Isolation,Identification And Application Of Carotenoids-Producing Bacteria From Intestine Of "Nan’ao Golden Scallop"

The noble scallop Chlamys nobilis Reeve is an important mariculture shellfish species in the southern coast of China.The“Nan’ao Golden Scallop”Chlamys nobilis（hereinafter“Nan’ao Golden Scallop”）is a new national aquatic variety bred by our research group,which not only has a golden appearance,but has higher carotenoids content in adductor muscle,mantle,intestine and hemolymph,and more potential carotenoids-producing bacteria in the intestine than others.In addition,previous studies have shown that carotenoids synthesized by carotenoids-producing bacteria can be utilized by the host.In view of the important role of carotenoids,based on previous studies,carotenoids-producing bacteria will be isolated from the intestine of“Nan’ao Golden Scallop”,and the feasibility of the isolate to be used as probiotic for producing seedlings in scallop was studied.Therefore,in this paper,“Nan’ao Golden Scallop”was used as material.Firstly,several pigmented bacteria from the intestine were isolated by microbial isolation and culture technology,and their species were identified through 16S r RNA gene sequence analysis.Subsequently,the carotenoids biosynthesis by one of the carotenoids-producing bacteria was analyzed by the whole genome and non-targeted metabolome,and their metabolic pathways were speculated.Finally,a rapid determination method of total carotenoids content of this isolate was formulated,and the feasibility of it to be used as probiotic in scallop larvae and broodstock was evaluated.The main results and conclusions are as follows:1.Sixteen potential carotenoids-producing strains were isolated.The 16S r RNA gene sequence analysis showed that strain C13 and Erythrobacter pelagi shared 99.28%similarity,and the Average Nucleotide Identity（ANI）was 85.48%.Therefore,the strain was named Erythrobacter sp.C13.The Erythrobacter sp.C13 did not generate hemolysin and did not have potential pathogenicity.The suitable temperature,p H,and salinity for the growth of Erythrobacter sp.C13 was 20～35°C,7.0～8.0 and 25～40‰,respectively.2.The genome of Erythrobacter sp.C13 contained the key genes Crt B,Crt I,Crt Y,Crt Z,and Crt W for carotenoids biosynthesis,which could produce C₄₀ carotenoids,including beta-cryptoxanthin,lutein,and metabolite of fucoxanthin——Halocynthiaxanthin（CAS:81306-52-7）,and it is speculated that the strain syntheses beta-cryptoxanthin and lutein through MEP pathway.3.A new method for the rapid determination of TCC in bacterial solution was established.The linear regression equation of strain Erythrobacter sp.C13 was y=0.99427x-0.04612（R²=0.9164）.The absorbance value（x）atλ=600 nm was measured by UV spectrophotometer,and TCC（y）in the bacterial solution could be quickly calculated by substituting the equation.4.The survival rate,growth and development rate of D-larvae in the experimental group were significantly higher than those in the control group（P<0.05）when Erythrobacter sp.C13 solution with the final concentration of 1×10⁴cfu/m L was added to the seedling water,and the level of Vibrio genus in the water of experimental group was continuously declined.We believe that this strain can be utilized as a potential probiotic by scallop larvae.5.Erythrobacter sp.C13 solution with the final concentration of 1×10⁵cfu/m L was added to the water,the hemolymph TCC of juvenile scallops with a shell height of3～4 cm in the experimental group was significantly higher than that in the control group（P<0.001）after three weeks of indoor culture,indicating that the carotenoids synthesized by the strain may be absorbed by the host.In addition,the level of Vibrio genus in the intestine of scallop in the experimental group was significantly lower than that in the control group（P<0.05）.In summary,several potential carotenoids-producing bacteria were screened from the intestine of“Nan’ao Golden Scallop”.Among them,Erythrobacter sp.C13 has been confirmed to synthesize three carotenoids,and the metabolic pathways of two carotenoids（beta-cryptoxanthin and lutein）were speculated.A new method for rapid determination of TCC in the bacterial solution was developed.Moreover,the present experimental results showed that Erythrobacter sp.C13 was able to be used as a potential probiotic for scallop seedling production,and carotenoids synthesized by Erythrobacter sp.C13 might be utilized by the host.Therefore,the present study has an important role in theory and application.