| Swine wastewater has caused severe environmental damages due to its high concentrations of pollutants.The development of methods to treat swine wastewater has become the focus of attention.Green algae-based N and P removal technology represents an eco-friendly solution,which reduce the environmental cost by decreasing land used for this purpose,and recover N and P.However,the low C to N ratio,unbalanced N to P ratio and biologically toxic compounds in swine wastewater will inhibit the growth of green algae.Due to the symbiotic interactions between microalgae and bacteria in nature,microalgae-bacteria symbiosis system can produce synergy and remove N and P from wastewater efficiently,which has potential for treating wastewater.Aiming at the bottleneck of green algae based swine wastewater treatment technology,green algae which can grow rapidly and tolerate swine wastewater were isolated and screened in this study,which verified the feasibility of green algae treatment of swine wastewater as a resource;Based on the symbiotic relationship between green algae and indigenous bacteria in swine wastewater,a green algae-bacteria symbiotic system was constructed to explore the factors affecting its N and P removal efficiency and its inhibitory effect on pathogenic bacteria and antibiotic resistance genes;The relationship between green algae and other microorganisms and the changes of microbial community in green algae-bacteria symbiosis system were analyzed in order to clarify the formation mechanism of green algae bacteria symbiosis.The correlation between microbial community and N and P removal was analyzed from the perspective of gene,and the contribution of green algae and bacteria to N and P removal in green algae-bacteria symbiosis system was clarified.It is verified that the green algae-bacteria symbiosis system is an effective and environmentally sustainable method for N and P removal from swine wastewater.Firstly,the feasibility study on the separation and screening of green algae and the resource utilization of N and P removal from swine wastewater was carried out.59 green algal strains were isolated and purified from fresh water in Harbin by plate scribing method,the growth rate of green algae was specific to algae strains.Finally,12 strains with fast growth rate were selected to study the removal of N and P from swine wastewater by green algae.Among them,P.kessleri QWY28,Chlamydomonas sp.QWY37,Chlorella sp.MA1 and Coelastrella sp.KE4 have a superior ability to remove N and P.The concentrations of PO43--P and total P are below the detection limit.The removal efficiencies of NO3--N,NH4+-N and total N were 47.06±1.02-68.20±1.45%,40.11±1.06-44.71±0.62%and38.42±0.98-41.89±0.86%,respectively.The biochemical composition of green algae is related to the concentration of N and P.At the end of simulated swine wastewater treatment,the concentration of N and P is low.The contents of pigment,protein,carbohydrate and lipid were 0.09±0.01-0.23±0.06%,30.27±0.95-34.82±1.63%,37.70±1.54-44.27±1.35%and 13.03±1.41%-16.74±1.35%,respectively.In order to remove N and P from swine wastewater,green algae-bacteria symbiosis systems based on green algal strains QWY28、QWY37、MA1 and KE4 were constructed,respectively,and the environmental conditions and operation mode were further studied and optimized to improve the stability and N and P removal efficiency of the green algae-bacteria symbiosis system.The green algae-bacteria symbiosis systems constructed at the initial inoculation concentration of 0.08 g/L has the highest N and P removal efficiency.It operated in the mixotrophic mode at 30oC and p H 7.0-8.0,which further improved the practical application potential of the green algae-bacteria symbiosis system.The maximum removal efficiencies of total N and NH4+-N were 70.30±3.76-96.41±2.63%and 74.82±4.56-99.56±2.56%,respectively.The concentrations of PO43--P and total P were below the detection limit.The effluent concentration of N and P in the green algae-bacteria symbiosis system dominated by strain KE4meets the discharge standard of pollutants for livestock and poultry breeding in China（GB18596-2001）,which is of research value.Therefore,in-depth sequencing was used to further study the dynamic changes of pathogenic bacteria and antibiotic resistance genes in influent,treatment stage and effluent during the treatment of swine wastewater by green algae-bacteria symbiosis system dominated by strain KE4,and to evaluate the efficiency of N and P removal,and inhibition of pathogenic bacteria and antibiotic resistance genes by green algae-bacteria symbiosis system.The results showed that Pseudomonas sp.and Acinetobacter sp.were always the most dominant,and the antibiotic resistance genes encoding efflux pump（Mac B,Mef A,Bcr A,Mex F,Mex D,Ceo B and tet C）were the dominant genes.Pathogenic bacteria and antibiotic resistance genes inactivation were induced via microalgae,driven by high dissolved oxygen conditions.In this green algae-enhanced green algae-bacteria symbiotic system,the purpose of inhibiting pathogenic bacteria and antibiotic resistance genes was achieved.The changes of biological activity during the formation of green algae-bacteria symbiosis system were further studied,the microbial community structure was analyzed,and the formation mechanism of green algae-bacteria symbiosis system was revealed.Due to the biotic interactions between the green algae and bacteria,the photosynthesis of strain KE4 is enhanced,and the abundance of genes related to green algae oxidative stress and cell growth were found to increase.There are great differences in microbial communities before and after swine wastewater treatment,and the microbial community diversity is reduced,which is conducive to the formation of a stable green algae-bacteria symbiosis system.which may come from the concentrations of dissolved oxygen,N and P,and the strain KE4 biomass.The dominant niche of Coelastrella sp.in eukaryotic community confirmed the high tolerance of strain KE4 to pollutants,which can ensure its long-term dominance and effective swine wastewater treatment.At the same time,strain KE4 induced the dominant niche of Stenotrophomonas sp.and Sphingobacterium sp.related to N and P removal,and promoting the growth of green algae.Network analysis illustrates the formation process of green algae-bacteria symbiosia system.That is,in the early stage,it is mainly probiotics of green algae,the middle stage is mainly the bacteria related to N and P removal,and in the later stage,green algae and symbiotic bacteria achieve ecological balance.Finallt,the relationship between green algae and other microorganisms in green algae-bacteria symbiosis system and the contribution of green algae and bacteria to N and P removal were analyzed,and the mechanism of N and P removal in green algae-bacteria symbiosis system was clarified.The high removal efficiency of N and P was mainly related to green algae.Due to the biotic interactions between the green algae and bacteria,the removal efficiencies of N and P in green algae-bacteria symbiosis system is higher than that in green algae only group.The green algae-bacteria symbiosis system mainly removes N and P through assimilation.The assimilation ratios of green algae to total N,NH4+-N and total P were 61.73%,about 54%and 89.40%,respectively.The remaining 38.27%of N was mainly removed by bacterial nitrification and denitrification.High concentration of dissolved oxygen,C deficiency and light inhibited the denitrification process,while O2 induced nitrification removed about 32%of NH4+-N.It shows that green algae play a positive role in nitrifying bacteria,which makes green algae-bacteria symbiosis system have strong synergistic N and P removal ability. |
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