Effect of β-Lactam-Based Bacterial Residue Fertilizer on Lettuce Rhizosphere Soil Bacteria and Antibiotic Resistance Genes
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摘要: β-内酰胺类菌渣的资源化处理已成为生物制药企业急需解决的问题. 为探究β-内酰胺类菌渣资源化产物是否满足《有机肥料》(NY/T 525—2021)标准，施加后是否会对作物根际土壤微生物产生显著影响以及是否会导致ARGs(抗生素抗性基因)的富集等问题，该研究选用经水热干燥喷雾(HT+SD)处理后的头孢菌渣和板框压滤(MD)处理后的青霉素菌渣为研究对象，以空白组、化肥处理组为对照，各施肥处理均采用高施用量(1%)和低施用量(0.5%)两种肥料投加量进行盆栽试验. 通过16S rRNA及HT-qPCR方法研究了菌渣肥对生菜根际土壤细菌多样性、物种组成、ARGs以及MGEs(可移动遗传元件)的影响. 结果表明：在经过无害化处理后的菌渣中，残留抗生素远低于HPLC-MS检出限(<6.78 μg/kg)，含水率降低95%，重金属、有机质等指标均能满足《有机肥料》(NY/T 525—2021)要求；相较于施肥量，施肥类型对土壤细菌群落结构的影响更明显，其中，两种菌渣处理组Shannon-Wiener指数显著高于化肥处理组(P<0.05)；两种菌渣的施用均没有改变土壤细菌组成，各处理组中最丰富的菌门皆为变形菌门(相对丰度为34.8%~48.1%)；高施用量青霉素菌渣组和低施用量头孢菌渣组的ARGs数目和总相对丰度均显著高于对照组(P<0.05)，而低施用量的青霉素菌渣和高施用量的头孢菌素菌渣处理组中ARGs数目和相对丰度与空白对照组均无显著性差异(P>0.05)，但两种菌渣的施用都没有改变土壤细菌的抗性机制；ARGs的变化与外源性DNA的引入以及土壤特征菌相关，其中假单胞菌属与mexE、ttgA和ttgB三种ARGs均表现出极强的相关性(相关系数分别为0.93、0.90、0.84). 研究显示，经HT+SD和MD技术处理后的抗生素菌渣可以资源化为安全有效的农业替代肥.Abstract: Innovative treatment of β-lactam residues has become a pressing concern for biopharmaceutical firms. This study determined whether resource-based products of β-lactam residues meet the standards of Organic Fertilizer (NY/T 525-2021) and whether the application significantly affects crop inter-root soil microorganisms and leads to the enrichment of antibiotic resistance genes (ARGs). Cephalosporin residues treated by hydrothermal spray-drying (HT+SD) and penicillin residues treated by multi-plate dryer (MD) were selected for the study. A blank group and fertilizer treatment group served as controls. High fertilizer application rate (1%) and low application rate (0.5%) were used to carry out potting experiments. Using 16S rRNA and HT-qPCR, the effects of mycorrhizal fertilization on lettuce inter-root soil bacterial diversity, species composition, ARGs, and mobile genetic elements (MGEs) were investigated. The results indicated that the residual antibiotics in the harmlessly treatment residue were well below the detection limit of HPLC-MS (<6.78 μg/kg), the water content was reduced by 95%, and the indexes of heavy metals and organic matter met the requirements of Organic Fertilizer (NY/T 525-2021). Compared with the amount of fertilizer applied, the effect of fertilizer application type on soil bacterial community structure was more significant, and the Shannon-Wiener indices of both residue treatment groups were significantly higher than those of the chemical fertilizer treatment group (P<0.05). Neither residue affected the bacterial composition of the soil, with Proteobacteria being the most abundant phylum (34.8%-48.1%) in all treatment groups. The number and total relative abundance of ARGs were significantly higher in the HP. The low application rate cephalosporin residue groups were compared to the control group (P<0.05). In contrast, the number and relative abundance of ARGs in the low application rate penicillin residue and HP treatment groups were not significantly different from the blank control group (P>0.05). However, none of the residue applications altered the soil bacterial resistance mechanisms. The changes in ARGs were associated with introducing exogenous DNA and soil characteristic bacteria, among which Pseudomonas spp. showed extremely strong correlations with three ARGs of mexE, ttgA and ttgB (correlation coefficients of 0.93, 0.90 and 0.84, respectively). The study shows that antibiotic residues treated with HT+SD and MD technologies can be repurposed as safe and effective alternative agricultural fertilizers.
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图 1 微生物α多样性分析与β多样性分析
Figure 1. Microbial alpha diversity analysis and beta diversity analysis
图 2 不同施肥处理对土壤中细菌物种组成的影响
Figure 2. Effect of different fertilization treatments on bacterial species composition in soil
图 3 不同施肥处理对土壤抗生素抗性基因和可移动原件的影响
Figure 3. Effects of different fertilization treatments on soil antibiotic resistance genes and mobile genetic elements
图 4 土壤特征菌与抗生素抗性基因相关性热图
Figure 4. Heat map of the correlation between soil characteristic bacteria and antibiotic resistance genes
表 1 青霉素和头孢菌渣盆栽试验施肥处理
Table 1. Fertilization treatments for penicillin and cephalosporin residue pot experiments
序号 肥料 命名 肥料质量/g 处理1 未施肥 CK 0 处理2 化肥(复合肥)，施用比例为0.5% IL 2 处理3 化肥(复合肥)，施用比例为1% IH 4 处理4 青霉素菌渣，施用比例为0.5% PL 10 处理5 青霉素菌渣，施用比例为1% PH 20 处理6 头孢菌渣，施用比例为0.5% CL 10 处理7 头孢菌渣，施用比例为1% CH 20 
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表 2 处理前后抗生素菌渣理化性质
Table 2. Physicochemical properties of antibiotic residues before and after treatment
项目 pH 含水率/% 抗生素
残留量/(mg/kg)重金属含量/（mg/kg） 有机质
含量/%总养分/% 粪大肠菌
群数/(个/kg)蛔虫卵
死亡率/%As Cd Cr Hg Pb 青霉素鲜菌渣 6.27 78.85 21.5 0.53 ND 1.28 0.01 0.28 — — — 100 青霉素无害化菌渣 5.77 3.55 ND 0.2 <0.01 <1 0.006 0.2 93.1 15.06 <3 100 头孢菌素鲜菌渣 4.1 90.68 152.3 0.19 0.03 ND ND 0.23 — — — 100 头孢菌素无害化菌渣 4.96 2.76 ND 1.3 2.8 <0.01 0.02 0.3 89 10.57 <3 100 NY/T 525—2021 5.5~8.5 ≤30 未规定 ≤15 ≤3 ≤150 ≤2 ≤50 ≥30 ≥4 ≤100 ≥95 注：—表示未检测；ND表示未检出.
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