The Performance Of Ecological Microflora Constructed For The Anaerobic Treatment Of High Organic Salinity Wastewater

High salt organic wastewater mainly comes from related industries such as chemical,pharmaceutical,food,and pesticide.This type of wastewater not only contains organic pollutants,but also contains high concentrations of soluble inorganic ions(Cl^-,SO₄^2-,Na⁺,Ca²⁺).The wastewater from the production of heparin sodium is a typical high salt organic wastewater,with a salinity usually above 2.5%and a high COD concentration（mainly based on protein derived organic matter）.The anaerobic biological approach is better suited for the treatment of organic wastewater with a high concentration,but how to improve the treatment capacity in high salt environment is crucial.Based on the concept of bioaugmentation,constructing a salt-tolerant ecological microbiota to improve treatment efficiency is one of the feasible options,but there are currently few relevant research reports.In view of this,this paper takes simulated heparin sodium wastewater as the research object.Firstly,an anaerobic microbial community with certain salt tolerance was obtained through a"top-down"method of microbial community domestication.Regarding the inhibition of protein hydrolysis under high salt stress,targeted screening of salt-tolerant protease producing bacteria and investigated their metabolic characteristics at varying levels of salinity.Based on this,a new ecological microbial community was constructed by combining salt-tolerant protease producing bacteria with domesticated bacteria,effectively improved the anaerobic treatment efficiency of heparin sodium production wastewater.The main research findings of the paper are as follows:（1）Based on the"top-down"method of microbial community domestication,an anaerobic ecological microbial community that can tolerate 3.2%salinity was successfully constructed by gradually increasing salt stress pressure.Under operating conditions with a salinity of 3.2%,the salt-tolerant anaerobic bacterial community achieved an average protein degradation rate of 90.11%,an average COD removal rate of 80.21%,and an average biogas production intensity of 318.68 L/m³/d,respectively.When the salinity increased from 3.2%to 3.6%,the COD removal rate gradually decreased to52.50%-55.20%,and the protein concentration in the effluent increased by nearly 1.08 g/L,while the VFAs concentration only increased by about 0.49 g/L,and the protease activity significantly decreased from 116.15 U/m L to 89.05 U/L.Therefore,it can be inferred that at a salinity of 3.6%,The methane generation pathway was less severely hampered than the protein hydrolysis stage,which eventually became the rate-limiting phase.The results of microbial high-throughput sequencing showed that salt stress had a more significant impact on the bacterial population than the archaea population.After salinity gradient domestication,unclassified＿Bacteroidetes、unclassified＿Clostridiales、unclassified＿Porphyromonadaceae and Acetoanaerobium are dominant bacterial populations.Metanothrix,Metalomassilicoccus,and Metanobacterium are dominant populations of archaea.（2）To address the issue of protein hydrolysis inhibition under high salinity stress,salt-tolerant bacteria with high protease production were selectively screened from a domesticated bacterial population that can tolerate 3.2%salinity.Firstly,16 strains were isolated and purified on a peptone solid culture medium with a salinity of 4.0%.After initial screening through transparent circles and gelatin liquefaction experiments,as well as rescreening through salt tolerance experiments,three protease producing bacteria（A1,A2,A7）that can tolerate 8.0%salinity were ultimately obtained.According to molecular biology identification,the most similar strain among these three strains is Arthrobacter sp.,but their sequences are not completely the same.The results of shake flask experiments at different salinity levels showed that as salinity increased,the growth performance and protein degradation rate of each strain decreased.At 2.0%salinity,the protein degradation rates of A1,A2,and A7 were 45.41%,43.64%,and 41.45%,respectively;When the salinity increased to 8.0%,the protein degradation rate decreased to 26.04%,21.51%,and 20.40%,respectively.In addition,the protease activity and bacterial biomass under various salinity conditions generally reached their peak on the second day,indicated that the production of proteases from the three strains was coupled with cell growth.（3）The combination of salt-tolerant protease producing bacteria and domesticated bacterial communities to construct a new ecological microbial community effectively improved the anaerobic treatment efficiency of heparin sodium production wastewater.Under operating conditions of 3.6%salinity,the COD removal rate gradually increased from 54.00%to 76.50%;Furthermore,by further increased the salinity to 4.6%,the COD removal rate under steady-state conditions can still remain above 70%.Within the stress range of 3.6%-5.2%salinity,the protein degradation rate can reach over 90%.However,under operating conditions of 5.2%salinity,the COD removal rate significantly decreased to 36.00%-39.60%,and the steady-state VFAs concentration increased to between4.07-4.50 g/L.It can be seen that the introduction of salt-tolerant protease producing bacteria can effectively solve the problem of protein hydrolysis inhibition under high salt stress,but the methane production stage has become a speed limiting step.The effect of salinity on the methanogenic pathway of ecological flora was further analyzed using sodium acetate and H₂/CO₂as the sole carbon sources,respectively,and the results showed that salinity had a more significant inhibition on the acetic acid type methane production pathway.The introduction of salt-tolerant protease producing bacteria changed the dominant population structure of bacteria and archaea in the reactor.The anaerobic bacterial community was also prompted by higher salinity to generate more bound extracellular polymers（B-EPS）to shield the cells.