Structure and function of the soil 1242 microbial community in a long-term fertilizer experiment

Ministry of Agriculture, the People's Republic of China (MOAC), 2012. Standard of 1245 Organic fertilizer

People's republic of China (MEEC)

National Standards of People's Republic of China

Quality-Risk control standard for soil contamination of agricultural land

National Development and Reform Commission, the People's Republic of China 1257 (NDRC), 2012. China Annual Reports of Resources Comprehensive Utilization 1258 in 2011

National Development and Reform Commission, the People's Republic of China 1260 (NDRC), 2013a. China Annual Reports of Resources Comprehensive Utilization 1261 in 2013

Circular on strengthening the comprehensive utilization and 1264 prohibition of open field burning of crop stalks

National Health Commission, the People's Republic of China (NHFPC), 2017. 1268 National food safety standard limits of contaminants in food

Closing the loop on organic waste management: 1270 biochar for agricultural land application and climate change mitigation

Organic solid waste management in a circular economy perspective -A 1274 systematic review and SWOT analysis

Sustainable soil 1284 management: an emerging soil science challenge for global development

Waste-to-resources: 1287 green preparation of magnetic biogas residues-based biochar for effective heavy 1288 metal removals

Polysome fractionation to analyze 1291 mRNA distribution profiles

Biochar 1294 reduces the bioavailability and phytotoxicity of heavy metals

Gene cassettes and cassette 1297 arrays in mobile resistance integrons

1300 and eco-friendly applications for climate change mitigation, waste management, 1305 and wastewater treatment: A review

Impact of fertilizing with raw or 1309 anaerobically digested sewage sludge on the abundance of antibiotic-resistant

coliforms, antibiotic resistance genes, and pathogenic bacteria in soil and on 1311 vegetables at harvest

Resolving governance issues to achieve priority 1314 sustainable development goals related to solid waste management in developing 1315 countries

Soil Pollution: a 1317 hidden reality

4 per 1,000' initiative will boost soil 1319 carbon for climate and food security

A review of survival of pathogenic bacteria in organic waste used 1322 in biogas plants

Solid-solution partitioningof metals in 1325 contaminated soils: Dependence on pH , total metal burden, and organic matter

Agricultural utilization of 1332 biosolids: A review on potential effects on soil and plant grown

Studies on distribution and 1335 fractionation of heavy metals in Gomti river sediments

Standardization Administration ,the People's Republic of China (SAC)

Standardization Administration, the People's Republic of China (SAC

Determination of oxytetracyline, tetracyline, chlortetracycline and doxycycline 1341 content for organic fertilizers-HPLC method

Antibiotic resistome and its association with bacterial communities during 1344 sewage sludge composting

Composting manure and sludge

Environmental and potential health 1349 effects of growing leafy vegetables on soil irrigated using sewage sludge and 1350 effluent: a case of Zn and Cu

Textural and 1357 chemical properties of swine-manure-derived biochar pertinent to its potential 1358 use as a soil amendment

Screening biochars for heavy metal 1361 retention in soil: role of oxygen functional groups

Diversity and activity of 1364 cellulose-decomposing bacteria, isolated from a sandy and a loamy soil after 1365 long-term manure application

Speciation of heavy 1368 metals in soils and sediments: an account of the improvement and harmonization 1369 of extraction techniques undertaken under the auspices of the BCR of the 1370 Commission of the European Communities

United States, Environmental Protection Agency, Integrated Risk 1375 Information System. www.epa.gov/iris/subst. assessment of heavy metals in sewage sludge during hydrothermal treatment 1381 combined with pyrolysis

Health risks of heavy metals to the general 1384 public in Tianjin, China via consumption of vegetables and fish

The role of the natural environment in the emergence of 1389 antibiotic resistance in gram-negative bacteria

Codex Alimentarius Commission. General standard for 1392 contaminants and toxic in food and feed

Changing trends of market demand for Pak choi cultivars in

Chinese with English 1396 summary

Leachability and vegetable absorption of heavy metals 1398 from sewage sludge biochar

Concentrations of 1401 organo-phosphorus pesticides in fresh vegetables and related human health risk management: lessons learned and guidelines

Speciation and bioavailability of heavy metals in pyrolytic 1409 biochar of swine and goat manures

Transfer of antibiotic resistance from manure-amended soils to vegetable 1413 microbiomes

Short-term biochar 1417 manipulation of microbial nitrogen transformation in wheat rhizosphere of a 1418 metal contaminated Inceptisol from North China plain

Changes in microbial biomass 1422 and the metabolic quotient with biochar addition to agricultural soils: A

Chinese swine farms

Economics of 1434 sustainable development and the bioeconomy

wheat residue, swine manure and sewage sludge respectively before and after CK 78.15 ±4

Data of the mean ± standard deviation. CK, control; UWS and WSB, USM and SMB, and USS and SSB, wheat residue, swine manure and 1544 sewage sludge, respectively before and after pyrolysis. Different uppercase letters following a single pair of amendment in a single column 1545 represents a significant difference of the bio-waste between before and after pyrolysis while different lowercase letters in a single column 1546 represent a significant difference among all the treatments, respectively at p<0.05(One-way ANOVA followed by Turkey test). 1547 1548