Research On Optimizing Fungal Flocculation With An Indigenous Chlorella Vulgaris From Swine Sewage And Its Application In Undiluted Sewage Treatment

Microalgae are considered as potential renewable biodiesel feedback,due to their fast growth rate,high oil yield,no land competition with farmland and high carbon dioxide fixation rate.To date,microalgal biodiesel has not been widely used commercially due to its high expense of algae cultivation and harvesting process comparing with traditional petrochemical diesel.The use of swine sewage for low-cost microalgae biomass production is likely to be one of the most viable strategie to reduce the production cost of algae-based biodiesel.However,an urgent problem to be solved is that how to effectively harvest microalgae cultured in swine sewage by a simple and low-cost method.Among the develop harvesting methods,fungal pellet-assisted microalgal harvesting methods has the following advantages:(1)Fungal pellet possesees high flocculation efficiency,short havest time,and easy to solid-liquid separation,so the energy consumption of havesting process can be further reduced;(2)Fungal pellet is widely applied to the bioremediation of heavy metal sewage and swine sewage,showing strong resistance that can maintain the high flocculation activity in swine sewage and improve the nutrients removal efficiency.In this paper,through indicators including flocculation efficiency,biomass and fatty acid yield,a pellet-forming filamentious fungus,Aspergillus oryzae FSD181214,with high flocculation efficiency on Chlorella vulgaris MBFJNU-1 which can be cultured in primitive swine sewage was screened among six fungi.The optimal condition conditions for algal-fungal flocculation in BG11 medium,sterilized swine sewage and non-sterilized swine sewage culture system were optimized using single-factor analysis.The kinetic model of flocculation time and flocculation efficiency under the optimal conditions was established and the flocculation process and the purification effects of the three kinds of algal-fungal pellets were further studied.Eventually,the biodiesel properties from the oils of fungal pellets,microalgae,flocculated algal-fungal pellets and the collected algal-fungal pellets after swine sewage treatment were predicated based on the compositions of fatty acid in those samples.The main contents and results of this paper are listed below:1.Six filamentous fungi strains isolated from corrupt plant stems,and their biomass,fatty acid content,fatty acid yields and the flocculation efficiency of fungal pellets on Chlorella chlorella MBFJNU-1 cultured in BG11 medium,sterilized swine sewage,and unsterilized swine sewage was compared and analyzed,from which a dominant fungi was selected for a further round of flocculation tests.Phylogenetic analysis of ITS gene sequences and morphological identification confirmed that it is Aspergillus oryzae(FSD181214).The biomass of Aspergillus oryzae pellets cultured in PDB medium was7.55 g/L after 60h cultivation,and the fatty acid yield was 0.78 g/L.The flocculation efficiency of A.oryzae assist chlorella MBFJNU-1 havesting from BG11 medium,sterilized swine sewage and unsterilized swine sewage could reach 99.96%,91.89%and96.60%respectively.It also exhibits outstanding flocculation activity with C.protothecoides,Scenedesmus,Porphyridium sp and Pseudochlorococcum sp.For Chlorella protothecoides and Scenedesmus,the flocculation efficiency was higher than 90%after 6h co-culture,for Porphyridium cruentum and Pseudochlorococcus the flocculation efficiency was higher than 85%within 6h.2.In order to improve the flocculation efficiency of A.oryzae pellets assist Chlorella vulgaris MBFJNU-1 havesting and futher shorten the harvesting time,the fungal pellets culture stage parameters(fungal cultivation time,spore inoculums size)and the co-cultivation stage parameters(p H,temperature,agitation speed,dry weight ratio of fungal to algae)were optimized.The results showed that in BG11 culture system,the fast and efficient harvesting efficiency could be obtained under the optimal conditions:p H 5.0,temperature 31?,agitation speed 140rpm,dry weight ratio of fungal to algae 2:1,fungal pellets culture time 60h,and inoculum size 1.5×10⁵spore/m L(pellet diameter was2.04mm);In the culture system of sterilized swine sewage,the optimal flocculation conditions were:p H 4.75,temperature 37?,agitation speed 140rpm,fungal-algal ratio2:1,and spore inoculation 1.5×10⁵ spore/m L(pellet diameter 2.04 mm).In unsterilized swine sewage system,the optimal flocculation conditions were:p H 4.75,temperature31?,agitation speed 120rpm,fungal-algal ratio,dry weight ratio of fungal to algae 2:1,fungal pellets culture time 60h,and inoculum size 1.5×10⁵ spore/m L(pellet diameter5.56mm).Under the optimal conditions,the flocculation efficiency of Aspergillus oryzae in culture systems of BG11,sterilized swine sewage and unsterilized swine sewage could reach 96.94%,94.26%,91.74%in 3.5h and 99.66%,98.97%,98.23%in 6h,respectively.The kinetic model was used to fit and analyze the flocculation data under the optimal conditions,and it was found that the model fitting goodness R² of the flocculation curves about the three media systems could be above 0.98 by Hill equation.The results therefore suggested that Hill equation could fit well with the flocculation curves and the relationship between flocculation time and flocculation efficiency under the optimal conditions could be quantitatively described by Hill equation.3.The effects of Aspergillus oryzae pellets assist Chlorella vulgaris havesting from primitive swine sewage in response to the treatment days of swine sweage purification by Chlorella vulgaris were studied,and the nutrients profile of swine sewage before and after bio-flocculation were compared and analyzed.The results showed that fungal pellets have flocculation capacity in both sterilized swine sewage and non-sterile swine sewage with different treatment days,and the longer treatment days could achieve the higher flocculation efficiency.At the end of flocculation process,It was found that concentrations of turbidity,SS(solid suspended solids),COD,TN,TP,and NH₃-N in swine sweage in different treatment days all were decreased significantly except the unsterilized swine sewage after 5 days treatment,which COD concentration showed a slight increase.The results showed that the fungal pellets assist C.vulgaris flocculation could further improve the purification effect of swine sewage.4.The harvested fungi-algae pellets were further reused as efficient bioremediation agents for primitive swine sewage disposal using a two-step culture method.The results showed that the NH₃-N,TN,turbidity,and solid suspended solids removal of SSW(sterilized swine sewage)fungi-algae pellets group and USW(unsterilized swine sewage)fungi-algae pellets group in sterilized swine sewage could reach 91.02%,87.67%,35.03%,76.03%and 98.70%,84.31%,85.33%,69.03%respectively after 15days treatment.In unsterilized swine sewage,after 15days treatment,the NH₃-N,TN,turbidity,and solid suspended solids recovery were 98.25%,79.10%,35.03%,81.82%and 96.97%,77.77%,73.78%,77.27%respectively.Compared with single algae treatment and BG11fungi-algae pellets group,it has obvious advantages in recovery of NH₃-N,TN,turbidity,and solid suspended solids under primitive swine sewage.5.The gas chromatographic technique and mathematical model were used to evaluate the properties of biodiese from the oils of A.oryzae,C.vulgaris MBFJNU-1cultured in three mediums(BG11,sterilized swine sewage and unsterilized sewage)and their fungal-algae pellets.Except for the iodine value(IV)of C.vulgaris MBFJNU-1 does not meet the requirements of European standard(EN 14214),the fatty acids in the remaining experimental groups all meet the requirements of American biodisel standard(ASTM D6751-08)and European standard(EN 14214)for Polyunsaturated fatty acid composition,Cetane number(CN),Kinematic viscosity(Kv),Cloud point(CP),Iodine value(IV),Higher heating value(HHV).