Condition Optimization of an Efficient Phosphate-dissolving Bacterial Strain and Its Phosphate-dissolving Characteristics

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

Abstract

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

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.

Figures/Tables 15

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

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

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

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

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

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

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

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

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

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	**

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

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

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.

Fig. 10 Colonization of bacteria 1616X1 in soil

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

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