Isolation And Fermentative Condition Of High Yield Ectoine-producing Bacterium

Ectoine can be potentially applied in a variety of industries such as enzymes,bio-medicine and skin care manufactory.Currently it is synthesized mainly by biological methods.The objectives of the present study were 1)to isolate a moderately halophilic bacterium capable of synthesizing ectoine as main compatible solute,2)to investigate the effects of hypo/hyper-osmotic stress on release and re-synthesis of ectoine in the isolated bacterium,and 3)to explore the feasibility of producing ectoine through "bacteria milking" by using low molecular weight organic acids and food waste hydrolyte as carbon sources.A moderately halophilic bacterial strain W2 was successfully enriched and isolated from a saltern soil sample collected from Lianyungang city,Jiangsu Province.Strain W2 was characterized by synthesizing ectoine as its main compatible solute and preliminarily identified as a species of genus Halomonas according to its morphological characteristics,physiological,biochemical properties and similarity analysis of 16S rDNA sequence.The effects of several factors on the growth and ectoine synthesis were investigated by shake-flask experiments.Results showed that optimal growth conditions for strain W2 were:35?,pH 7.0?8.0,NaCl 100 g/L,liquid volume 50?200 mL/500 mL and inoculum size?10%(v/v),C/N?7:1 respectively.Optimal conditions for the ectoine synthesis in strain W2 were:35?,pH 6.0?8.0,NaC1 100?125 g/L,liquid volume 50?200 mL/500 mL and inoculum size 1%or ?20%(v/v),C/N?7:1,respectively.It can be considered to be an excellent ectoine-producer as indicated by a high yield up to 161 mg/g cdw when the strain was cultured under optimal conditions.When cultivated in the medium containing different NaCl concentrations four compatible solutes,namely betaine,ectoine,glutamate and aspartate were detected in the cell extracts of strain W2.NaCl concentrations have a significant effect on the content of those four compatible solutes.Their percentages were betaine(60.8%),ectoine(10.5%),glutamate(16.4%)and aspartate(12.3%),respectively when it was cultivated in the medium containing 25 g/L NaCl and they were changed to ectoine(70.8?85.9%),betaine(10.7-16.1%),glutamate(1.6-8.8%)and aspartate(1.8-4.2%),respectively when cultivated in the medium containing 50?100 g/L NaCl.This indicated different mechanisms for its adaptation to osmotic conditions.Betaine was synthesized as the major compatible solute for balancing osmotic pressures across bacterial cell membrane when it was cultivated in the medium containing low levels of NaCl(25 g/L)while ectoine was synthesized as the major compatible solute when it was cultivated in the medium containing high levels of NaCl(100 g/L).Strain W2 was a secreting ectoine-producer as indicated by rapid ectione release in hypotonic solutions and resynthesis under hypertonic conditions.The optimum NaCl concentration for hypotonic shock was 30 g/L,which led to a release rate of 59.7%within 5 min.By transferring the biomass to hypertonic medium containing 100 g/L NaCl intracellular ectoine could be resynthesized to its highest level(161 mg/g cdw)in just 3 h.Above-mentioned hypotonic/hypertonic shock conditions can also be applied to W2 biomass harvested from the medium with acetate,propionate,butyrate or lactate as sole carbon sources.However,slightly longer time was required for achieving highest rate of release or synthesis when higher concentrations of W2 cells were performed under the same osmotic shock conditions.For W2 cells harvested in the medium with acetate,propionate,butyrate or lactate as sole carbon sources,alternative hypertonic/hypotonic shock could be repeated by 11 times without significant reduction in ectoine synthesis and release although both synthesis and release decreased significantly during the 12th osmotic shock period.Total amount of synthesized ectoine was 7.99?9.03?8.58 and 7.95 g/L respectively,and total amount of released ectoine was 4.33?4.47?4.60 and 4.15 g/L,respectively after 12 cycles of osmotic shock.Average release rate was 54%?49%?52%and 54%respectively.Similar results were obtained for W2 cells harvested from the medium prepared using food waste hydrolyte as carbon source.The amount of synthesized and released ectoine remained quite stable during 11 cycles of hypertonic/hypotonic shocks.However,ectoine yield decreased significantly during the 12th osmotic shock period.Total amount of synthesized ectoine was 10.13?10.8 and 9.53 g/L respectively,and total amount of released ectoine was 5.09?5.21 and 5.09 g/L,respectively after 12 cycles of osmotic shock.The average release rate was 50%?48%and 53%,respectively,thus verifying the feasibility of ectoine production through“bacterial milking”using organic acid or food waste hydrolyte as carbon sources.