Application And Interaction Mechanism Of Microalgae And Bacteria Co-culture In Artificial Biogas Slurry Treatment

Biogas slurry from pig farms is rich in nitrogen,phosphorus and organic carbon,which poses serious environmental risks if not disposed properly.Coupling microalgae culture with biogas slurry treatment could reduce the cost of microalgae culture and produce high value-added biomass products while recycling biogas slurry.The exchanges of O₂/CO₂ and metabolites between microalgae and aerobic bacteria could provide favorable conditions for their growth and enable them to share environmental pressures.Using microalgae and bacteria co-culture could further improve the efficiency of wastewater treatment.In this study,the bacteria isolated from biogas slurry were firstly screened to investigate their growth capacities when co-cultured with different microalgae,so as to establish a mutualistic symbiosis co-culture.Then the conditions of artificial biogas slurry treatment by co-culture were optimized and the lipid content and fatty acid composition were determined to evaluate its potential in biogas slurry treatment and biofuel production.Based on transcriptome technology,the interaction mechanisms of microalgae and bacteria in artificial biogas slurry treatment were analyzed.Finally,the photobioreactors suitable for microalgae-bacteria immobilization were designed to verify the potential for large-scale artificial biogas slurry treatment.This study provides theoretical support for revealing the interaction mechanism between microalgae and bacteria,and provides basic data for evaluating biogas slurry treatment by other biofilm reactors.The main results are as follows:1.Five strains of bacteria that could be co-cultured with microalgae were selected from biogas slurry,and two symbiotic co-culture systems were constructed.The specific results are as follows.Five strains of bacteria that could use the metabolites of different microalgae for their own growth were obtained.Among them,two strains（Staphylococcus sciuri and Kurthia gibsonii）could use Chlorella vulgaris metabolites to grow,two strains（K.gibsonii and Rhizobium sp.MN13）could use Chlorella pyrenoidosa metabolites to grow,three strains（S.sciuri,Staphylococcus saprophyticus and Bacillus megaterium）could use Desmodesmus sp.metabolites to grow,three strains（S.sciuri,K.gibsonii and R.MN13）could use Desmodesmus sp.G41-M metabolites to grow.The five strains were co-cultured with their corresponding microalgae,and two symbiotic co-cultures were obtained.The C.vulgaris and S.sciuri co-culture（Cv-Ss）and the Desmodesmus sp.and B.megaterium co-culture（Ds-Bm）.2.By comparing the effects of Cv-Ss co-culture and Ds-Bm co-culture in treating artificial biogas slurry and optimizing the treatment conditions,an efficient Ds-Bm co-culture for biogas slurry treatment was established,and the C/N ratio was found to be the key factor regulating NH₄⁺-N removal.The specific results are as follows.Compared with pure C.vulgaris,the removal rates of TP and NH₄⁺-N in Cv-Ss co-culture were improved by 18%and 53%,respectively.Compared with pure Desmodesmus sp.,the removal rates of TP and COD in Ds-Bm co-culture were improved by 43%and 142%,respectively.The Ds-Bm co-culture was selected to optimize the conditions.When the C/N ratio was 106/16 and the initial microalgae/bacteria inoculation ratio was 9/1,the co-culture attained the maximum COD,TP and NH₄⁺-N removal rates of 86%,81%and 65%,respectively.The biogas slurries with different nutrient concentrations were used to analyze the key factor affecting NH₄⁺-N removal by the Ds-Bm co-culture.The Ds-Bm co-culture could not remove NH₄⁺-N in biogas slurry with low C/N ratio and high NH₄⁺-N concentration（C/N=106/80,NH₄⁺-N=277.56mg/L）,but could remove NH₄⁺-N in biogas slurry with high C/N ratio and higher NH₄⁺-N concentration（C/N=106/16,NH₄⁺-N=333.8 mg/L）.It indicated that the C/N ratio was the key factor regulating NH₄⁺-N removal.Compared with the microalgae-activated sludge co-culture,the Ds-Bm co-culture had more advantages in nitrogen and phosphorus removal.The NH₄⁺-N removal effects of pure microalgae,pure bacteria and the co-culture under different C/N ratio conditions were compared.The NH₄⁺-N removal efficiency by pure microalgae was not affected by the C/N ratio.Under the high C/N ratio condition,B.megaterium significantly enhanced the absorption of NH₄⁺-N in the co-culture,while under the low C/N ratio condition,B.megaterium secreted NH₄⁺-N into the biogas slurry,suggesting that the changes of NH₄⁺-N metabolism of B.megaterium and the relationship between microalgae and bacteria might be the key factors regulating NH₄⁺-N removal in Ds-Bm co-culture under different C/N ratio conditions.3.The transcriptional reactions of pure microalgae/bacteria culture and the co-culture were analyzed to reveal the interaction mechanism between microalgae and bacteria and the mechanism of NH₄⁺-N removal by microalgae and bacteria.The specific results are as follows.Under both the high C/N ratio and the low C/N ratio conditions,B.megaterium upregulated the genes related to glycolysis,Calvin cycle and TCA cycle in Desmodesmus sp.,which promoted the mixed nutrient utilization of carbon source and activated the energy metabolism in Desmodesmus sp.In addition,the genes related to IAA synthesis were upregulated in both co-cultured Desmodesmus sp.and co-cultured B.megaterium.Therefore,co-culturing promoted the growth of microalgae and bacteria and the removal of pollutants.Under the high C/N ratio condition,Desmodesmus sp.downregulated the metabolic pathway of bacterial flagellar assembly,resulting in slower bacterial motility,which was conducive to the stability of microalgae-bacteria co-culture.The microalgae-bacteria interaction caused changes in NH₄⁺-N related metabolisms in the co-culture.Under the high C/N ratio condition,the genes related to urea hydrolysis in B.megaterium were significantly downregulated,while the genes related to urea hydrolysis and NH₄⁺-N assimilation in Desmodesmus sp.were upregulated.The results indicated that the co-culture under the high C/N ratio condition inhibited the degradation of urea by bacteria,promoted the utilization of urea by microalgae,enhanced the assimilation of NH₄⁺-N by microalgae,and achieved the synergistic removal of NH₄⁺-N by microalgae and bacteria.4.By investigating the lipid content and fatty acid composition of Ds-Bm co-culture after biogas slurry treatment,it was found that the co-culture had the potential of being used as biofuel and revealed the regulation mechanism of lipid metabolism in the co-culture.The specific results are as follows.The lipid content and saturated fatty acid content of Ds-Bm co-culture were 30%and 76%,respectively,which were 12%and 46%higher than that of pure Desmodesmus sp.The transcriptional information showed that the increase of C/N ratio upregulated the genes related to fatty acid biosynthesis and downregulated the genes related to fatty acid degradation and unsaturated fatty acid biosynthesis in co-cultured Desmodesmus sp.Under the high C/N ratio condition,B.megaterium downregulated the genes related to unsaturated fatty acid biosynthesis in Desmodesmus sp.Therefore,the high C/N ratio condition was more conducive to lipid accumulation of Ds-Bm co-culture,and the fatty acid composition of Ds-Bm co-culture under the condition was more suitable for biofuel production compared with pure Desmodesmus sp.5.On the basis of recycling waste,using pine sawdust and waste cotton cloth as immobilized carriers of microalgae and bacteria,two photobioreactors with different structures were designed to realize the large scale treatment of biogas slurry while harvesting the biomass of microalgae and bacteria.The specific results are as follows.Using pine sawdust as immobilized carrier and vertical flat photobioreactor to treat biogas slurry,the biomass reached 2.05 g/L,and the removal rates of COD,TP and NH₄⁺-N were 91%,89%and 74%,respectively.Using waste cotton cloth as immobilized carrier and slant flat photobioreactor to treat biogas slurry,the biomass reached 3.45 g/L,and the removal rates of COD,TP and NH₄⁺-N were 100%,98%and 93%,respectively.In conclusion,the Desmodesmus sp.and B.megaterium co-culture was used to effectively treat biogas slurry,and the interaction mechanism of Desmodesmus sp.and B.megaterium in biogas slurry treatment was revealed.The photobioreactor combined with immobilized technology could efficiently treat biogas slurry and recycle biomass and waste resources.This study provided technical support for the large-scale application of microalgae-bacteria co-culture to treat wastewater in the future.