Parameters Optimization And Process Analysis Of Humic Acid Bioconversion

Hunic acid(HA) is abundant and ubiquitous in nature, it hasn’t been used effectively because of the complex structure and macromolecular. Fulvic acid(FA) is a group of humic substances with smaller molecular, more simple structure, more active groups and higher physiological activitycomparing with HA, and it is soluble in water. Due to FA could be used more easily, obtaining FA from HA is a way to broaden the use of resources. Comparing with physical and chemical methods, microbial transformationissafer and more reliable, and the production process is more environmental.This article researched on the lignite-derived HA with white rot fungus AH.First of all, the growth characteristics and enzymatic properties of WRF-AH were studied. Growth cycle of fungus AH was researched with SMB liquid medium. Fungus AH was moving into logarithmic phase on the third day after inoculation and the stable phase started from the 6th day. The tests with Tannic acid, guaiacol and Azure-B solid culture separately showed fungus AH could release ligninolytic enzymes, In the meantime, It came to a conclusion through the quantitative experiment that the enzymatic activity of Mn P was the highest, Li P came second and lowest with Lac. Instead of being inhibited, the production of peroxidase and polyphenol oxidasewas accelerated by humic acid in the control test.Secondly, liquidation of HA with PDA, SDA and SMA solid medium was investigated, the results showed that HA could be liquidated only in the SMA solid medium. Some of other results revealed that 1 g of purified HA added into a petri dish on the third day after inoculation could be better liquidated.Biotransformation control tests of the HA before and after purificat ion in SMB medium was studied, the result showed the content of FA converted from the purified HA was highly improved, approximately two times than before being purified, at the same time, with the increase of FA the degradation rate of HA was also increased.Elemental analysis, Fourier transform cyclotron resonance(FTCR) and 1HNMR were employed to the primary research of the humic acid transformation process with WRF-AH, as well as the tests of acidic groups and E4/E6. The purified HA, liquefied product and residue were tested. The Elemental analysis revealed a decrease in the C percentage and an increase in the O percentage, meanwhile, the 1HNMR analysis showed the signals of oxygen-containing functional groups in the product were enhanced, indicated the occurrence of oxidation reaction of HA was caused by fungus AH during the biotransformation process. The increase of H percentage was also being observed in the product, suggested hydrogenation reactiontook place, consequently decreased the unsaturation of product. Based on the ratios of FTCR absorbance and the results of acidic groups detection, it was shown that the carboxyl reduced, which would be attributed by the fungus AH. According to the FTCR spectra, 1HNMR spectra and the E4/E6 ratio, there were more aliphatic chains and less aromatic structure in the product, which indicated the ring opening reaction of some of the aromatic structure, and then would be further converted and used. The E4/E6 value of residue was smaller than that of HA, indicated that aromaticity of residue increased along with the increase of molecular weight, this suggested that HA was not only depolymerized but also polymerized by fungus AH.At last, optimization of biotransformation from HA to FA with fungus AH was studied. Based on SMB liquid medium, three major effect factors sifted among 9 factors of additive amount of nitrogen source, Cu2+ concentration, Mn2+ concentration, guaiacol concentration, culture temperature, initial p H, rotation speed, inoculum dosage and additive amount of HA, they were additive amount of nitrogen source, Cu2+ concentration and additive amount of HA. Then Box-Benhnken experiment was designed with Design-Expert 8.0.6. It was confirmed through the analysis of quadratic regression and response surface that the optimum conversion conditions were 0.29 mmol/L of Cu2+ concentration, 0.68 g/80 ml of additive amount of HA and 12.68 g/L of additive amount of nitrogen source. The predicted value of the maximum FA content was 14.16 mg/ml. In the verification test, the maximum FA content was 13.35 mg/ml which was quite close to the predicted value.