Mechanisms of heavy metal immobilization and resistance genes reduction following phosphate-modified biochar application during anaerobic digestion of swine manure

Abstract

This study investigated the effects of biochar (BC), H₃PO₄-modified biochar (BP) and KH₂PO₄-modified biochar (BK) on heavy metals (HMs) passivation and the distribution of antibiotic resistance genes (ARGs) and metal resistance genes (MRGs) during anaerobic digestion (AD). Characterization results revealed that the application of BK significantly decreased the mobility of Cr, Cu, and Pb, attributing to metal phosphate precipitation formation. EEM-PARAFAC and 2D-FTIR-COS analysis showed that the C–O stretching, polysaccharide-like structures, N-contain substance or carboxylic reacted faster after adding biochar. Biochar addition promoted the synthesis of fluorescent and humic-like components, and accelerated humification process, thus showing a greater complexation ability for HMs. In BK treatments, most detected ARGs abundance were reduced with efficiencies of 85%–96%, and MRGs abundance, such as phrT, yieF, chrR, etc., were significantly declined by more than 90%. Biochar addition directly reduced MRGs abundance by improving the immobilization degree of HMs. Biochar addition inhibited horizontal gene transfer (HGT) facilitated by mobile genetic elements (MGEs) and reduced co-selective pressure by HMs, thereby decreasing ARGs abundance. Biochar stimulated growth of HMs tolerant bacteria, such as Bacillus, Romboutsia, Clostridiales, etc., which functioned well in HMs immobilization. P-loaded biochar as additive is recommended in AD to mitigate ARGs distribution and reduce HMs risks.

Graphical abstract

Keywords

P-modified biochar / Heavy metals passivation / Resistance genes / Humification / Anaerobic digestion

Highlight

	● P-modified biochar passivated HMs via phosphate precipitation & surface complexation.
	● Biochar promoted the synthesis of HCs and accelerated humification process.
	● Biochar addition directly reduced MRGs by improving immobilization degree of HMs.
	● Biochar inhibited MGEs mediated co-selection of HGT and HMs, reducing ARGs spread.
	● P-loaded biochar offers a dual strategy to mitigate HM and ARGs risks in AD systems.

References

[1] Bao H Y, Chen Z Q, Wen Q X, Wu Y Q, Fu Q Q. (2022). Effect of calcium peroxide dosage on organic matter degradation, humification during sewage sludge composting and application as amendment for Cu(II)-polluted soils. Journal of Hazardous Materials, 439: 129592

[2] Bao H Y, Chen Z Q, Wen Q X, Wu Y Q, Fu Q Q. (2024). Effects of oxytetracycline on variation in intracellular and extracellular antibiotic resistance genes during swine manure composting. Bioresource Technology, 393: 130127

[3] Brito M J P, Veloso C M, Santos L S, Bonomo R C F, Fontan R D C I. (2018). Adsorption of the textile dye Dianix® royal blue CC onto carbons obtained from yellow mombin fruit stones and activated with KOH and H₃PO₄: kinetics, adsorption equilibrium and thermodynamic studies. Powder Technology, 339: 334–343

[4] Chen Z Q, Bao H Y, Wen Q X, Wu Y Q, Fu Q Q. (2022). Effects of H₃PO₄ modified biochar on heavy metal mobility and resistance genes removal during swine manure composting. Bioresource Technology, 346: 126632

[5] Cui H Y, Zhang S B, Zhao M Y, Zhao Y, Wei Z M. (2020). Parallel faction analysis combined with two-dimensional correlation spectroscopy reveal the characteristics of mercury-composting-derived dissolved organic matter interactions. Journal of Hazardous Materials, 384: 121395

[6] Duman O, Tunç S. (2009). Electrokinetic and rheological properties of Na-bentonite in some electrolyte solutions. Microporous And Mesoporous Materials, 117(1−2): 331–338

[7] Fan X J, Liu C, Yu X F, Wang Y, Song J Z, Xiao X, Meng F D, Cai Y B, Ji W C, Xie Y. . (2021). Insight into binding characteristics of copper(II) with water-soluble organic matter emitted from biomass burning at various pH values using EEM–PARAFAC and two-dimensional correlation spectroscopy analysis. Chemosphere, 278: 130439

[8] Fan Y H, Wang H, Deng L Y, Wang Y, Kang D, Li C Z, Chen H. (2020). Enhanced adsorption of Pb(II) by nitrogen and phosphorus co-doped biochar derived from Camellia oleifera shells. Environmental Research, 191: 110030

[9] Gao J, Wang L C. (2018). Ecological and human health risk assessments in the context of soil heavy metal pollution in a typical industrial area of Shanghai, China. Environmental Science & Pollution Research, 25(27): 27090–27105

[10] Gao R L, Hu H Q, Fu Q L, Li Z H, Xing Z Q, Ali U, Zhu J, Liu Y H. (2020). Remediation of Pb, Cd, and Cu contaminated soil by co-pyrolysis biochar derived from rape straw and orthophosphate: speciation transformation, risk evaluation and mechanism inquiry. Science of the Total Environment, 730: 139119

[11] Guo T, Zhang X Y, Liu T Y, Wu Z Z, Wang D Z. (2020). N, K Co-activated biochar-derived molybdenum carbide as efficient electrocatalysts for hydrogen evolution. Applied Surface Science, 509: 144879

[12] Huang R Q, Gao S F, Wang W L, Staunton S, Wang G. (2006). Soil arsenic availability and the transfer of soil arsenic to crops in suburban areas in Fujian Province, Southeast China. Science of the Total Environment, 368(2−3): 531–541

[13] Huang S B, Wang Y X, Ma T, Tong L, Wang Y Y, Liu C R, Zhao L. (2015). Linking groundwater dissolved organic matter to sedimentary organic matter from a fluvio-lacustrine aquifer at Jianghan Plain, China by EEM-PARAFAC and hydrochemical analyses. Science of the Total Environment, 529: 131–139

[14] Huang Y, Tian Y Y, Xie L H, Liu Y D, Dai B L, Guo X J, Yang Y J. (2020). The application of two-dimensional correlation spectroscopy for the binding properties of heavy metals onto digestate-derived DOM from anaerobic digestion of chicken manure. Ecotoxicology and Environmental Safety, 204: 111129

[15] Kong Y L, Ma R N, Li G X, Wang G Y, Liu Y, Yuan J. (2022). Impact of biochar, calcium magnesium phosphate fertilizer and spent mushroom substrate on humification and heavy metal passivation during composting. Science of the Total Environment, 824: 153755

[16] Lan J R, Zhang S S, Dong Y Q, Li J H, Li S Y, Feng L, Hou H B. (2021). Stabilization and passivation of multiple heavy metals in soil facilitating by pinecone-based biochar: mechanisms and microbial community evolution. Journal of Hazardous Materials, 420: 126588

[17] Laurent C, Bravin M N, Crouzet O, Pelosi C, Tillard E, Lecomte P, Lamy I. (2020). Increased soil pH and dissolved organic matter after a decade of organic fertilizer application mitigates copper and zinc availability despite contamination. Science of the Total Environment, 709: 135927

[18] Li K C, Fu M X, Ma L C, Yang H X, Li Q L. (2023). Zero-valent iron drives the passivation of Zn and Cu during composting: fate of heavy metal resistant bacteria and genes. Chemical Engineering Journal, 452: 139136

[19] Li L C, Xu Y L, Zhong D J, Zhong N B. (2020). CTAB-surface-functionalized magnetic MOF@MOF composite adsorbent for Cr(VI) efficient removal from aqueous solution. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 586: 124255

[20] Lian F, Yu W C, Zhou Q X, Gu S G, Wang Z Y, Xing B S. (2020). Size matters: nano-biochar triggers decomposition and transformation inhibition of antibiotic resistance genes in aqueous environments. Environmental Science & Technology, 54(14): 8821–8829

[21] Memon A G, Xing Y P, Zhou X H, Wang R Y, Liu L H, Zeng S Y, He M, Ma M. (2020). Ultrasensitive colorimetric aptasensor for Hg²⁺ detection using Exo-III assisted target recycling amplification and unmodified AuNPs as indicators. Journal of Hazardous Materials, 384: 120948

[22] NodaIOzaki Y (2005). Two-Dimensional Correlation Spectroscopy: Applications in Vibrational and Optical Spectroscopy. Chichester: John Wiley & Sons, Ltd.

[23] Qiao H T, Qiao Y S, Sun C Z, Ma X H, Shang J, Li X Y, Li F M, Zheng H. (2023). Biochars derived from carp residues: characteristics and copper immobilization performance in water environments. Frontiers of Environmental Science & Engineering, 17(6): 72

[24] Sanchez-Monedero M A, Cayuela M L, Roig A, Jindo K, Mondini C, Bolan N. (2018). Role of biochar as an additive in organic waste composting. Bioresource Technology, 247: 1155–1164

[25] Tang J, Zhuang L, Yu Z, Liu X M, Wang Y Q, Wen P, Zhou S G. (2019). Insight into complexation of Cu (II) to hyperthermophilic compost-derived humic acids by EEM-PARAFAC combined with heterospectral two dimensional correlation analyses. Science of the Total Environment, 656: 29–38

[26] Tang R, Yuan S J, Wang Y L, Wang W, Wu G X, Zhan X M, Hu Z H. (2020). Arsenic volatilization in roxarsone-loaded digester: insight into the main factors and arsM genes. Science of the Total Environment, 711: 135123

[27] Tessier A, Campbell P G C, Bisson M. (1979). Sequential extraction procedure for the speciation of particulate trace metals. Analytical Chemistry, 51(7): 844–851

[28] Wang C, Jia Y X, Li J P, Li P, Wang Y, Yan F F, Wu M H, Fang W Z, Xu F, Qiu Z P. (2023). Influence of microbial augmentation on contaminated manure composting: metal immobilization, matter transformation, and bacterial response. Journal of Hazardous Materials, 441: 129762

[29] Wang L, Yuan L, Li Z H, Zhang X, Sheng G P. (2021). Quantifying the occurrence and transformation potential of extracellular polymeric substances (EPS)-associated antibiotic resistance genes in activated sludge. Journal of Hazardous Materials, 408: 124428

[30] Wang M M, Wu Y C, Zhao J Y, Liu Y, Gao L, Jiang Z K, Zhang J B, Tian W. (2022). Comparison of composting factors, heavy metal immobilization, and microbial activity after biochar or lime application in straw-manure composting. Bioresource Technology, 363: 127872

[31] Wang Y, Geng M K, Jia H, Cui J C, Zhang M, Zhao Y X, Wang J. (2024). Removal of antibiotic resistant bacteria and antibiotic resistance genes: a bibliometric review. Frontiers of Environmental Science & Engineering, 18(12): 146

[32] Wu W D, Li J H, Niazi N K, Müller K, Chu Y C, Zhang L L, Yuan G D, Lu K P, Song Z L, Wang H L. (2016). Influence of pyrolysis temperature on lead immobilization by chemically modified coconut fiber-derived biochars in aqueous environments. Environmental Science and Pollution Research, 23(22): 22890–22896

[33] Xing J, Xu G R, Li G B. (2020). Analysis of the complexation behaviors of Cu(II) with DOM from sludge-based biochars and agricultural soil: effect of pyrolysis temperature. Chemosphere, 250: 126184

[34] Xu J W, Liu C, Hsu P C, Zhao J, Wu T, Tang J, Liu K, Cui Y. (2019). Remediation of heavy metal contaminated soil by asymmetrical alternating current electrochemistry. Nature Communications, 10(1): 2440

[35] Yang S, Chen Z Q, Wen Q X. (2021a). Impacts of biochar on anaerobic digestion of swine manure: methanogenesis and antibiotic resistance genes dissemination. Bioresource Technology, 324: 124679

[36] Yang S, Wen Q X, Chen Z Q. (2021b). Effect of KH₂PO₄-modified biochar on immobilization of Cr, Cu, Pb, Zn and as during anaerobic digestion of swine manure. Bioresource Technology, 339: 125570

[37] Yang S, Wen Q X, Chen Z Q. (2022). Biochar induced inhibitory effects on intracellular and extracellular antibiotic resistance genes in anaerobic digestion of swine manure. Environmental Research, 212: 113530

[38] Zarei-Baygi A, Wang P, Harb M, Stadler L B, Smith A L. (2020). Membrane fouling inversely impacts intracellular and extracellular antibiotic resistance gene abundances in the effluent of an anaerobic membrane bioreactor. Environmental Science & Technology, 54(19): 12742–12751

[39] Zhang H, Shao J G, Zhang S H, Zhang X, Chen H P. (2020). Effect of phosphorus-modified biochars on immobilization of Cu (II), Cd (II), and As (V) in paddy soil. Journal of Hazardous Materials, 390: 121349

[40] Zhang J J, Shao J A, Jin Q Z, Li Z Q, Zhang X, Chen Y Q, Zhang S H, Chen H P. (2019). Sludge-based biochar activation to enhance Pb(II) adsorption. Fuel, 252: 101–108

[41] Zhang M, Wang Y C. (2020). Effects of Fe-Mn-modified biochar addition on anaerobic digestion of sewage sludge: biomethane production, heavy metal speciation and performance stability. Bioresource Technology, 313: 123695

[42] Zhao M, Dai Y, Zhang M Y, Feng C, Qin B J, Zhang W H, Zhao N, Li Y Y, Ni Z B, Xu Z H. . (2020). Mechanisms of Pb and/or Zn adsorption by different biochars: biochar characteristics, stability, and binding energies. Science of the Total Environment, 717: 136894

[43] Zhao N, Zhao C F, Lv Y Z, Zhang W F, Du Y G, Hao Z P, Zhang J. (2017). Adsorption and coadsorption mechanisms of Cr(VI) and organic contaminants on H₃PO₄ treated biochar. Chemosphere, 186: 422–429

[44] Zhong J W, Yin W Z, Li Y T, Li P, Wu J H, Jiang G B, Gu J J, Liang H. (2017). Column study of enhanced Cr(VI) removal and longevity by coupled abiotic and biotic processes using Fe⁰ and mixed anaerobic culture. Water Research, 122: 536–544

[45] Zhu L, Qi H Y, Lv M L, Kong Y, Yu Y W, Xu X Y. (2012). Component analysis of extracellular polymeric substances (EPS) during aerobic sludge granulation using FTIR and 3D-EEM technologies. Bioresource Technology, 124: 455–459

[46] Zhu Y C, Jin Y, Liu X S, Miao T L, Guan Q K, Yang R, Qu J J. (2021). Insight into interactions of heavy metals with livestock manure compost-derived dissolved organic matter using EEM-PARAFAC and 2D-FTIR-COS analyses. Journal of Hazardous Materials, 420: 126532

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