Study On The Resource Utilization Of Anaerobic Digestion Piggery Effluent Based On Fungi-Microalgae Synergism

The biogas slurry produced by large-scale biogas projects under the“specialized energy utilization of manure waste”model,which is promoted by the Ministry of Agriculture,has the characteristics of large output,complex composition,high pollutant concentration,instability,and poor biodegradability.The existing farmland is unable to meet the demand for biogas slurry consumption.The disposal of biogas slurry has become one of the bottlenecks limiting the development of biogas technology in China.Finding and developing suitable ways for biogas slurry resource utilization plays an important role in promoting the sustainable development of the livestock and poultry breeding industry.Fungi-microalgae system can not only alleviate the inhibition of biogas slurry on algal growth but also promote the recycling and utilization of nutrients.The purpose of this study is to achieve resource utilization of anaerobic digestion piggery effluent（ADPE）with a fungi-microalgae synergistic system.Firstly,the microalgae and fungal strains were isolated and selected,subsequently,the selected algal strain was optimized for growth in ADPE.Secondly,the trophic mode and nitrogen sufficiency conversion strategy was employed to enhance the growth of algal strain and ammonium nitrogen removal from ADPE,and the related mechanism was investigated.Then,fungal pretreatment was used to improve algal growth in ADPE,and fungal decolorization conditions and mechanisms were optimized and explored.Finally,the feasibility of using fungal pellet to flocculate and harvest microalgae from ADPE was explored,then,the flocculating conditions for microalgal harvesting with fungal pellet were optimized and the specific mechanism was revealed.The main conclusions obtained are as follows:（1）One microalgal strain NCU-7 with strong adaptability to ADPE was isolated and selected.The biomass yield,Chl a concentration,and specific growth rate in sterilized 10%ADPE were 1.06 g L^-1,21.1 mg L^-1,and 0.24 d^-1,respectively,and it was identified as Chlorella sorokiniana.NCU-7 showed the best growth performance in raw 10%ADPE with a light intensity of 4000 Lux.After 7 days of cultivation,NCU-7obtained the biomass yield of 1.26 g L^-1 and achieved the removal efficiency of 10.4%,67.3%,61.1%,and 84.3%for COD,NH₄⁺-N,TN,and TP,respectively.（2）Under the optimal conversion strategy from nitrogen-deprived mixotrophic culture（DN,M）to nitrogen-sufficient photoautotrophic culture（SN,P）,NCU-7achieved the maximum biomass yield（OD:1.18,1.59）and NH₄⁺-N removal rate(2.5,4.2 mg L^-1 d^-1)in wastewater at NH₄⁺-N concentration of 160 and 320 mg L^-1.The nitrogen and protein contents of NCU-7 cells in DN,M culture were lower than those in other cultures,while the lipid content was higher than other cultures,and the contents of N,protein,and lipid were 3.3%,20.6%,and 34.0%,respectively.Proteomics analysis showed that NCU-7 reduced intracellular nitrogen/protein content in response to N deprivation stress by regulating a range of physiological metabolisms including enhancement of proteolysis,reduction in syntheses of protein,chloroplast,and amino acid,along with overexpression of lipid synthesis pathway.After transferring from DN,M culture to N-sufficient wastewater,the metabolic pathways related to photosynthesis and protein synthesis in algae cells were completely restored,which increased the demand of algae for nitrogen,resulting in the increase in protein content of algal cells and high NH₄⁺-N removal from wastewater.Furthermore,the strategy by DN,M culture improved the growth of NCU-7 and nutrient removal from sterilized 10%ADPE,verifying the feasibility of this strategy in microalgae cultivation with ADPE.（3）Two fungal strains A-17 and H-1 with good adaptability and decolorization performance to ADPE were isolated and selected out of ten Trichoderma strains,and they were identified as Trichoderma harzianum and Trichoderma afroharzianum,respectively.The optimal conditions for fungal decolorization and NH₄⁺-N removal from ADPE by A-17 and H-1 were:20%ADPE,8 g L^-1 glucose,initial p H 6,160 rpm,25-30℃,0.15 g L^-1 initial dry weight,with decolorization and NH₄⁺-N removal efficiencies of 85%,46%,and 90%,46%,respectively.Humic acid substance（HS）was the main reason for chromaticity in ADPE,and ADPE decolorization was mainly caused by fungal biodegradation of color-related HS through Mn P enzyme secretion.Removed nitrogen was completely converted into fungal biomass by nitrogen assimilation,of which ca.90%was attributed to NH₄⁺-N removal.Alkanes,ethers,esters,and phenols were the main macromolecular organic compounds in ADPE.Fungal pretreatment significantly reduced the chromaticity,NH₄⁺-N concentration,and macromolecular organic matter content in ADPE,and alleviated the inhibitory and toxic effects on algal cells,improving the growth of NCU-7 and nutrient removal in pretreated 20%ADPE.（4）The optimal conditions for NCU-7 flocculation by fungal pellet of H-1 and Aspergillus oryzae were:p H 3,ratio of fungus to algae（dry weight）:1.5:1,120 rpm,30℃;and p H 3,pellet diameter 8 mm,pellet amount 30（ratio of fungus to algae:0.9:1）,130 rpm,with maximum flocculation efficiency of 85%and 98%,respectively.The zeta potential value of A.oryzae was positive at p H 3,while algal cells showed a negative value,indicating that charge neutralization might be the main reason for algal cell flocculation by fungal pellet.Compared to p H 8,the content of protein and polysaccharide in EPS of fungal pellet（FP）and fungal-algal pellet（FA）significantly increased at p H 3.The functional groups including C=O,N-H,O-H of proteins and polysaccharides in EPS of A.oryzae promoted microalgae flocculation.At low p H,FP and FA reduced the content of hydrophilic amino acids and monosaccharides including glutamic acid,glutamine,mannose,and glucose in EPS,while increased the content of hydrophobic amino acids and monosaccharides including leucine,proline,γ-aminobutyric acid,alanine,and galactose.The flocculation mechanism of fungal pellet on NCU-7 at low p H was attributed to charge neutralization,adsorption of extracellular proteins and polysaccharides,and hydrophobic interaction.At p H 3,A oryzae pellet achieved a flocculation efficiency of 99%on algal cells in ADPE,and the composition of fungal-algal pellet biomass showed great potential in feed or biofuel production.This study provides theoretical data support for the development of a large-scale process for the sustainable resource utilization of ADPE using a fungi-microalgae synergistic system.