CN115786122A - Phosphorus-dissolving cadmium-reducing microbial agent and preparation method and application thereof - Google Patents
Phosphorus-dissolving cadmium-reducing microbial agent and preparation method and application thereof Download PDFInfo
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Abstract
本发明公开了一种解磷‑降镉微生物菌剂及其制备方法和应用。制备方法包括如下步骤:S1:微生物发酵液的制备:配置发酵培养液;S2:微生物的扩大培养:将解磷‑降镉微生物菌剂添加到发酵培养液中;S3:微生物菌剂的制备:待上述发酵液发酵培养18h后,将投入生物质材料进行混合,混合后,前8个小时,每隔2小时对菌剂进行搅拌,使得混合物内各组分状态均一,8小时后,待其自然稳定到24h,完成解磷‑降镉微生物菌剂的制作;本专利菌剂制作方式简单,利用吸附方式将微生物固定化,所选材料对环境的扰动性小,在降低土壤有效态镉的同时也降低了植物对镉的吸收,而且提高土壤中有效磷的含量。
The invention discloses a phosphorus-decomposing-cadmium-reducing microbial bacterial agent as well as a preparation method and application thereof. The preparation method comprises the following steps: S1: preparation of microbial fermentation broth: configuring fermentation broth; S2: expanded cultivation of microorganisms: adding phosphorus-reducing cadmium microbial bacterial agent to fermentation broth; S3: preparation of microbial bacterial agent: After the above-mentioned fermentation broth is fermented and cultivated for 18 hours, put in the biomass materials and mix them. After mixing, for the first 8 hours, stir the bacterial agent every 2 hours to make the state of each component in the mixture uniform. After 8 hours, wait for the It is naturally stable for 24 hours, and the production of phosphorus-reducing cadmium microbial bacterial agent is completed; the preparation method of this patented microbial agent is simple, and the microorganisms are immobilized by adsorption. At the same time, it also reduces the absorption of cadmium by plants and increases the content of available phosphorus in the soil.
Description
技术领域technical field
本发明涉及农用微生物技术与土壤修复技术领域,尤其涉及一种解磷-降镉微生物菌剂及其制备方法和应用。The invention relates to the field of agricultural microorganism technology and soil restoration technology, in particular to a phosphorus-dissolving-cadmium-reducing microbial bacterial agent and its preparation method and application.
背景技术Background technique
镉是一种银白色的重金属,是一种人体非必需元素。镉的毒性较大,半衰期长。粮食与我们的生活密切相关,粮食作物的生产离不开土壤。土壤镉污染来源广泛,最终会通过实物链在人体内富集,给人造成各种危害。当前针对土壤镉污染修复主要有化学处理法、物理处理法与生物处理法。化学处理法能够在短期内见效,但对环境的扰动性较大;物理处理法所需要的成本投入大,生物处理利用一些生物质材料或者微生物对污染场地进行修复,虽然在时间上可能花费较长,但生物处理是一种环境友好的处理方式,越来越受到人们的关注。Cadmium is a silvery-white heavy metal and a non-essential element for the human body. Cadmium is highly toxic and has a long half-life. Food is closely related to our life, and the production of food crops cannot be separated from the soil. Soil cadmium pollution comes from a wide range of sources, and will eventually accumulate in the human body through the physical chain, causing various harms to people. At present, there are mainly chemical treatment methods, physical treatment methods and biological treatment methods for the remediation of soil cadmium pollution. Chemical treatment can be effective in a short period of time, but it is more disturbing to the environment; physical treatment requires a large cost investment, and biological treatment uses some biomass materials or microorganisms to restore the contaminated site, although it may take a long time in terms of time. However, biological treatment is an environmentally friendly treatment method, which has attracted more and more attention.
现有技术中,大多只用单个菌群进行处理,每个微生物都有一定的适宜生长环境,使用单个菌群在某些土壤环境不起作用,不具有普遍的适用性。In the prior art, most of them only use a single flora for treatment, and each microorganism has a certain suitable growth environment. Using a single flora does not work in some soil environments and does not have universal applicability.
发明内容Contents of the invention
本发明的目的在于,针对现有技术的上述不足,提出一种解磷-降镉微生物菌剂及其制备方法和应用。The object of the present invention is to propose a phosphorus-dissolving-cadmium-reducing microbial bacterial agent and its preparation method and application in view of the above-mentioned deficiencies of the prior art.
本发明的一种解磷-降镉微生物菌剂的制备方法,包括如下步骤:A kind of preparation method of phosphorus solubilizing-cadmium reducing microbial bacterial agent of the present invention, comprises the steps:
S1:微生物发酵液的制备:配置发酵培养液;S1: preparation of microbial fermentation liquid: configuring fermentation culture liquid;
S2:微生物的扩大培养:将解磷-降镉微生物菌剂添加到发酵培养液中;S2: Expanded cultivation of microorganisms: adding phosphorus-dissolving-cadmium-reducing microbial bacterial agents to the fermentation broth;
S3:微生物菌剂的制备:待上述发酵液发酵培养18h后,将投入生物质材料进行混合,混合后,前8个小时,每隔2小时对菌剂进行搅拌,使得混合物内各组分状态均一,8小时后,待其自然稳定到24h,完成解磷-降镉微生物菌剂的制作;S3: Preparation of microbial inoculum: After the above-mentioned fermentation broth is fermented and cultivated for 18 hours, the biomass material will be put in for mixing. After mixing, for the first 8 hours, the inoculum is stirred every 2 hours to make the state of each component in the mixture Uniform, after 8 hours, wait for it to be naturally stable for 24 hours, and complete the production of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent;
解磷-降镉微生物菌剂的富集过程如下:制备菌悬液,好氧菌群土壤菌悬液制备方式如下:称取新鲜土壤置于装有无菌水中振荡,静置后取上清液制得好氧菌群土壤菌悬液;取上述好氧菌群土壤菌悬液加入到培养基中,进行微生物菌剂的富集,获取得到富集产物,而后再传代转接两次获得第三代的富集产物解磷-降镉微生物菌剂。The enrichment process of phosphorus-solubilizing-cadmium-reducing microbial bacterial agent is as follows: prepare the bacterial suspension, and the preparation method of the soil bacterial suspension of aerobic flora is as follows: weigh the fresh soil and place it in sterile water for shaking, then take the supernatant after standing The soil bacteria suspension of aerobic flora was prepared from the above liquid; the above-mentioned soil suspension of aerobic flora was added to the medium to enrich the microbial agent, and the enriched product was obtained, and then subcultured twice to obtain The enrichment product of the third generation is a phosphorus-decomposing-cadmium-reducing microbial bacterial agent.
进一步的,所述培养基为:葡萄糖10g/L,(NH4)2SO4 0.5g/L,NaCl 0.3g/L,MgSO40.3g/L,MnSO4 0.03g/L,FeSO4 0.03g/L,K2SO4 0.3g/L,Ca3(PO4)2 5g/L,pH 7.0-7.5。Further, the medium is: glucose 10g/L, (NH 4 ) 2 SO 4 0.5g/L, NaCl 0.3g/L, MgSO 4 0.3g/L, MnSO 4 0.03g/L, FeSO 4 0.03g /L, K 2 SO 4 0.3g/L, Ca 3 (PO 4 ) 2 5g/L, pH 7.0-7.5.
进一步的,所述配置发酵培养液为牛肉膏:3g、蛋白胨:10g、氯化钠:5g、pH:7.4-7.6。Further, the fermented culture medium is configured as beef extract: 3g, peptone: 10g, sodium chloride: 5g, pH: 7.4-7.6.
进一步的,发酵培养液中的总接种量为2%。Further, the total inoculation amount in the fermentation broth is 2%.
进一步的,生物质材料包括稻壳粉,生物质材料与混合的发酵培养液的固液比为1g:2-10mL。Further, the biomass material includes rice husk powder, and the solid-to-liquid ratio of the biomass material to the mixed fermentation broth is 1g:2-10mL.
一种上述制备方法制备的解磷-降镉微生物菌剂。A phosphorus-solubilizing-cadmium-reducing microbial bacterial agent prepared by the above-mentioned preparation method.
上述的解磷-降镉微生物菌剂的应用,用于降低土壤中有效态镉含量,提高土壤中有效磷的含量。The application of the above-mentioned phosphorus-dissolving-cadmium-reducing microbial bacterial agent is used to reduce the content of available cadmium in the soil and increase the content of available phosphorus in the soil.
本发明的解磷-降镉微生物菌剂能够降低土壤中有效态镉含量,从而达到减少作物吸收的作用,提高土壤中有效磷的含量,进一步改变土壤中的其它理化因子。具体为解磷-降镉微生物菌剂能够将土壤中难以被植物吸收利用的磷转化为易于被植物吸收的有效磷,有效磷主要是以磷酸根的形式存在,镉能够与重金属反应生成沉淀。xMm++yPO4 3-→My(PO4)x。The phosphorus-solubilizing-cadmium-reducing microbial bacterial agent of the present invention can reduce the content of available cadmium in the soil, thereby achieving the effect of reducing crop absorption, increasing the content of available phosphorus in the soil, and further changing other physical and chemical factors in the soil. Specifically, the phosphorus-solubilizing-cadmium-reducing microbial bacterial agent can convert phosphorus in the soil that is difficult to be absorbed by plants into available phosphorus that is easily absorbed by plants. Available phosphorus mainly exists in the form of phosphate, and cadmium can react with heavy metals to form precipitation. xM m+ +yPO 4 3- →My(PO 4 )x.
与现有技术相比,本菌剂具有如下有益效果:本专利菌剂制作方式简单,利用吸附方式将微生物固定化,省去了其它微生物固定化方式复杂的制作过程;本专利借助价廉易得的生物质材料作为载体,利用微生物修复土壤镉污染,与其它处理方式相比,投入成本小;本专利较其它化学处理方式相比,所选材料对环境的扰动性小,在降低土壤有效态镉的同时也降低了植物对镉的吸收,而且提高土壤中有效磷的含量,具有推广应用价值。Compared with the prior art, this bacterial agent has the following beneficial effects: the preparation method of the patented bacterial agent is simple, and the microorganisms are immobilized by adsorption, which saves the complicated production process of other microorganism immobilization methods; this patent uses cheap and easy The obtained biomass material is used as a carrier to repair soil cadmium pollution by using microorganisms. Compared with other treatment methods, the input cost is small; compared with other chemical treatment methods, the selected materials are less disturbing to the environment and are effective in reducing soil pollution. While reducing cadmium, it also reduces the absorption of cadmium by plants, and increases the content of available phosphorus in the soil, which has the value of popularization and application.
附图说明Description of drawings
图1制备的固氮微生物菌剂的照片;The photo of the nitrogen-fixing microbial bacterial agent prepared in Fig. 1;
图2为土壤体系有效态镉变化图(图中a、b、c、d标准代表是否出现显著性差异);Figure 2 is a diagram of the change of available cadmium in the soil system (the standards a, b, c, and d in the figure represent whether there is a significant difference);
图3为土壤镉形态分析图(Cd(F1)-酸可提取态镉Cd(F2)-可还原态镉Cd(F3)-可氧化态镉Cd(F4)-残渣态镉,CK为空白组,DP为菌剂组);Figure 3 is the soil cadmium form analysis diagram (Cd(F1)-acid extractable cadmium Cd(F2)-reducible cadmium Cd(F3)-oxidizable cadmium Cd(F4)-residual cadmium, CK is the blank group , DP is bacterial agent group);
图4为体系中部分理化展示图;Figure 4 is a partial physical and chemical display diagram in the system;
图5为植株体内镉含量与植株生长状况监测图;Fig. 5 is the monitoring diagram of cadmium content and plant growth status in the plant body;
图6为植株体内镉含量测定结果图(大田试验,P<0.01,**代表具有显著性差异)。Fig. 6 is a graph showing the measurement results of cadmium content in plants (field test, P<0.01, ** means significant difference).
具体实施方式Detailed ways
以下是本发明的具体实施例并结合附图,对本发明的技术方案作进一步的描述,但本发明并不限于这些实施例。The following are specific embodiments of the present invention and in conjunction with the accompanying drawings, the technical solutions of the present invention are further described, but the present invention is not limited to these embodiments.
制备解磷-降镉微生物菌剂Preparation of Phosphorus Solubilizing-Cadmium-Reducing Microbial Inoculant
首先制备菌悬液,好氧菌群土壤菌悬液制备方式如下:称取10g新鲜土壤置于装有90mL无菌水的三角锥形瓶中,150r/min振荡2后,静置后取上清液制得土壤菌悬液(好氧);取上述好氧土壤悬液1mL加入到解磷培养基中(葡萄糖10g/L,(NH4)2SO4 0.5g/L,NaCl0.3g/L,MgSO4 0.3g/L,MnSO4 0.03g/L,FeSO4 0.03g/L,K2SO4 0.3g/L,Ca3(PO4)2 5g/L,pH7.0-7.5),进行解磷功能菌群的富集,获取得到解磷富集产物,而后再传代转接两次获得第三代的富集产物(P)。然后将其置于20%甘油中保存,于-80℃冰箱中贮存,用于后续研究。First prepare the bacterial suspension. The preparation method of the aerobic flora soil bacterial suspension is as follows: Weigh 10g of fresh soil and place it in a triangular conical flask filled with 90mL sterile water, vibrate at 150r/min for 2 minutes, then take Soil bacteria suspension (aerobic) was obtained from the clear liquid; 1 mL of the above-mentioned aerobic soil suspension was added to the phosphorus-dissolving medium (glucose 10 g/L, (NH 4 ) 2 SO 4 0.5 g/L, NaCl 0.3 g/L L, MgSO 4 0.3g/L, MnSO 4 0.03g/L, FeSO 4 0.03g/L, K 2 SO 4 0.3g/L, Ca 3 (PO 4 ) 2 5g/L, pH7.0-7.5), Enrichment of the phosphorus-dissolving functional flora was carried out to obtain a phosphorus-dissolving enrichment product, and then subcultured and transferred twice to obtain the enriched product (P) of the third generation. It was then stored in 20% glycerol and stored in a -80°C refrigerator for subsequent studies.
配置牛肉膏蛋白胨培养基:分别称取牛肉膏3g、蛋白胨10g、氯化钠5g,用氢氧化钠溶液调节至7.4-7.6。Prepare beef extract-peptone medium: weigh 3g of beef extract, 10g of peptone, and 5g of sodium chloride, and adjust to 7.4-7.6 with sodium hydroxide solution.
将活化后的菌株进行隔夜扩大培养,待培养基体系中OD600达到1.0-1.2,停止培养。在冷冻离机中(设置离心条件:8000r/min,5min)进行离心,弃上清液,然后用灭菌的去离子水在涡旋仪上进行重悬,收集菌体。The activated strain was expanded overnight, and the culture was stopped when the OD 600 in the medium system reached 1.0-1.2. Centrifuge in a refrigerated centrifuge (setting centrifugation conditions: 8000r/min, 5min), discard the supernatant, and then resuspend on a vortex apparatus with sterilized deionized water to collect the bacteria.
将上述菌体与灭菌的稻壳粉进行混合,在摇床上进行振荡(设置条件180r/min,24h),使稻壳粉对微生物进行吸附固定,完成微生物菌剂的制备。参见附图1,为本实施技术方案制备的解磷-降镉微生物菌剂。Mix the above bacteria with sterilized rice husk powder, shake on a shaker (setting condition: 180r/min, 24h), make the rice husk powder adsorb and fix the microorganisms, and complete the preparation of microbial inoculum. Referring to accompanying
解磷-降镉微生物菌剂修复盆栽试验Potted Plant Restoration Test of Phosphorus Solubilizing and Cadmium Reducing Microbial Agents
开展室内盆栽试验(花盆上口直径23cm,高度13cm,底径13cm),每盆填充老化的镉污染土壤2kg。按照1%的添加量添加解磷微生物菌剂。监测体系中镉的形态、有效磷与其它理化因子。Indoor pot experiments were carried out (the top diameter of the flower pot was 23cm, the height was 13cm, and the bottom diameter was 13cm), and each pot was filled with 2kg of aged cadmium-contaminated soil. Phosphorus solubilizing microbial inoculum was added according to the addition amount of 1%. Monitor the form of cadmium, available phosphorus and other physical and chemical factors in the system.
①土壤体系中有效态镉监测①Monitoring of available cadmium in soil system
对体系中的有效态镉进行监测,镉的有效性越高,代表镉的活性越强,越易被植物吸收利用。实验结果见图2。实验结果表明,解磷菌剂的添加使土壤中有效态镉含量出现显著性下降,下降趋势在25d后趋于稳定。Monitor the available cadmium in the system. The higher the availability of cadmium, the stronger the activity of cadmium and the easier it is absorbed and utilized by plants. The experimental results are shown in Figure 2. The experimental results showed that the addition of phosphorus solubilizing bacteria significantly decreased the available cadmium content in the soil, and the decreasing trend tended to be stable after 25 days.
②体系中镉形态监测②Cadmium speciation monitoring in the system
对土壤中体系的镉形态进行进一步监测,用来解释镉生物有效性下降的原因,从左到右,从上到下,依次代表酸可提取态镉、可还原态镉、可氧化态镉、残渣态镉。镉的生物有效性依次降低,活性越来越弱。实验结果表明,体系中酸可提取态、可还原态镉含量出现显著性降低,与之相反的是体系中可氧化态镉、残渣态镉含量出现显著性升高。这解释了体系中土壤有效态镉含量下降,镉生物有效性降低是由于体系中镉的形态从不稳定的状态向体系中稳定状态转化。实验结果见图3。Further monitoring of cadmium species in the soil system is used to explain the reasons for the decline in cadmium bioavailability. Residual cadmium. The bioavailability of cadmium decreases successively, and the activity becomes weaker and weaker. The experimental results show that the content of acid-extractable and reducible cadmium in the system is significantly reduced. On the contrary, the content of oxidizable cadmium and residue cadmium in the system is significantly increased. This explains the decrease of soil available cadmium content in the system, and the decrease of cadmium bioavailability is due to the transformation of cadmium form in the system from an unstable state to a stable state in the system. The experimental results are shown in Figure 3.
③体系中部分理化监测③ Some physical and chemical monitoring in the system
图4数据结果表明,添加微生物菌剂后,土壤中的pH值与空白处理组相比有显著性上升;有机质含量虽未出现显著性差异,但在均值上有所上升;体系中的亚铁含量有明显上升,亚铁离子的含量可以反应体系中的氧化还原环境,当亚铁离子含量越高时,体系越趋向于还原环境。以往的研究表明,当土壤愈发趋向于碱性环境,土壤中镉的活性会有所降低,有机质含量的升高,会与重金属镉进行结合,可氧化态镉的含量会升高,环境愈发趋向还原环境,体系中CdS等沉淀状态镉的含量会越高。此外,值得注意的是,加入解磷菌剂后,土壤体系中有效磷的含量在前期有明显上升,出现显著性差异,这表明微生物解磷菌剂提高了土壤中的有效磷含量,一定程度上解释了体系中镉含量的降低是由于镉与磷酸根反应生成了磷酸镉沉淀,也解释了后面植株生长状况的改善与土壤养分的提高有关。The data results in Fig. 4 show that after adding the microbial agent, the pH value in the soil has significantly increased compared with the blank treatment group; although the organic matter content has no significant difference, it has increased on the mean value; the ferrous iron in the system The content of ferrous ions has increased significantly, and the content of ferrous ions can reflect the redox environment in the system. When the content of ferrous ions is higher, the system tends to be more reducing environment. Previous studies have shown that when the soil tends to be more alkaline, the activity of cadmium in the soil will decrease, and the content of organic matter will increase, which will combine with heavy metal cadmium, and the content of oxidizable cadmium will increase. The hair tends to reduce the environment, and the content of cadmium in the precipitated state such as CdS in the system will be higher. In addition, it is worth noting that after adding the phosphate-dissolving bacteria agent, the content of available phosphorus in the soil system increased significantly in the early stage, and there was a significant difference, which indicated that the microbial phosphate-dissolving bacteria agent increased the available phosphorus content in the soil to a certain extent. The above explains that the decrease of cadmium content in the system is due to the reaction of cadmium and phosphate to form cadmium phosphate precipitation, and also explains that the improvement of plant growth is related to the improvement of soil nutrients.
④镉形态与土壤理化的相关性分析④ Correlation analysis between cadmium form and soil physicochemistry
表1镉形态与土壤理化的斯皮尔曼相关性分析Table 1 Spearman correlation analysis between cadmium speciation and soil physicochemistry
**p<0.01,*p<0.05. ** p<0.01, * p<0.05.
(正值代表正相关,负值代表负相关,*代表是否具有显著性差异)(Positive value represents positive correlation, negative value represents negative correlation, * represents whether there is a significant difference)
实验数据表明,酸可提取态镉、可还原态镉与可氧化态镉、残渣态镉呈现显著负相关;镉的生物有效性与环境呈现显著负相关,镉的生物有效性与有机质含量呈现显著负相关,镉的生物有效性与T、H2O等指标变化不明显。Experimental data show that acid extractable cadmium, reducible cadmium, oxidizable cadmium, and residue cadmium are significantly negatively correlated; the bioavailability of cadmium is significantly negatively correlated with the environment, and the bioavailability of cadmium is significantly negatively correlated with the content of organic matter. Negative correlation, the bioavailability of cadmium did not change significantly with T, H 2 O and other indicators.
⑤植株体内镉含量与植株生长状况监测⑤Monitoring of cadmium content in plants and plant growth status
采用湿式消解法对植株体内镉含量进行监测,采用直尺测量植株高度,采用天平测定植株鲜重。实验结果见图5,实验结果表明,解磷菌剂处理组较空白处理组植株体内镉含量出现显著性下降,下降幅度达到36.24%,值得注意的是,在植株体内镉含量下降的同时,植株的高度,鲜重等出现显著性升高,这说明,解磷菌剂的添加缓解了重金属镉对植株的毒害作用。The cadmium content in plants was monitored by wet digestion method, the plant height was measured by ruler, and the fresh weight of plants was measured by balance. The experimental results are shown in Figure 5. The experimental results showed that the cadmium content in the plants of the phosphate-dissolving bacteria treatment group decreased significantly compared with the blank treatment group, and the decline rate reached 36.24%. It is worth noting that when the cadmium content in the plants decreased, the plants The height, fresh weight, etc. were significantly increased, which indicated that the addition of phosphate solubilizing bacteria alleviated the toxic effect of heavy metal cadmium on plants.
⑥野外大田试验应用探究⑥Discussion on the application of field experiments
将上述制备的微生物菌剂投加到野外镉污染土壤中,选取生菜作为研究对象。测定植株体内镉含量。测定结果见图6。数据表明,解磷微生物菌剂的添加能够出现与实验室基本一致的结果,能够显著降低植株体内的镉含量,降低程度达到32.16%。表明微生物菌剂也具有一定的应用开发前景。The microbial agent prepared above was added to cadmium-contaminated soil in the field, and lettuce was selected as the research object. Determination of cadmium content in plants. The measurement results are shown in Figure 6. The data showed that the addition of phosphorus-solubilizing microbial agents can produce results that are basically consistent with those in the laboratory, and can significantly reduce the cadmium content in plants, with a reduction rate of 32.16%. It shows that the microbial agent also has a certain application development prospect.
以上未涉及之处,适用于现有技术。What is not involved above is applicable to the prior art.
虽然已经通过示例对本发明的一些特定实施例进行了详细说明,但是本领域的技术人员应该理解,以上示例仅是为了进行说明,而不是为了限制本发明的范围,本发明所属技术领域的技术人员可以对所描述的具体实施例来做出各种各样的修改或补充或采用类似的方式替代,但并不会偏离本发明的方向或者超越所附权利要求书所定义的范围。本领域的技术人员应该理解,凡是依据本发明的技术实质对以上实施方式所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围。Although some specific embodiments of the present invention have been described in detail by examples, those skilled in the art should understand that the above examples are only for illustration, rather than for limiting the scope of the present invention. Various modifications or additions or similar substitutions can be made to the described specific embodiments without departing from the direction of the present invention or exceeding the scope defined by the appended claims. Those skilled in the art should understand that any modifications, equivalent replacements, improvements, etc. made to the above implementations based on the technical essence of the present invention shall be included in the protection scope of the present invention.
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