Study On Chloroaniline Removal By Aerobic Graules And High-Efficiency Chloroaniline-Degrading Strains

Aerobic granular sludge was successfully developed in a sequencing batch reator (SBR) with flocculent activated sludge as the seeding sludge and CAs as a sole carbon and nitrogen sources via controlling the operation parameters. SEM observation indicates that the mature granules consisted of a wide variety of bacteria, mainly including brevibacterium and cocci-like bacterium. After the granulation, the influent concentration of CAs was increased to 400mg/L, the removal efficiencies of 2-CA, 3-CA, 4-CA and TOC by the aerobic granule system were close to 85%, 100%, 100%, and over 80% respectively. Chloride anion removal up to 83% was achieved, indicating aerobic granules possessed high removal and mineralization efficiencies on CA compounds. This system could effectively degrade CAs as high as 800mg/L.PCR-DGGE fingerprints indicated that the high-efficiency aerobic granules were inhabited by a wide spectrum of bacterial species with stable community structure during the stable operation period. The sequencing results showed that the dominant organisms responsible for CAs degradation closely related to Ralstonia,Pseudomonas,Acidocella,Dyella,Comamons and Acidovorax.A novel bacterial strain (H1), able to degrade 2-,3-,and 4-CA compounds was isolated from a CA-degrading the developed aerobic granules. Based on analysis of the strain's morphological characteristics, physiological and biochemical characteristics, 16S rDNA sequences and the Biolog identification, this strain was identified as Delftia which was possibly a new species able to degrade 2-, 3-, and 4-CA compounds. The optimal conditions for the growth of the strain were at 30℃and pH 7.0. Yeast extract could remarkably promote the growth and chloroaniline-degradation rate of H1. In addition, an increasing growth rate of H1 was recorded with the increasing addition of YE in the cultures. The additions of citrate or succinate appeared to accelerate strain growth rate and CAs degradation. In contrast, NH₄Cl and aniline strongly inhibited the CAs degradation. 3-CA and 4-CA could be degraded efficiently by H1 when their concentrations were lower than 600mg/L, while 2-CA'tolerance concentration is only 100mg/L to the H1. Following CA consumption, chloride anion, NH₄⁺-N and NO₃^- were released simultaneously and stoichemically to CA degradation during the degradation. When CA compounds were degraded effectively, chloride anion concentration was close to the theoretical concentration, indicating H1 possessed high removal and mineralization efficiencies on CA compounds. At the same time, the changes of TOC/TOC0 and carbon dioxide concentration also show that the strain H1 could degraded CAs completely. Carbon balance illustrated that the carbon recovery rate of more than 85% was obtained when CAs were biodegraded completely, indicating all components of the mixture were simultaneously degraded and further mineralized or incorporated into cells by this isolate.The utilization patterns of CA compounds in a mixture showed that H1 had high removal efficiency for 3-CA and 4-CA than that for 2-CA. The biodegradation rate of these compounds was 3-CA>4-CA>2-CA. The degrading process of single substrates by strain H1 followed Andrew kinetic model. In multiple-substrate studies, the degrading process of CAs by the strain followed the SKIP kinetic model. When 3-CA and 4-CA co-existed, competition inhibition interactions were observed. Meanwhile the presence of 3-CA and 4-CA had an enhancing effect on 2-CA degradation. In contrast, 2-CA exhibited a slightly inhibitory effect on 3-CA and 4-CA when they existed as a mixture. The decay codfficients and yield codfficient of CA degradation by H1 in all possible combination followed the following orders: 2-CA+4-CA>2-CA+3-CA>4-CA>2-CA+ 3-CA+4-CA>3-CA+4-CA>3-CA, 3-CA>3-CA+4-CA>2-CA+3-CA+ 4-CA>4-CA>2-CA+3-CA>2-CA+4-CA, respectively.To investigate which degradation pathways are responsible for the degradation of CA compounds by the strain H1, the enzymes involved in the meta-, ortho-, and modified ortho-cleavage pathways were determined in crude extracts prepared from cells grown on single CA compounds or a mixture. For all the extracts made of cells grown on CA compounds, the activities of C12O (catechol 1,2-dioxygenase) and CC12O (chlorocatechol 1,2-dioxygenase) involved in the ortho-cleavage pathway and modified ortho-cleavage pathway were significantly high, while the activities of the enzymes of meta-cleavage pathway including C23O (catechol 2,3-dioxygenase) and CC23O (chlorocatechol 2,3-dioxygenase) would be negligible under the similar conditions. These results indicate that CAs were metabolized by H1 mainly through ortho-cleavage and modified ortho-cleavage pathways.