Screening And Optimization Of Endogenous Alkaline Protease And Its Effect On Production Of Volatile Fatty Acids By Anaerobic Fermentation Of Sludge

Sludge is a by-product of wastewater treatment.With the rapid development of industrialization and urbanization,the discharge of the sludge has increased significantly,which posed a great threat to the environment and human health.In this context,the resource utilization and reduction of the sludge has been a common challenge facing mankind.Anaerobic digestion（AD）is a common method for the treatment of the sludge,which effectively convert organics into valuable products while removing odor and pathogens.Especially,volatile fatty acids（VFA）was a high value-added intermediate products of AD and VFA production from the sludge have attracted widespread attention.However,the complex structure of extracellular polymeric substance（EPS）and strong cell walls limited the overall rate of AD,which is not conducive to large-scale sludge treatment.Hence,how to accelerate the disintegration of EPS became one of the current research focus.Enzymatic treatment has greater prospects due to its less by-products and mild operating conditions.Protein is the main organics in sludge,so it’s reasonable that protease could accelerate the hydrolysis of sludge.However,the use of commercial protease was not economically feasible due to its instability in sludge environment.Therefore,in this paper,we have screened out an endogenous protease stable in the sludge environment and determined its basic enzymatic properties.In additon,we have explored the effect of the addition of the enzyme on VFA production.It was expected to provide a theoretical basis for the enzymatic treatment of the sludge and VFA production.Firstly,we screened a protease-producing bacteria from the sludge,whose colony was off-white,with a round or irregular surface with folds.Combined with the physio-biochemical characteristics and 16S r DNA sequences,the bacteria was identified as Bacillus amyloliquefaciens HJ1.According to response surface methodology,casein and initial p H had significant effects on enzyme production,and the optimal fermentation medium composition was determined as follows:casein 4 g/L,soluble starch 20 g/L,KH₂PO₄4 g/L,Mg SO₄·7H₂O 1 g/L,CMC 4 g/L,initial p H 11.3.The yield of protease in the optimal medimue was up to 713 U/m L,which was about 15 times higher than the initial medium.Further,the protease was purificated and it was determined as a serine metalloprotease with molecular weight of 30 k Da.It had the highest activity at p H 9.3 and 50℃,and was stable in the range of p H 7.0-11.0 and 30℃-50℃,which indicated that it belonged to alkaline protease.Sencondly,the stability of the screened protease in the sludge was investigated.The result showed that the protease remained a relatively high activity（33%）within 72 h.Due to the high tolerance of the protease to the sludge environment,it could maintain high efficiency of sludge hydrolysis for a long time.thus effectively increasing the degree of hydrolysis.Soluble chemical oxygen demand（SCOD）of sludge increased rapidly from 3759.38 mg/L to 5112.75 mg/L within 12 h,signifying the improved biodegradability of the sludge.Thirdly,the screened protease was applied to promote VFA production by AD of the sludge.The addition of the protease destroyed the internal structure of the sludge（EPS and Pellet）,causing the inner organic matter transfered to the outer layer（supernatant）.In addition,it promoted the degradation and transformation of protein in the sludge.Protein in the supernatant rose rapidly from 220.76 mg/L to 884.92mg/L within 2 d and NH₄⁺,as a product of protein degradation,increased from 16.56 mg/L to 218.00 mg/L in the end of AD.The addition of the protease elevated the yield of VFA from 1397.66 mg COD/L in the control group to 1778.16 mg COD/L.Additionally,it caused significant changes of the community structure of the sludge,which bring down the high dominance of individual microorganism in the community.The addition of the screened protease increased the abundance of hydrolytic functional bacteria Bacill,Exiguobacterium and Saccharibacteria and the relative abundance of Clostridia（Firmicutes）responsible for VFA production was raised.