Studies On Cultivation Of Porphyridium Cruentum And Integrated Processes To Obtain Active Substances

Porphyridium cruentum(Rhodophyta)is a unicellular microalga.The cells of P.cruentum show the spherical or oval shape,with the diameter of 5-24?m,does not have the typical cell wall structure.The cell wall of P.cruentum has a mucinous sheath structure composed of some soluble polysaccharides.P.cruentum can produce high contents of phycobilin,polyunsaturated fatty acids and polysaccharides(PUFAs)and polysaccharides.Therefore,cultivating P.cruentum and exploiting some techniques for extracting its bioactive substances have become one of the hot topics in microalgae.In this project,P.cruentum was selected.Firstly,the different kind of nitrogen resources and the nitrogen concentration of the culture medium for P.cruentum were optimized to obtain the most suitable nitrogen source and nitrogen concentration for the growth of P.cruentum and bioactive substances biosynthesis.Then,in the optimized medium,the photobioreactor was used to culture P.cruentum:after collecting the data,the kinetic models of growth and bioactive substance synthesis of P.cruentum were established.Finally,a green and efficient technology was developed for separating phycoerythrin and PUFAs from P.cruentum by the direct enzymatic of P.cruentum biomass.The main research contents and the results are as follows:(1)The effects of four different nitrogen sources(potassium nitrate,sodium nitrate,ammonium sulfate and urea)on the growth and bioactive substances biosynthesis of P.cruentum were investigated using 1.5 L open column bubble-type photobioreactors.The results showed that,comparing with the other nitrogen sources,urea was the best nitrogen source for the growth of P.cruentum,after the cultivation,the highest biomass of 3.23 g/L was obtained.The highest concentrations of phycoerythrobin(B-PE),phycocyanin(R-PC),phycocyanin(APC)and extracellular sulfate polysaccharide(EPS)were synthesized under urea nitrogen source,and their concentrations were 45.90 mg/L,6.08mg/L,5.74 mg/L and 529.67 mg/L,respectively.In the four nitrogen sources,microalgal cells showed the similar change trends of fatty acid biosynthesis;meantime,the content of arachidonic acid(ARA)was increased with the prolongation of culture time,but the content of eicosapentaenoic acid(EPA)had little change.(2)The effects of different urea concentrations on cell growth and biosynthesis of bioactive substances of P.cruentum were studied.The results showed that P.cruentum could achieve the maximum biomass yield,and the highest contents of total protein,EPS,intercellular polysaccharide(IPS)and chlorophyll a at 200 mg/L urea concentration,with the values of 3.2 g/L,769.5 mg/L,542.55 mg/L,1647.79 mg/L and 11.76 mg/L,respectively.At the 200mg/L urea concentration,microalga attained the highest concentrations of the highest concentrations of R-PC(9.43 mg/L),APC(4.64 mg/L)and B-PE(54.11 mg/L).The contents of ARA and EPA by microalga were positively correlated with the initial urea nitrogen concentration.Thus,based on the results,the optimal urea concentration of for the growth and bioactive substances biosynthesis of P.cruentum was 200 mg/L.(3)Under the optimal urea concentration conditions,a 25 L plate photobioreactor was used to cultivate P.cruentum,and the kinetic models for the growth and synthesis of bioactive substances were established.The obtained results showed that the nonlinear fitting of Logistic equation could match the data about the growth of P.cruentum,due to the fact that the R~2 of the function was 0.984.Moreover,the Luedekig-Piret equation was used to fit the experimental data of bioactive product protein,chlorophyll a,phycobilin and polysaccharide.All the expression functions R~2 were all greater than 0.9 for the aforementioned bioproducts.These results further stated that the established kinetic models could illustrate the growth and bioactive substances biosynthesis of P.cruentum in the plate photobioreactors.(4)After the cultivation in the plate photobioreactors,the cells and the extracellular polysaccharides were directly separated by centrifugation.The P.cruentum powders containing polyunsaturated fatty acids and phycobilin were obtained by freeze-drying.The cells containing phycobilin and PUFAs were used as the raw materials.In order to achieve the separation of phycobilin and PUFAs of P.cruentum,a green direct enzymatic reaction of P.cruentum biomass was developed and the best lipase was firstly screened.The results showed that Lipozyme CALB was the most suitable lipase for the direct enzymatic reaction of P.cruentum cells.To achieve the highest PUFAs yield by this developed process,the enzymatic reaction parameters were optimized.The optimal reaction parameters of direct enzymatic reaction of P.cruentum cells were as follows:water content was 5%,enzyme load was 10%,solid-liquid ratio was 3:1,reaction temperature was 45?,stirring speed was 300 rpm,and reaction time was 8 h.Under the optimal reaction conditions,the highest conversion rate of arachidonic acid ethyl ester(ARA-FAEEs)and eicosapentaenoate ethyl ester(EPA-FAEEs)catalyzed by lipase were88.28%and 45.19%,respectively.These obtained results suggested that the developed process mediated with liquid Lipozyme CALB was a promising approach to effectively separate the PUFAs and phycobilin of P.cruentum cells.Moreover,the method was regarded as a green way,because ethanol was used as the acyl acceptor.This process will be further evaluated in the large scale for the co-production of PUFAs and phycobilin by P.cruentum cells in future.In this paper,the optimal nitrogen source is urea,and the optimal concentration of urea nitrogen is 200mg/L.The application potential of the optimal nitrogen source and nitrogen concentration in the culture of P.cruentum was verified,and the kinetic equations related to the growth and product synthesis of P.cruentum were constructed,which could describe the growth and product synthesis process of P.cruentum.The results of amplification culture were used to construct ethanol hydrolysis reaction catalyzed by lipase,and to provide a practical basis for the co-production and separation of intracellular polyunsaturated fatty acids and phycobilin from P.cruentum.