| Anaerobic digestion(AD)can recover organic matter from excess sludge,provide renewable biogas energy,and realize the harmlessness,reduction and recycling of sludge.However,there are still some problems,such as low organic matter conversion rate and slow anaerobic digestion treatment rate.High temperature thermal hydrolysis technology can accelerate the hydrolysis step and improve the reaction rate.However,the released dissolved humic acids(HAs)will affect the AD process.With the large-scale application of temperature thermal pretreatment technology,the effect of humic acids on sludge AD cannot be ignored.In addition,the hydrolysis of bacteria,lignocellulose and humic acid in sludge greatly affects the efficiency of AD and transformation of sludge.Among them,humic acids are more special.It not only cannot be biodegraded,but also affect the degradation and transformation of other organic substances in the process of AD.In order to solve these problems,it is very important to study the effect of humic acids on sludge AD.Therefore,in this paper,the effects of different concentrations,types and molecular weights of humic acids on sludge AD were studied,the effects of humic acids on the function of biological enzymes,the kinetics of humic acids inhibiting alkaline protease and its effects on the activity and structure of alkaline protease were explored.The main research contents and conclusions are as follows:(1)Humic acids have a significant inhibitory effect on sludge anaerobic digestion and biological enzyme activity,which is closely related to its concentration and type.Three kinds of humic acids were selected in the experiment:humic acids purchased commercially(called SAHA),humic acids extracted from sludge(called WNHA)and humic acids extracted from hydrolysate after thermal hydrolysis at 180℃(called THHA).All three types of humic acids contain phenolic functional groups.Most of the functional group bands of SAHA appear in the short-wave number range,and mainly contains phenols,phenolic carboxyl groups and phenolic hydroxyl groups.While THHA and WNHA mainly contain aliphatic and alicyclic substances.WNHA has the highest humification degree and the highest aromatic condensation degree.It was also found that the higher the concentration of humic acids,the stronger the inhibition on acid production.This is due to the combination of humic acid and alkaline protease,which inhibits its activity,so that the protein cannot be completely hydrolyzed.All three types of humic acids inhibited methane production at high concentration,but low concentration of WNHA promoted the cumulative methane production.Humic acids had no significant effect on F420 enzyme activity,but had a significant inhibitory effect on acetyl CoA synthase(ACS)activity.In general,humic acids can inhibit both acidogenic and methanogenic processes,and reduce the activities of specific hydrolases and key methanogenic enzymes.(2)There were significant differences in the effects of humic acids with different molecular weights on sludge fermentation acid production and methane production.The distribution weight of three selected humic acids in different molecular intervals is different,and the structure of humic acids is significantly related to their molecular weight.The structural characteristics of humic acids in small molecular interval are relatively less content of functional groups,higher E4/E6 value and stronger aromaticity.With the gradual increase of molecular weight,the hydrogen content gradually increases,the nitrogen content first decreases and then increases,the H/C ratio increases from 0.81 to 1.01,and the E4/E6 value gradually decreases from 13.60 to 5.70.The result showed that high molecular humic acids could significantly reduce the activity of alkaline protease and inhibit the decomposition of soluble protein.While small molecular weight humic acid had no significant effect on the degradation of soluble protein.Humic acids with molecular weight less than 30 kDa significantly can inhibit methanogenesis,and the highest inhibition rate can reach 92.5%,which may be caused by the inhibition of ACS activity.Humic acids in each molecular interval had little effect on the concentration of soluble polysaccharides and the activities of glucosidase and F420 enzyme.It can be seen that the structures of humic acids with different molecular weights and their effects on anaerobic digestion and biological enzymes are significantly different.(3)The mechanism of humic acids affecting biological enzyme activity is that they combine with biological enzymes,resulting in changes in enzyme particle size,environmental polarity and structure of amino acids.The reaction kinetics of alkaline protease under the action of humic acids showed that there was a close and slow binding process between humic acids and enzyme,and this process was reversible.In order to understand the structural changes of humic acids combined with alkaline protease,the particle size distribution of alkaline protease and humic acids complex was studied.By analyzing the particle size distribution of protease and humic acid complex,it was found that the particle size of the complex increased significantly.The combination of humic acids and alkaline protease changed the microenvironment near the tryptophan residue of protease,the environmental polarity of the residue increased,the hydrophobicity decreased,and the structure of protease became loose.In addition,by analyzing the fluorescence spectra of three humic acids combined with alkaline protease,it was found that humic acids had fluorescence quenching effect on the specific fluorescence peak of alkaline protease,and the fluorescence quenching effect was directly proportional to its concentration.The FTIR spectra of the complex were analyzed.It was concluded that the coating effect of humic acids on alkaline protease masked its infrared absorption peaks at 1540 cm-1 and 1650 cm-1,resulting in the structural changes of carbon chain skeleton,amide I band and amide II band.It can be seen that humic acids and enzyme can combine reversibly and closely,form large particle size complexes,and affect their structure and main functional groups. |
