Fermentation And Purification Technology Of Algicidal Components Of Algicidal Bacteria L7

From three aspects which are bacteria cell culture, induced production and separation of algicidal components, explore fermentation technology and purification of algicidal components of an indigenous algicidal bacteria L7. The investigation was aim at achieving technical and process parameters during the development process of biological algicide. The author carried out the research following three aspects, and the conclusions as below.1. Cell culture technology of algicidal bacteria L7In this stage, select the appropriate cell culture medium of bacteria L7 (carbon, nitrogen, C/N ratio, pH value), bacterial load, DO, agitation speed.In five kinds of carbon sources and four kinds of nitrogen sources tested, glucose was the optimum carbon source and ammonium chloride was the optimum nitrogen sources.Compared with the starch medium, L7 cell growth could increase 34.86% and 37.74%, respectively. The optimum medium pH = 7.5, C/N =3:1 (6g: 2g), and bacterial load was 3.1×107 cfu ? mL-1. The suitable cell culture condition was 30% DO (±10%) and stirring rate of 160 rpm (±10%). In the medium, inoculum and culture conditions, bacterial cell growth could reach to 5.30×108 cfu ? mL-1, which was about 19 times the initial concentration of bacteria.2. Induced production of algicidal components of algicidal bacteria L7In this stage, preliminary study addition concentrations of various factors (Ca2 +, Fe3 +, amino acids and intact algal solution of Anabaena flos-aquae), which may induce algicidal bacteria L7 produce extracellular algicidal components.(1) In starch medium containing CaCl2 ? 2H2O, concentration of Ca2 + of which is 0.01 g/L, the removal rate of chlorophyll a ( Chla ) of sterile filtrate could rise by 11.66%.(2) Cysteine and methionine could significantly promote the growth of algicidal bacteria, but they had strong killing ability to algae. Glutamic acid inhibited the growth of algicidal bacteria, and it could kill algae. Tyrosine had a role in promoting growth of L7, but whether it could promote the secretion of extracellular algicidal components need further study.(3) With the addition concentration of intact algal solution of Anabaena flos-aquae increased (5%, 10%, 20%), the algicidal effect of sterile filtrate of L7 had increasing trend. Compared to the control group, addition 20% of the intact algal solution could increase algicidal effect by 49.98%.3. Purification technology of algicidal components of algicidal bacteria L7Separate and identify algicidal components of L7 by alcohol precipitation, dialysis, TLC, silica gel column chromatography, gel chromatography, HPLC, MS and et al. The study was aim at exploring low-cost and efficient means of separation and process route. Apply cylinder plate method for the detection of algicidal effect of small amount of sample.(1) In the study stage of crude purification, extraction, ethanol precipitation were all available means of selected crude separation means. The specific alcohol precipitation conditions: temperature 30℃, pH=9, the dosage of alcohol: sterile filtrate volume=6:1. The influence of different factors is the temperature> pH> the amount of alcohol.(2) In the study stage of fine purification, before separating algicidal components by Sephadex LH-20 column chromatography, dialysis (with the dialysis bag whose intercepted molecular weight is 3500Da) which was used to extraction of samples could significantly improve the purity of the sample, then collect and concentrate the sample outside the bag. Different elution fractions had UV absorption peak at 220 nm, 270 nm independently or both of the two wavelengths. Finally we received two algicidal components, the molecular weights were 565.2 and 365.0 respectively, and the algicidal effect of the latter one was better.(3) We could use thin layer chromatography ( TLC ) as a mean of fine purification. When the expand agent was ethyl acetate: acetic acid: water = 3:5:1 or n-butanol: acetic acid: water = 5:3:1, the isolation effect was good, and the separation points were obvious. Because n-butanol was more viscous, more separation time was needed. The third point whose Rf value was third had significant algicidal effect.