【作者】 王效昌；

【导师】 高丽；

【作者基本信息】 烟台大学 ， 海洋科学， 2022， 硕士

【摘要】 近年来,我国黄海等水域绿潮暴发的频率逐年增大,大型藻华的暴发与衰亡对水环境造成了严重影响。磷是大多数水体富营养化和藻华暴发的重要诱因,而解磷菌是影响沉积物内源磷释放的一重要因素,在水体磷的生物地球化学循环中发挥着关键作用。本文以硬毛藻频繁暴发的荣成天鹅湖为研究对象,通过室内筛选和模拟试验,分析了藻类暴发区和非暴发区沉积物中土著微生物的解磷能力,对表层沉积物中的无机解磷菌进行了分离鉴定后,在单独和混合接种条件下比较了不同菌株的解磷效果,探讨无机解磷菌对天鹅湖沉积物磷释放的影响及解磷作用机制。主要研究结果如下:（1）天鹅湖各湖区沉积物中微生物均表现出对磷酸钙的溶解能力,其中西部、北部和湖中心土著微生物的解磷能力较强。培养过程中,秋季和春季沉积物微生物培养液的p H分别降至4.78～4.87和4.35～5.31,其中西部湖区2个季节均为最低;而培养液可溶性磷含量分别升至217.44～256.09 mg L^-1和201.01～333.02 mg L^-1,其中秋季北部湖区含量最高,春季则中心湖区较高。（2）平板筛选过程中,无机解磷菌菌落呈现不同的形态和不同大小的溶磷圈。本研究共从天鹅湖沉积物中分离到10株无机解磷菌,并通过16S r DNA序列分析对其种属进行了鉴定。分离出的10株无机解磷菌分别属于芽胞杆菌科（Bacillaceae）,芽胞杆菌属（Bacillus sp.）和地芽胞杆菌属（Geobacillus sp.）2个属,弯曲芽胞杆菌（Bacillus flexus）、巨大芽胞杆菌（Bacillus megaterium）、地衣芽胞杆菌（Bacillus licheniformis）和嗜热噬脂肪地芽胞杆菌（Geobacillus stearothermophilu）4个种。芽胞杆菌属是天鹅湖沉积物中无机解磷菌的主要菌属。（3）利用液体培养法,对筛选出的无机解磷菌菌株（IPB1-IPB10）的解磷效果进行评估。结果发现,所有的无机解磷菌菌株均能促进磷酸钙的溶解,但不同菌株间解磷能力差异很大。培养期间,各解磷菌处理培养液的p H降至4.10～5.06,而可溶性磷含量明显升高,最大值变幅为99.75～209.55 mg L^-1。巨大芽孢杆菌具有较强的解磷能力,培养液中可溶性磷含量最高。分泌酸性物质是天鹅湖无机解磷菌的主要解磷机制,培养液的p H与可溶性磷含量呈显著负相关（P<0.01）。在菌株混合培养过程中,无机解磷菌的解磷能力得到了增强。（4）选取巨大芽胞杆菌、地衣芽胞杆菌和弯曲芽胞杆菌3个菌株分别接种到天鹅湖沉积物中,通过室内摇瓶试验研究无机解磷菌对不同湖区沉积物的解磷能力。结果表明,无机解磷菌接种后2个湖区沉积物中钙结合态磷（HCl-P）含量均明显降低,尤其是中心湖区,相较初始含量降低了17.84～25.90 mg kg^-1。培养过程中,北部和中心湖区沉积物3个接菌处理的水体p H分别降至4.17～4.55和4.56～4.61,可溶性磷含量最大值为0.18～0.37 mg L^-1和0.09～0.18 mg L^-1。巨大芽胞杆菌对天鹅湖2个湖区沉积物均表现出了较强的解磷能力。（5）以3株无机解磷菌和湖中心沉积物为试材进行室内模拟试验,发现试验前期各接菌处理水体p H均不同程度的下降,其中沉积物灭菌组和非灭菌组分别降至6.46～6.76和6.66～6.78。试验期间各处理水体氧化还原电位均不断下降,结束时沉积物-水界面呈还原状态。灭菌条件下解磷菌的接种明显促进了沉积物磷的释放,试验后期各加菌处理水体的总磷含量远高于对照(差值为0.06～0.30 mg L^-1);未灭菌条件下水体总磷上升较慢且总体水平较低,不同解磷菌处理间差异较小。试验结束时,大部分加菌处理沉积物中HCl-P含量明显下降,降幅为19.92～50.08 mg kg^-1,BD-P含量略有下降,而Na OH-P并未下降。研究表明,无机解磷菌的接种可明显降低上覆水p H,从而使得沉积物中HCl-P溶解释放;在天鹅湖,HCl-P为沉积物磷的主要赋存形态,其在解磷菌作用下对上覆水体磷水平的潜在贡献不容忽视。更多还原

【Abstract】 Green tides have occurred frequently in the Yellow Sea in recent decades.The outbreak and decline of macroalgae blooms have caused a serious impact on the water environment.Phosphorus（P）is an important inducement of eutrophication and algal blooms in most waters.Inorganic phosphate-solubilizing bacteria（IPB）is an important factor affecting P release from the sediments and play a key role in P biogeochemical cycle in waters.Chaetomorpha linum have outbreak frequently in recent years in Rongcheng Swan Lake.Through the laboratory screening and simulation,the phosphate-solubilizing ability of indigenous microorganisms in sediments was studied in algae outbreak area and non-outbreak area,and the IPB strains were screened and identified from the surface sediments in Swan Lake.In addition,the phosphate-solubilizing ability of different IPB strains was analyzed under separate and mixed inoculation conditions.The effects and mechanism of IPB strains on P release from the sediments were discussed.The main results are as follows:（1）In Swan Lake,the indigenous microorganisms in sediments were able to dissolve tricalcium phosphate,whose phosphate-solubilizing ability were stronger at the western,northern and central lake.At the treatments of autumn and spring sediments,the p H in liquid medium decreased to 4.78～4.87 and 4.35～5.31,while the maximum concentrations of soluble reactive phosphorus（SRP）ranged at 217.44～256.09 mg L^-1and 201.01～333.02mg L^-1,respectively.At different lake areas,the highest SRP in autumn and spring sediments were observed in the north and central lake area,respectively,while the lowest p H both appeared at the western lake.（2）During the plate screening process,the IPB isolates showed great differences in their morphological characteristics and the size of phosphate-solubilizing halos.In this research,ten IPB strains were isolated from the sediments of Swan Lake,and their taxonomy was determined by 16S r DNA sequence analysis.The ten IPB strains isolated belonged to one family（Bacillaceae）,two genera（Bacillus sp.and Geobacillus sp.）and four species（Bacillus flexus,Bacillus megaterium,Bacillus licheniformis and Geobacillus stearothermophilu）.Bacillus was the main genus of IPB in the sediments from Swan Lake.（3）The phosphate-solubilizing characteristics of obtained IPB strains（IPB1-IPB10）were evaluated using liquid medium.In the liquid medium culture,all IPB strains were able to dissolve tricalcium phosphate,but the phosphate-solubilizing ability varied greatly among different IPB strains.During the culture period,the p H of medium reduced to4.10～5.06.The concentrations of SRP in medium was obviously increased,and the maximum concentrations of SRP in every medium ranged at 99.75～209.55 mg L^-1.Bacillus megaterium strain showed the strongest phosphate-solubilizing ability,resulting in the highest SRP concentration in the liquid medium cultures.In this research,the secretion of acids was the main mechanism of phosphate solubilization by IPB of Rongcheng Swan Lake,and there was a significant negative correlation between SRP concentration and p H value（P<0.01）.The phosphate-solubilizing ability of the IPB was promoted when strains were grown in mixed-strain cultures.（4）Through inoculating Bacillus megaterium,Bacillus licheniformis,Bacillus flexus into the sediments,the ability of IPB strains to dissolve phosphate from sediments of different lake area was investigated by shaking flask culture.And the results showed that the calcium-bound phosphorus（HCl-P）concentration in the sediments decreased greatly under different IPB conditions.Compared with the initial value,HCl-P concentration decreased by 17.84～25.90 mg kg^-1in sediments from the central lake.At the water of three bacterial inoculated sediments from the north and central Lake areas,the p H decreased to4.17～4.55 and 4.56～4.61,and the maximum concentrations of SRP ranged at 0.18～0.37mg L^-1and 0.09～0.18 mg L^-1,respectively.Bacillus megaterium showed a strong phosphate-solubilizing ability in sediments of two lake areas.（5）The indoor simulation test was carried out with three IPB strains and the sediments of central lake area.During the early stage of the experiment,water p H value decreased at the treatments inoculated with IPB,and the p H of sterilized-sediment group and non-sterilized sediment group decreased to 6.46～6.76 and 6.66～6.78,respectively.Water Eh at different treatments decreased continuously with time during the experiment,and the interface of sediment and water was anaerobic at the end.For the sterilization group,the inoculation of IPB promoted the release of phosphorus from sediments significantly.During the later stage,water total phosphorus（TP）concentration at the inoculation treatments was much higher than that of the control,with the difference being0.06～0.30 mg L^-1.In the non-sterilization group,TP concentrations increased slowly with time,and there was small difference among different IPB treatments.At the end of the experiment,the concentrations of HCl-P in most sediments inoculated with IPB decreased obviously,with a decline of 19.92～50.08 mg kg^-1,and BD-P concentrations decreased by1.81～4.19 mg kg^-1.However,Na OH-P concentration did not decrease.The results indicate that the inoculation of IPB can reduce water p H greatly,which led to the dissolution and release of HCl-P from the sediments.In Rongcheng Swan Lake,HCl-P is the main form of sediment phosphorus,under the action of IPB,whose effect on the P level in overlying water can not be ignored.更多还原