The Effect Of Bacillus Subtilis On The Growth And Population Dynamics Of Brachionus Plicatilis

Bacillus subtilis is one kind of gram-positive bacteria,colored purple with gram staining,which are commonly used as probiotics in aquaculture.It has the functions of regulating water quality,improving the growth of aquatic animals,the structure of intestinal flora and immunity,etc.It is one of 16 feed-grade probiotics approved by the ministry of agriculture of China.Brachionus plicatilis belong to the rotifers of the class rotifers,has the phenomenon of generation alternation,and is an indispensable biological bait for large-scale cultivation in the coastal crab breeding industry.This strain is one of the most common Marine rotifers in sheyang coastal area of jiangsu province.It mainly feeds on Chlorella pyrenoidesa.It has a wide distribution,strong tolerance and high yield.The main research contents and results of this thesis are as follows:1.Determination of Bacillus subtilis growth curve and its effect on Chlorella pyrenoidesa growthBacillus subtilis was shaken and cultured at a shaking table with a rotating speed of 220 r/min and a temperature control of 37±1℃.Samples were taken at time points of 0,1,3,5,7,10,12,15,20,24 and 28h.Solid medium was inoculated at a temperature of 37±1℃and colony count was performed after 24h of culture.The results showed that the growth of Bacillus subtilis was exponential,with four growth stages:slow period（0-4h）,logarithmic period（4-10h）,stable period（10-24h）and decline period（24h）.The initial density of bacterial solution was 3×10⁵cfu/m L,and reached the maximum density of 1.14×10¹⁰ cfu/m L in 15h.The light intensity was 4000lx,the day length ratio（L:D）was 16:8,and the temperature was 25±1℃.Control group,live bacteria group and broken cell group were set,and the initial density of chlorella was 1.0×10⁵cells/m L.In the viable group,live bacteria were added to chlorella culture medium,and in the crushing group,cell breaking medium was added to chlorella culture medium.On the second day of the experiment,the algal density of the living bacteria group was higher than that of the other two groups（P<0.05）,and the algal density was lower than that of the other two groups（P<0.05）.During the experiment period,the density of algal cell fragmentation group was always higher than that of control group.In general,the inhibitory effect of Bacillus subtilis on chlorella growth was limited,and the inhibitory effect was derived from reproduction to produce extracellular secretions and hypoxia.The cell fragments of bacteria have algal-promoting effect but no inhibition effect.Combined with the actual production situation,this chapter confirms that chlorella can maintain exponential growth under the influence of Bacillus subtilis,which can be used as a micro-ecological additive of chlorella,a rotiger-based feed.2.Effects of different temperature and growth period of Bacillus subtilis on individual reproduction and life span of Brachionus plicatilisThe experimental temperatures were set at 20℃,25℃and 30℃,the light intensity was set at 4000lx,and the day length ratio L:D=16:8.The mother fluids of Bacillus subtilis with activation time of 1h,5h,10h and 24h were added to the rotifer culture medium,respectively.Five experimental groups were set up,including the retarding phase,logarithmic phase,stable phase,decline phase and control group,with 6 parallel groups in each group.Individual culture was carried out,and rotifer life,progenerative period and offspring individual number were recorded statistically.The results showed that high temperature could shorten the progenerative period and increase the number of progeny.Low temperature can significantly prolong rotifer life.The effect of Bacillus subtilis on individual rotifers increased with the increase of temperature.At the same temperature,compared with the control group,the logarithmic Bacillus subtilis could shorten the progenerative period of rotifers,increase the number of offspring and prolong the life of rotifers.The maximum shortening time of progenerative period reached 6.5h（P<0.05）,the average increase of the number of offspring of rotifers was 2（P<0.05）,and the maximum extension of life was 21h（P<0.05）.During the stable period,the rotifers reproduction and life expectancy decreased relative to the logarithmic period,and the difference between the rotifers and the control group gradually decreased.In the decline period,the indexes were significantly lower than those in the control group,which had a negative effect on the life span of rotifers,early reproductive period and the number of offspring.The experimental results showed that the optimal activation and addition time of Bacillus subtilis was controlled at about 5h at all temperatures.3.Effects of Bacillus subtilis combining Chlorella pyrenoidesa on population of Brachionus plicatilis under different temperatureThe experimental temperatures were set at 20℃,25℃and 30℃,the light was4000lx,the day length ratio（L:D）was 16:8,rotifers culture medium was prepared with logarithmic Bacillus subtilis,and chlorella density was 3×10⁶ cell/m L.According to the preliminary experiment,the bacterial concentration was 0（control group）,50mg/L,100mg/L,200mg/L,300mg/L and 500mg/L experimental solutions were prepared,and the rotifers were cultured in a colony with 6 parallel in each group.The total population,population growth rate and population density were calculated.The results showed that the maximum density of rotifers in the 20℃,25℃and 30℃treatment groups reached 26.53±1.57,39.50±4.80 and 42.23±5.69 ind.At all temperatures,the population growth rate of the concentration group was significantly higher than that of the control group in the first 7 days（P<0.05）.The conclusion of this chapter is that the maximum density of rotifers increases significantly with the increase of temperature,but the growth potential of rotifers decreases with the increase of temperature.At the same temperature,with the increase of bacterial concentration,the growth of rotifers was first promoted and then inhibited.The optimal concentration of added bacteria in chlorella was 100mg/L.4.Effects of Bacillus subtilis combining yeast on population of Brachionus plicatilis under different temperatureThe yeast concentration of rotifer culture medium was 6×10⁶ cell/m L.The logarithmic Bacillus subtilis was used.According to the preliminary experiment,the experimental concentration was 0（control group）,100mg/L,200mg/L,300mg/L,500mg/L and 800mg/L.The total population,population growth rate and population density were calculated.The experimental results showed that the maximum density of rotifera in the 20℃,25℃and 30℃groups could reach 13.4±1.24,28.1±2.44 and19.5±1.01 ind./m L,which were 24%,152%and 123%higher than those in the control group（P<0.05）.The conclusion of this chapter is that under the condition of yeast-based bait,the higher the temperature is,the faster the reproduction will be,and the maximum population growth rate will be 0.63 at 30℃.The maximum capacity can be obtained by adding a concentration of 500 mg/L at 25℃.