Optimization Of Heterotrophic Growth Of Two Bait Microalgae And Its Application In Clam Culture

At present,the quantity and quality of bait microalgae directly affect the success or failure of shellfish production（its survival and growth quality）.With the continuous expansion of the scale of industrial culture,it is difficult for microalgae to achieve high-density culture under the traditional photoautotrophic mode,resulting in a shortage of supply.However,heterotrophic culture has gradually emerged in recent years due to its fast speed and high yield.This study understood the effects of various organic carbon sources on microalgae growth,and created a kind of biological feed rich in active small molecules for microalgae culture.It is not only fertilizer,but also shellfish can directly absorb the nutrients from the water through the gills or skin,promoting the growth of shellfish,and has the effect of improving the aquaculture water environment.First,two kinds of common food microalgae,Phaeodactylum tricornutum and Isochrysis galbana,were selected as experimental subjects.Organic acids（acetic acid,lactic acid,citric acid,malic acid,succinic acid,tartaric acid,formic acid,propionic acid,etc.）,glucose,alcohols（ethanol,methanol for example）,amino acids and small peptides were used as carbon sources for microalgae heterotrophic culture.Through microscopic counting method to understand the micro algae growth rule,the results showed that the carbon source for two kinds of micro algae growth has obvious promoting effect.Lactic acid,malic acid and citric acid had significant promoting effects on Phaeodactylum tricornutum,and the highest algal density was 2.12 times,1.65 times and 1.60 times of that in the control group.Through formulation design,the biomass of Phaeodactylum tricornutum was used as the main index to optimize the formulation of complex organic acids.The optimized combinations were lactic acid 0.1g/L,citric acid 0.05g/L and malic acid 0.05g/L,and the maximum algal density reached 1.07×10⁷cells/m L.Lactic acid,succinic acid and citric acid had significant promoting effects on Isochrysis galbana,and the highest algal density was2.06 times,1.68 times and 1.60 times of that in the control group.Through formulation design,the composition of complex organic acids was optimized with the biomass of Isochrysis galbana as the main index.The optimized combinations were:lactic acid 0.1g/L,succinic acid 0.1g/L and citric acid 0.05g/L,and the highest algal density reached 8.60×10⁶cells/m L.Secondly,the influence of mineral ions（Cu,Fe,Mn,Zn,Si,Ca）on the growth of microalgae was explored by single factor method.The changes in the biomass of microalgae were detected using a microscopic counting method to determine the optimal ion concentration in the culture solution.The experimental results showed that,The concentration of Cu²⁺was 7.68μg/L,Fe³⁺was 1mg/L,Mn²⁺was 24μg/L,Zn²⁺was 15.36μg/L,Ca²⁺was 50μg/L and Si⁴⁺was 7mg/l.The concentration of Cu²⁺was 3.84μg/L,Fe³⁺0.88mg/L,Mn²⁺24μg/L,Zn²⁺15.36μg/L,Ca²⁺2.7mg/L.Then,two mineral elements（Fe and Ca）with more obvious effects on microalgae were selected and compared with chelate type.The experimental results showed that chelate type ions were more easily absorbed and utilized by microalgae.Based on the above experimental guidance,a kind of biological feed which can be used as carbon source for microalgae culture and shellfish feed was developed.At first,the lactic acid production capacity of lactic acid bacteria was analyzed,and two kinds of lactic acid bacteria with strong lactic acid production capacity were screened,namely Lactobacillus fermentans and Lactobacillus plantarum,as subsequent fermentation strains.And then use the bacterial liquid of yeast and bacillus to culture Phaeodactylum tricornutum and Isochrysis galbana,respectively.With the biomass of microalgae as the index,strains that could promote the growth of Phaeodactylum tricornutum and Isochrysis galbana were selected as fermentation strains.Among them,Bacillus subtilis had significant growth promotion effects on Phaeodactylum tricornutum and Isochrysis galbana.The cell density of algae increased by 1.15 times and 0.49 times,respectively.The effects of yeast on Phaeodactylum tricornutum were ranked as follows:Saccharomyces cerevisiae>Candida utilis>Pichia pastoris>Candida tropicalis>Wine yeast.The effects of yeast on Isochrysis galbana were ranked as follows:Candida utilis>Saccharomyces cerevisiae>Candida tropicalis>Wine yeast>Pichia pastoris.Therefore,Candida utilis and saccharomyces cerevisiae were selected as the fermentation strains.After the fermentation strains were determined,the fermentation process was optimized from five factors,namely fermentation time,fermentation temperature,water content,inoculation amount and enzyme addition amount,and a group of fermentation conditions for the maximum increase of small and medium peptide content in fermented feed were obtained by single factor and response surface method:The fermentation time was 7 days,the inoculation amount was 7%（V/W）,the enzyme amount was 1%（W/W）,the fermentation temperature was 30℃,and the water content was 45%.Under this fermentation condition,the small peptide reached the maximum of 34.09%on the 7th day of fermentation.Then,the soybean meal feed was fermented by this process,and the dynamic changes of each substance during fermentation were measured.The results showed that the content of small peptide increased from 10.15%to 34.09%on the 7th day.The organic acids increased from 24.74mg/g at the beginning to 148.98mg/g on the7th day of fermentation.The p H value of the feed decreased from 6.16 on the 1st day to 4.10 on the 7th day.The results of protein electrophoresis showed that the large proteins in fermented soybean meal were hydrolyzed to small proteins and peptides below 27 k Da.Then,the above fermented biological feed was used for microalgae culture,with algal biomass as the main index.The experimental results showed that in addition to organic acids,amino acids and other active small molecules in the biological feed can effectively promote the growth of microalgae,among which beneficial bacteria can also act on microalgae,that is,the biological feed can be directly used for microalgae culture.Then,the single factor method was used to explore the effects of different supplemental levels of biological feed on the growth of microalgae.According to the algal biomass index,biological feed had significant effects on the growth of Phaeodactylum tricornutum and Isochrysis galbana.When the supplemental level was 1g/L,the algal biomass reached 9.79×10⁶cells/m L and 9.03×10⁶cells/m L,respectively.They were 1.72 times and 1.91 times higher than the control group.At the same time,some parameters affecting the water quality were determined.The results showed that after the microalgae cultured in biological feed,the ammonia nitrogen in the culture medium was completely eliminated,and the removal rates of nitrite nitrogen and total nitrogen were also significantly decreased,reaching 82%and97.6%,respectively.However,the residue of nitrite nitrogen and total nitrogen in f/2culture medium was more.Finally,the growth conditions of the two microalgae were observed under different temperature and rotation speed.The results showed that the optimum temperature of Phaeodactylum tricornutum and Isochrysis galbana was between 20℃and 24℃,and the suitable water flow disturbance had a significant effect on the growth of microalgae.Finally,the biological feed is used in clam breeding.The experimental results of different salinities of seawater showed that salinities had an important effect on the growth and survival of clams.When salinities ranged from 10 to 25,clams had a higher survival rate,and the daily increase of body mass was the largest when salinities were 20.The results showed that when the microalgae density was0.5×10⁶cells/m L,the growth rate of clam body mass was up to 3.25%,and the ammonia nitrogen content in water changed greatly and decreased obviously under this feeding rate.The survival rate and growth of clams were better when 0.5‰shellfish powder and 1‰biological feed were fed.The results of feeding experiment with different diets showed that the body weight of clams increased by 5.58%when feeding algal solution and biological feed at the same time,which was 2.79 times that of the algal solution group alone,and the ammonia nitrogen content of the biological feed group was lower.These results indicate that the biological feed as bait has certain effect on the growth and water environment of clams.This study understood the effects of organic carbon sources and mineral ions on the abnormal health growth of microalgae,and developed a kind of biological feed rich in active small molecules for microalgae culture.It is not only fertilizer,but also shellfish can directly absorb its nutrients from water through gills or skin,promoting the growth of shellfish,and has the effect of improving the aquaculture water environment.