Optimizing Cultural System Of Porphyridium Sp,Polysaccharide Preparation And Effects As Bio-stimulant In Promoting Crop Tolerance To Salt Stress

Objectives This study was conducted to investigate the production of Porphyridium cruentum biomass,pigment contents,and cell wall fraction and soluble fraction of polysaccharides under different environmental conditions.Additionally,the bacterial communities in phycosphere of Porphyridium purpureum were identified under different sources of nitrogen at different days.The growth performance and biochemical composition of Porphyridium purpureum were examined when this red alga was co-cultured with the beneficial bacterium isolated from the phycosphere.Furthermore,the polysaccharides and liquid culture from the alga were prepared as natural biostimulants.Finally,the effects of these biostimulants on plant growth,particularly salt stress tolerance were systematically explored,including plant growth characteristics at various stages,pigments,cellar activities,antioxidant enzymes and substances in tomato and strawberry plants under salt stresses.Methods 1.Optimizing culture conditions to increase biomass and polysaccharide production in microalga Porphyridium cruentum.A series of single-factor experiments were performed to examine effects of pH,temperature,light,nitrogen and carbon sources,as well as nitrogen(KNO₃)and Phosphate(KH₂PO₄)concentration on algal cell growth,cell wall fraction and soluble fraction of polysaccharides.2.Metagenomic analysis was employed to investigate dynamics of microbial communities in phycosphere of Porphyridium purpureum cultured in different nitrogen sources and at different days.3.A beneficial bacterium strain Pseudoalteromonas sp,was isolated from the phycosphere.Co-culture system of the red alga and Pseudoalteromonas sp,was constructed at different initial ratios of algae vs bacteria,followed by detecting growth performance,photosynthesis,and biochemical profiles in Porphyridium sp during culture progress.4.To investigate effects of polysaccharides and liquid culture from P.cruentum and C.vulgaris on growth characteristics and metabolism of tomato plants,100 m M NaCL was selected as the salt stress treatment on plants,and a number of physio-biochemical characters were examined for tomato plant grown under such salt stress,including growth performance,contents of chlorophyll,malondialdehyde(MDA),proline,total phenolics,ascorbic acid(As A),protein,and activities of antioxidant enzyme system.5.Similarly,100 m M NaCL was also selected as the salt stress treatment on strawberry plants,and a number of examinations were taken to explore the effects of polysaccharides and liquid culture from P.cruentum on growth performance and metabolism of strawberry plants under the salt stress,including the plant growth rate,levels of chlorophyll,malondialdehyde(MDA),proline,total phenolic,ascorbic acid(As A),and protein.Main Results 1.The optimized culture conditions for algal growth were pH 7.5,25? temperature and blue light.The maximum cell growth was obtained in KNO₃(2.85 dry-wt g/L)and Na HCO₃(2.87 dry-wt g/L)as nitrogen and carbon source,respectively.KNO₃ and KH₂PO₄ at a concentration of(0.8 to 1 g/L and 0.8 to 1 ml/L)could increase algal cell growth up to high biomass(2.73 dry-wt g/L)and(2.78 dry-wt g/L).Furthermore,the maximum levels of chlorophyll a(3.01 ?g/ml)and carotenoids(1.9 ?g/ml)as well as soluble exo-polysaccharides(2.89 ?g/L)were achieved in the medium containing(1 g/L)KNO₃ and(1 ml/L)KH₂PO₄.Whereas,(0.8 g/L)of KNO₃ and(0.8 ml/L)of KH₂PO₄ were favourable for accumulation of cell wall polysaccharides(4.06 ?g/L and 4.26 ?g/L).2.Metagenomic analysis revealed dynamics of microbial flora in phycosphere of P.purpureum cultured under different N sources.Changes of microbial community abundance were closely correlated with the culture time and conditions.Although the peak growth rate and the maximum level of polysaccharides were observed in P.purpureum cultured in the medium contain KNO₃ as N source.The highest ?-diversity index of microbial communities was obtained in phycosphere of the alga cultured for 16 days in the medium contain ammonia as N source.The main bacterial flora detected in the phycosphere included Oxyphotobacteria,Bacteroidia,Alphaproteobacteria,Chloroplast,Cytophagales,and Phycisphaerales,which dominated microbial abundance by 78%.Abundance of these microbial flora were changed with the culture time.In taxonomic phylum level,Proteobacterai,Becteroidetes and Firmicutes are mostly abundant phylum,with OTUs(operational taxonomic units)detected account for 65% of total OUTs.At the genus level,Bacteroides,P.purpureum and Algorisphages were main bacterium flora whose OUTs occupied 20% of total OTUs detected.The Venn diagram showed that number of the unique OTUs was 3,566 in the phycosphere of this alga cultured for 16 days using ammonia as N source,again indicating that the high degree of microbial diversity was presented in the phycosphere of P.purpureum.3.A beneficial bacterium strain Pseudoalteromonas sp,was successfully isolated from the phycosphere of the red alga(P.purpureum),showing growth-promoting effect for the alga.This bacterium was used to prepare different initial inoculum ratios of algae vs bacteria,and then these initial alga-bacterium inoculum were screened to develop the efficient co-culture system of the alga and bacterium for increase algal biomass and polysaccharide yield.Analysis on algal growth and metabolism in these co-culture systems showed that the coculture system with initial inoculum ratio of 1:20(alga:bacterium)could significantly promote algal growth and photosynthesis.After 26 days of co-culture using this system,algal biomass(2.13 dry-wt g/L),chlorophyll a(2.04 ?g/ml),and carotenoids(1.41 ?g/ml)were increased significantly.Similarly,phycoerythrin(PE),phycocyanins(PC)and allophycocyanins(AP)were also increased up to(1.1 %dw,0.56 %dw and 0.53(%dw)in algal cells,respectively,under this co-culture system.Moreover,this co-culture system resulted in the highest levels of lipids(0.86 g/L),proteins(0.86 g/L)and carbohydrates(2.16 g/L)produced in algal cells.However,the co-culture system with initial inoculum ratio of 1:10(alga:bacterium)greatly benefited accumulation of cell wall fraction(4.03 ?g/L)and soluble fraction(2.55 ?g/L)of polysaccharides,respectively,after 26 days of co-culture.4.Polysaccharides and liquid culture application of P.cruentum and C.vulgaris were applied as biostimulants.Different dosages(40 mg.L^-1 and 60 mg.L^-1)of alga-derived biostimulants(polysaccharides or liquid culture of the alga)were applied to the hydroponic tomato seedlings,respectively.Compared to tomato plants grown under salt stress and without biostimulant application,the biostimulant-treated plants under salt stress exhibited normal growth and excellent fruit quality.For 30-day tomato seedlings,plant height,shoot length,root length,per plant dry weight and LAI as well as soil plant analysis development(SPAD)were increased up to 48.33 cm,18.93 cm,4.28 cm,4.38 g 1.32 m²m^-2 and 47.01 SPAD vales,respectively.In addition,these biostimulants could mitigate inhibitory effects of salt stress on tomato plants by reducing MDA and H₂O₂ levels,but increasing accumulation of chlorophylls,As A,phenolic,anthocyanin,proline and protein in plants.Notably,the biostimulants could activate plant antioxidant enzyme system.The most effectivity was obtained by treatment of 40 mg.L^-1 polysaccharide or 60 mg.L^-1 liquid algal culture.These results indicated that algaderived biostimulants can efficiently increase production of antioxidant and osmotic adjustment substances,and simultaneously,effectively clean excess H₂O₂ for relieving oxidative damage,consequently leading to enhancement of plant salt tolerance.5.Effects of P.cruentum polysaccharides and liquid cultures used as biostimulants on strawberry.Salt stress(5 g/L NaCL)significantly inhibited growth of strawberry plants,leading to serious physiological damage on plants.P.cruentum polysaccharides and liquid cultures both effectively alleviated plant injury caused by salt stress.Likely in cases of the treated tomato plants,such biostimulants also greatly up-regulated the activities of antioxidant enzyme system and accumulation of antioxidant substances,meantime reducing generation of MDA and H2O2 in strawberry plants.Overall,the effect of P.cruentum polysaccharide at(40 mg.L^-1)and liquid culture at(60 mg.L^-1)treatments both significantly benefited strawberry plants especially under salt stress.Conclusions 1.Optimized culture conditions for achieving larger biomass of P.cruentum are pH 7.5,blue light,25? temperature,and using KNO₃ and Na HCO₃ as nitrogen and carbon source.Furthermore(0.8 to 1 g/L)of KNO₃ and(0.8 to 1 ml/L)of KH₂PO₄ obtained maximum biomass production respectively.The highest yield of pigments and polysaccharides produced by P.purpureum is obtained under the optimized culture conditions including KNO₃(0.8 to 1 g/L)and KH₂PO₄(0.8 to 1 ml/L)as N,P sources,respectively.2.The dynamic of microbial community in the phycosphere of P.purpureum is highly correlated with nitrogen sources and culture time.The largest diversity of microbial flora in the phycosphere of the red alga is occurred when the alga is cultured for 16 days using ammonia as N source.P.purpureum biomass and polysaccharide yield are significantly affected by the dominant bacterial flora such as Oxyphotobacteria,Bacteroidia,Chloroplast and Cytophagales.One growth-promoting bacterial strain(Pseudoalteromonas sp)is isolated from the phycosphere,which could be used to construct the efficient co-culture system of alga and bacterium for increasing P.purpureum growth and polysaccharide enrichment.3.Biomass and polysaccharide yields of P.purpureum are different among the co-culture systems with different initial inoculum ratio of alga to bacterium.The co-culture system with initial inoculum ratio of 1:20(alga:bacterium)could significantly promote algal growth,photosynthesis,lipid and phycoerythrin accumulation.However,the co-culture system with initial inoculum ratio of 1:10(alga:bacterium)largely increase polysaccharide yield.The present data provide a new strategy for further increasing P.cruentum and commercial production of polysaccharides.4.P.cruentum and C.vulgaris polysaccharides and their liquid cultures can be prepared as valuable biostimulants for agricultural applications.Exogenous application of these algalderived biostimulants can significantly promote tomato plant growth and fruit quality.Such treatments can also result in activation of endogenous antioxidant enzyme system and reduction of physiological damage,consequently leading to enhancement of tomato plant tolerance to salt stress.These findings provide scientific supports for preparation and application of algal-derived biostimulants.5.Similarly as effects on tomato plants,exogenously applying P.cruentum polysaccharides and liquid cultures can greatly improve strawberry plant growth.Such algal-derived biostimulants also active the antioxidant enzyme system and enrichment of osmosis regulatory substances,increasing strawberry plant tolerance against salt stress.The present data lay the foundation for development and utilization of highly effective algal-derived biostimulants.In summary,an optimized culture condition is developed for increasing P.cruentum biomass and polysaccharide yield.Microbial community structure in the phycosphere of P.cruentum is examined and their diversities are associated with culture time and nitrogen source.One beneficial bacterium strain isolated from the phycosphere can be used to construct the efficient co-culture system of alga and bacterium for further enhancing algal biomass and polysaccharide production.Polysaccharides of P.cruentum and C.vulgaris and their liquid cultures can be prepared as biostimulants for agricultural applications.Tomato and strawberry are selected as the targets for investigating the effect of algal-derived biostimulants on crops.These biostimulants can significantly promote plant growth.Particularly increase plant tolerance against salt stress.The present findings provide a solid scientific basis for development new types of agal-derived biostimulants with high efficiency and broad applications in agriculture production.