發(fā)布時間：2018-05-09 10:32

  本文選題：紅樹林 + 磺胺間甲氧嘧啶��； 參考：《廣東海洋大學(xué)》2015年碩士論文

【摘要】：磺胺間甲氧嘧啶(sulfamonomethoxine,SMMX)屬于廣譜、高效和高選擇性的磺胺類抗生素,廣泛應(yīng)用于醫(yī)藥、畜禽與水產(chǎn)養(yǎng)殖業(yè)中的細(xì)菌、真菌或原生生物引起的感染疾病治療。市政廢水排放入海和海水養(yǎng)殖用藥,造成抗生素類藥物在包括紅樹林在內(nèi)的濱海濕地藥物殘留,可能削弱微生物的分解作用從而降低生態(tài)系統(tǒng)的功能。本文采集了湛江觀海長廊人工紅樹林區(qū)的沉積物樣品,以篩選SMMX為單一碳源的抗生素降解細(xì)菌為目標(biāo),采用馴化方法篩選到了6株具有降解潛力的初篩菌株,運用經(jīng)典和現(xiàn)代分子生物學(xué)方法對降解菌株進(jìn)行了系統(tǒng)分類鑒定,同時采用超高效液相色譜儀(UPLC)檢測方法結(jié)合正交試驗設(shè)計在實驗室內(nèi)研究了降解菌株(Aeromonas caviae,HA-J1)對SMMX的生物降解性能,以獲得最優(yōu)降解條件,為后續(xù)構(gòu)建生物修復(fù)工程菌株奠定基礎(chǔ)。具體研究結(jié)果如下:1、應(yīng)用搖瓶富集馴化的方法,通過熒光顯微計數(shù)法測定馴化菌株在培養(yǎng)前后的數(shù)量變化,從湛江觀海長廊人工紅樹林區(qū)的沉積物樣品篩選分離到6株以獸藥SMMX抗生素為單一碳源的初篩菌株,分別編號為HA-J1、HA-J4、HA-J5、HA-J7、HA-J8和HA-J9。在相同培養(yǎng)條件下,隨獸藥濃度的增大6株菌的數(shù)量先增加后減少,在800mg/L時,生長最適宜。通過生長曲線測定,菌株在15~24h處于生長旺盛的階段,可以作為測定生物降解量末端的合適取樣時間。2、根據(jù)細(xì)菌形態(tài)學(xué)觀察、生理生化試驗和細(xì)菌16s rDNA擴(kuò)增對6株SMMX降解菌進(jìn)行了系統(tǒng)分類鑒定,結(jié)果表明,HA-J1、HA-J4、HA-J5、HA-J8為氣單胞菌屬(Aeromonas),HA-J7、HA-J9為克雷伯氏菌屬(Klebsiella),再分別通過看家基因gyr B/gyr A基因進(jìn)一步鑒定,結(jié)果表明HA-J1、HA-J4、HA-J5、HA-J8為豚鼠氣單胞菌(Aeromonas caviae),HA-J7、HA-J9為肺炎克雷伯氏菌(Klebsiella pneumoniae)。3、通過在無機(jī)鹽培養(yǎng)液中添加色譜純SMMX室內(nèi)搖瓶培養(yǎng)研究6株降解菌進(jìn)行濃度遞增復(fù)篩,采用UPLC測定降解菌培養(yǎng)前后的SMMX的降解率,結(jié)合熒光顯微鏡測定培養(yǎng)前后SMMX降解菌的數(shù)量變化,獲得復(fù)篩結(jié)果,6株菌HA-J1、HA-J4、HA-J5、HA-J7、HA-J8與HA-J9的有效生物降解率依次為24.13%、6.8%、1.4%、4%、13%、3.75%,由此將HA-J1菌株確定為SMMX高效生物降解菌株。通過Pearson相關(guān)性分析6株初篩菌對磺胺間甲氧嘧啶降解率與降解前后細(xì)菌的增量,發(fā)現(xiàn)相關(guān)性不顯著(p0.05)。4、通過正交試驗設(shè)計測定了HA-J1菌株在不同pH值、溫度、鹽度、SMMX濃度的四因素五水平的25組實驗組合條件下對SMMX降解率及降解菌數(shù)量變化,獲得HA-J1菌株最優(yōu)降解條件組合為溫度25℃、pH5、鹽度為15、抗生素濃度為200mg/L,最優(yōu)組合條件下的生物降解率達(dá)14.47%。多因素方差分析結(jié)果顯示各因素間無顯著性差異(p0.05)。通過Pearson相關(guān)性分析,發(fā)現(xiàn)各組合下的降解率與降解前后的細(xì)菌增量有顯著的正相關(guān)(r=0.735,p0.05)。在最優(yōu)正交組合下測定細(xì)菌最佳接種量為16%,降解率為19.29%。5、為探討降解菌株對其他磺胺類藥物的降解性能,研究了6株初篩菌株在相同培養(yǎng)條件下分別在以100mg/L的磺胺間甲氧嘧啶(SMMX)和磺胺甲VA唑(SMX)為單一碳源的無機(jī)鹽培養(yǎng)液中的生物降解效率,HA-J1、HA-J4、HA-J5、HA-J7、HA-J8與HA-J9對SMMX的有效生物降解率依次為14.11%、1.55%、1.47%、4.87%、8.49%、3.33%,對SMX的生物降解率分別為0.64%、0.32%、0.47%、0.45%、0.14%。配對T檢驗結(jié)果表明,降解菌對SMX的降解率顯著低于其對SMMX的降解率(p0.05)。6、研究了添加100mg/L的葡萄糖、甲醇、蔗糖等不同碳源后高效降解菌株HA-J1對相同質(zhì)量體積濃度SMMX抗生素(即也為100mg/L)的降解率,結(jié)果表明,添加葡萄糖、甲醇、蔗糖這三種碳源后的降解效率依次分別為23.02%、18.36%、14.61%,無外加碳源的對照組降解率為13.45%。添加外加碳源不同程度的提高了高效降解菌株對SMMX的降解率,但兩者之間無顯著差異(ANOVA,p0.05)。本文的研究結(jié)果表明從紅樹林區(qū)沉積物中可以篩選得到高效降解磺胺類藥物磺胺間甲氧嘧啶的單一菌株,并鑒定為豚鼠氣單胞菌(Aeromonas caviae),但其降解功能基因及降解途徑尚不清楚,還有待于進(jìn)一步研究。
[Abstract]:Sulfamonomethoxine (SMMX) is a broad-spectrum, highly efficient and highly selective sulfonamide, widely used in medicine, in livestock and in the aquaculture industry, for the treatment of infectious diseases caused by bacteria, fungi, or protists. Municipal wastewater is discharged into the sea and aquaculture, causing antibiotic drugs in mangroves, including mangroves. The residues of drugs in the coastal wetlands may weaken the decomposition of microbes and reduce the function of the ecosystem. In this paper, the sediment samples from the artificial mangrove area of Zhanjiang Guan Hai corridor were collected in order to screen SMMX as a single carbon source of antibiotic degrading bacteria, and the biodegradation potential of 6 strains of screening were screened by acclimatization. The strain was systematically classified and identified by classical and modern molecular biology methods. At the same time, the biodegradability of the degraded strain (Aeromonas caviae, HA-J1) to SMMX was studied in the laboratory by the ultra high performance liquid chromatograph (UPLC) detection method combined with the orthogonal design in order to obtain the optimal degradation conditions for the subsequent construction. The basis of the bioremediation engineering strain was laid. 1, 1, using the method of shaking bottle enrichment and acclimation, the quantity changes of the domesticated strains before and after the culture were measured by the fluorescence microscopy, and the screening and separation of the sediment samples from the artificial mangrove area of the Zhanjiang Guan Hai corridor were screened and separated to the initial screening of the single carbon source with the veterinary drug SMMX antibiotics. The strains were numbered as HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9. in the same culture conditions. The number of 6 strains increased first and then decreased with the increase of the concentration of veterinary drugs. In 800mg/L, the growth was the most suitable. By measuring the growth curve, the strain was at the stage of vigorous growth, which could be used as a suitable sampling time for the determination of the end of biodegradation. .2, according to bacterial morphological observation, physiological and biochemical tests and bacterial 16S rDNA amplification, 6 strains of SMMX degrading bacteria were systematically classified and identified. The results showed that HA-J1, HA-J4, HA-J5, HA-J8 were Aeromonas (Aeromonas), HA-J7, HA-J9 were Klebsiella (Klebsiella), and further identified by the family gene. The results showed that HA-J1, HA-J4, HA-J5, HA-J8 were Aeromonas of guinea pig (Aeromonas caviae), HA-J7 and HA-J9 were Klebsiella pneumoniae (Klebsiella pneumoniae).3. The concentration of 6 strains of degraded bacteria were studied by adding a chromatographic pure SMMX chamber shake flask culture in the inorganic salt culture solution. The number of SMMX degradation bacteria before and after culture was measured by fluorescence microscopy, and the results of rescreening were obtained. The effective biodegradation rates of 6 strains of bacteria HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9 were 24.13%, 6.8%, 1.4%, 4%, 13%, 3.75%. Thus, HA-J1 strain was identified as a SMMX high efficiency biodegrading strain. The 6 strains of screening bacteria were analyzed by Pearson correlation. The correlation between the degradation rate of sulfadimethoxine and the increment of bacteria before and after degradation was not significant (P0.05).4. Through the orthogonal design, the SMMX reduction rate and the number of degrading bacteria were measured under the four factors and five levels of the pH value, temperature, salinity and SMMX concentration, and the optimal drop of the HA-J1 strain was obtained. The combination of the solution conditions was 25 C, pH5, the salinity 15, the antibiotic concentration of 200mg/L, and the biodegradation rate of 14.47%. under the optimal combination conditions was up to 14.47%.. The results showed that there was no significant difference between the factors (P0.05). The degradation rate of each combination and the increment of bacteria before and after degradation were found to be significant positive phase through Pearson correlation analysis. (r=0.735, P0.05). Under the optimal orthogonal combination, the optimum inoculation amount of bacteria was 16% and the degradation rate was 19.29%.5. In order to investigate the degradation performance of the degrading strains to other sulfonamides, the 6 initial screening strains were used as the single carbon source of sulfadiazine (SMMX) and sulfamethoxazole (SMX) under the same culture conditions. The biodegradability of HA-J1, HA-J4, HA-J5, HA-J7, HA-J8 and HA-J9 to SMMX in the inorganic salt medium was 14.11%, 1.55%, 1.47%, 4.87%, 8.49%, 3.33%. The biodegradation rate of SMX, respectively, 0.64%, 0.32%, 0.47%, 0.45%, and 0.14%., respectively, showed that the degradation rate of the degrading bacteria to SMX was significantly lower than that of SMMX. Degradation rate (P0.05).6, the degradation rate of High Efficient Degradation Strain HA-J1 for the same mass volume concentration SMMX antibiotic (also 100mg/L) was studied after different carbon sources, such as glucose, methanol and sucrose were added to 100mg/L, and the results showed that the degradation efficiency of the three carbon sources after adding glucose, methanol and sucrose, respectively, was 23.02%, 18.36%, 14.61%, respectively. The degradation rate of the carbon source in the control group was 13.45%. adding additional carbon source to improve the degradation rate of SMMX, but there was no significant difference between the two groups (ANOVA, P0.05). The results of this study showed that a single strain of highly efficient sulfadiazine sulfadiazine can be screened from the mangrove area sediments. It is identified as Aeromonas caviae, but its degradation function genes and degradation pathways are not yet clear.

【學(xué)位授予單位】：廣東海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：X172

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