一株高效溶磷细菌的条件优化及其溶磷特性研究

doi:10.13560/j.cnki.biotech.bull.1985.2022-0281

摘要/Abstract

摘要：

探究一株自湖泊沉积物中分离到的高效溶磷菌株1616X1的溶磷条件和特性。通过菌落形态、生理生化特性及16S rRNA基因序列分析进行菌种分类鉴定，采用钼锑抗比色法测定溶磷量，利用单因素和响应面试验对溶磷条件进行优化，土壤溶磷试验观察土壤中速效磷含量变化以及菌株1616X1在土壤中的定殖效果。菌株1616X1为Pseudomonas sp.；在NBRIP液体培养基中溶磷量与pH呈显著负相关，最佳溶磷条件为葡萄糖10 g/L、硫酸铵0.17 g/L、pH 5.70、接种量5%、装液量75 mL/250 mL，培养温度23.3℃，该条件下溶磷量为684.91 mg/L；1616X1在实验所用菜地以及花圃土壤中均能有效定殖且能显著增加土壤速效磷含量。菌株1616X1具有较好的溶磷能力，有望为开发溶磷微生物肥料提供优质菌种资源，为该溶磷菌的合理应用提供理论依据。

关键词: 溶磷, 条件优化, 响应面, 土壤定殖, 速效磷

Abstract:

In order to study the phosphate-dissolving conditions and characteristic of a highly effective phosphate-dissolving strain 1616X1 isolated from lake sediment, the classification and identification of bacterial species were carried out by colony morphology, physiological and biochemical characteristics and 16S rRNA gene sequence analysis. The method of Mo-Sb colorimetry was applied to measure the amount of dissolved phosphorus. The single factor and response surface experiment were applied to find optimal phosphate-dissolving condition. Through soil culture experiments, the changes of available phosphorus and the colonization of strains were observed. The result of identification revealed that phosphate-dissolving strain 1616X1 can be identified as Pseudomonas sp. The phosphate-dissolving ability of strain 1616X1 showed a negative correlation with pH in NBRIP liquid medium. The optimal phosphate-dissolving conditions were as follows：glucose 10 g/L, ammonium sulfate 0.17 g/L, pH5.70，inocula concentration 5.0%，loaded liquid 75 mL/250 mL, and fermentation temperature 23.3℃，under which the maximum phosphate-dissolving capacity was up to 684.91 mg/L. Concurrently，1616X1 effectively colonized in the experimental vegetable field and flowerbed soils, and significantly increased soil available phosphorus content. Strain 1616X1 presents a good ability of dissolving phosphorus, aiming to provide high-quality bacterial resources for the development of phosphate-dissolving microbial fertilizers, and to provide a theoretical basis for the rational application of the phosphate-dissolving bacteria.

Key words: phosphate-dissolving, optimization of conditions, response surface, soil colonization, available phosphorus

李思思, 张博源, 符运会, 周佳, 屈建航. 一株高效溶磷细菌的条件优化及其溶磷特性研究[J]. 生物技术通报, 2022, 38(12): 274-286.

LI Si-si, ZHANG Bo-yuan, FU Yun-hui, ZHOU Jia, QU Jian-hang. Condition Optimization of an Efficient Phosphate-dissolving Bacterial Strain and Its Phosphate-dissolving Characteristics[J]. Biotechnology Bulletin, 2022, 38(12): 274-286.

图/表 15

表1 响应面试验的因素及水平

Table 1 Factors and levels of response surface experiment

水平 Level	因素Factor
水平 Level	A（硫酸铵Ammonium sulfate）/（g·L^-1）	B（温度Temperature）/℃	C（pH）
-1	0.05	20	5
0	0.10	24	6
1	0.15	28	7

图1 菌株1616X1基于16S rRNA基因序列的系统发育树 Bootstrap次数设置为1 000，邻接法构建系统发育树；分支位置中的数字表示Bootstrap值；括号中为相关细菌的GenBank登录号；标尺为进化距离

Fig. 1 Phylogenetic tree of strain 1616X1 based on 16S rRNA sequences The Bootstrap times were set to 1 000，and the phylogenetic tree was constructed by adjacency method. The numbers at the branch nodes are the Bootstrap values. In parentheses is the accession number of 16S rRNA gene sequence of the strain in Genbank. The data of the ruler is the evolutionary distance

图2 1616X1溶磷曲线及溶磷量与pH相关性分析

Fig. 2 1616X1 phosphate-dissolving curve and correlation analysis of solubilized phosphorus amount and pH

图3 碳源种类和浓度对1616X1溶磷能力的影响

Fig. 3 Effects of carbon source type and concentration on the phosphate-dissolving ability of 1616X1

图4 氮源种类和浓度对1616X1溶磷能力的影响

Fig. 4 Effects of nitrogen source type and concentration on the phosphate-dissolving ability of 1616X1

图5 温度对1616X1溶磷能力的影响

Fig. 5 Effect of temperature on the ability of 1616X1 disso-lving phosphate

图6 初始pH对1616X1溶磷能力的影响

Fig. 6 Effects of initial pH on the ability of 1616X1 dissolv-ing phosphate

图7 接种量对1616X1溶磷能力的影响

Fig. 7 Effect of inoculum quantity on the phosphate-disso-lving ability of 1616X1

图8 装液量对1616X1溶磷能力的影响

Fig. 8 Effect of liquid loading on the ability of 1616X1 diss-olving phosphate

表2 方差分析

Table 2 Analysis of variance

来源Source	自由度Freedom	平方和Sum of squares	均方Mean square	F值F value	P值P value	显著性Significance
碳源浓度Carbon source concentration	5	908119.44	181623.88	3307.15	0.000	**
氮源浓度Nitrogen source concentration	5	112287.61	22457.52	98.18	0.000	**
温度 Temperature	4	94456.32	23614.08	287.36	0.000	**
接种量 Inoculum quantity	4	6210.98	1552.74	4.74	0.0210	*
装液量 Loaded liquid	4	1123.59	280.89	1.78	0.2090	-
pH	5	23076.69	4615.33	86.42	0.000	**

表3 Box-Behnken试验设计与结果

Table 3 Box-Behnken experiment design and results

序号Number	A：硫酸铵Ammonium sulfate/（g·L^-1）	B：pH	C：温度Temperature/℃	溶磷量Amount of dissolved phosphorus/（mg·L^-1）
1	-1	-1	0	537.63
2	1	-1	0	652.85
3	-1	1	0	587.73
4	1	1	0	618.79
5	-1	0	-1	514.59
6	1	0	-1	645.84
7	-1	0	1	573.70
8	1	0	1	621.79
9	0	-1	-1	654.86
10	0	1	-1	660.87
11	0	-1	1	630.81
12	0	1	1	643.84
13	0	0	0	667.88
14	0	0	0	685.92
15	0	0	0	686.92
16	0	0	0	653.86
17	0	0	0	676.90

表4 二次模拟的方差分析

Table 4 Analysis of variance in quadratic model

方差来源Source	平方和Sum of squares	自由度Freedom	均方Mean square	F值F value	P值P value	显著性Significance
回归模型 Regression model	38483.42	9	4275.94	18.45	0.0004	**
A	13253.97	1	13253.97	57.18	0.0001	**
B	153.71	1	153.71	0.6631	0.4423
C	4.52	1	4.52	0.0195	0.8929
AB	1770.79	1	1770.79	7.64	0.0279	*
AC	1728.88	1	1728.88	7.46	0.0293	*
BC	12.30	1	12.30	0.0530	0.8244
A²	18804.31	1	18804.31	81.12	<0.0001	**
B²	284.21	1	284.21	1.23	0.3048
C²	1438.79	1	1438.79	6.21	0.0415	*
残差 Residuals	1622.68	7	231.81
失拟项 Lack of fit	862.56	3	287.52	1.51	0.3401
纯误差 Pure error	760.12	4	190.03
合计 Total	40106.09	16

图9 各因素间对菌株1616X1溶磷能力影响的响应曲面与等高线 a：硫酸铵浓度与pH的响应面与等高线；b：硫酸铵浓度与温度的响应面与等高线；c：pH与温度的响应面与等高线

Fig. 9 Contour and response surface about the effects of two factors on the ability of strain 1616X1 dissolving phosphate a：The response surface and contour of ammonium sulfate concentration and pH；b：the response surface and contour of ammonium sulfate concentration and temperature；c：the response surface and contour of pH and temperature.

图10 细菌1616X1在土壤中的定殖情况

Fig. 10 Colonization of bacteria 1616X1 in soil

图11 细菌1616X1对土壤速效磷含量以及pH的影响

Fig. 11 Effects of bacterium 1616X1 on soil available phos-phorus content and pH

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