Bioaugmentation Of 3, 5-DNBA Degrading Bacteria And Biofilm-Forming Bacteria For Treating The Wastwater Containing Nitroaromatic Compounds

Microorganisms that can degrade xenobiotic,such as nitroaromatic compounds are quite scarce in natural environment.The study of bioaugmentation of adding indigenous or exogenous wild type microorganisms or genetically engineered microorganisms into contaminated waters has made some achievements in laboratory scale.But in reality,very often,due to the flowing-off of degrading bacteria,the bioaugmentation can not be remained.This paper is to explore the bioaugmentation strategies with the mixed inoculant of biofilm-forming bacteria and degrading bacteria to approach new ways of immobilizing degrading bacteria into the wastewater treatment system.The soil and sediments contaminated by nitroaromatic compounds and some other chemicals for a long time were inoculated into a bioreactor full of synthetic wastewater with nitroaromatic compounds.One 3,5-dinitrobenzoic acid-degrading bacterium A3, isolated from the wastewater treatment system which operated one month,was identified preliminarily as Comamonas testosteroni based on its morphological characters, physiological and biochemical analyses and 16S rDNA series same source analysis.This bacterium degradation rate of 12h could reach above 95%,using 200mg/L 3,5-dinitrobenzoic acid as sole carbon source.24h dissolved organic carbon(DOC) was reduced from 72.5mgC/L to 10.2mgC/L,indicated that 3,5-dinitrobenzoic acid was degraded through mineralization but not transformed into other organic matter.But the strain A3 transformed 3,5-dinitrobenzoic acid into a yellow product,cultivated liquor was not discolored all along in the next several days,when 200mg/L 3,5-dinitrobenzoic acid was used as the sole carbon and nitrogen sources.DOC being 56.5mgC/L in 24h,indicated that the yellow product was not further degraded easily.The suitable temperature for A3 degradation of 3,5-dinitrobenzoic acid was 20-30"C and pH value was 5-9.A total number of 18 bacterial isolates were obtained from the biofilms of wastewater treatment systems and of little carpolite in soil.Results of their in vivo detection showed a great difference in their abilities to form biofilms:the bacteria with the strongest biofilm-forming capacity can form biofilms biomass 28 times stronger than that formed by the weakest.The phylogeny affiliation of those isolates showing high biofilm formation capacity has been determined through a 16s rDNA sequencing and they were grouped into 7 bacterial species including Pseudomonas sp.,Pseudomonas putida,Aeromonas caviae, Bacillus cereus,Pseudomonas plecoglossicida,Aeromonas hydrophila and Comamonas testosteroni.Selected biological characteristics that are potentially related to biofilm forming capacity were investigated and the relative importance of these biological properties in biofilm formation was statistically assessed by path analysis.According to the coefficient of determination,the relative importance of the five biological characteristics to biofilm formation was in the order from greatest to least:exopolysaccharide＞flagella＞N-acyl-homoserine lactones(AHLs) signaling molecules＞extracellular protein＞swarming motility.In order to investigate the distribution of the degrading bacteria and biofilm-forming bacteria in a dual-species biofilms or in the biofilm of the wastewater treatment system and immobilization of A3 in biofilms,C.testosteroni A3,P.putida M9 and A.hydrophila M22 were tagged using fluorescence labeling,pTR102gfp plasmid in E.coli DH5a was introduced into C.testosteroni A3,P.putida M9 and A.hydrophila M22 by triparental mating and pJZ402rfp plasmid was introduced into C.testosteroni A3 by electroporation. Fluorescent measurement shows that both the bacteria and the colonies have bright green or red fluorescence.The degrading ability,the biofilm biomass,the resistance of biofilm to shock loading and the fxation of A3 were investigated in a bioreactor with mixed inoculation of high biofilm-forming bacteria and degrading bacterium A3.The results showed that the biofilm biomass formed by all the mixed inoculation was 1.76-5.95 times higher than that of the single inoculation of A3.The dual-species biofilms,A3 with M9 and A3 with M22,had a strong resistance to 3,5-DNBA shock loading during successive replacement of DCMM2 synchetic wastewater(3,5-dinitrobenzoic acid mineral medium containing 2%LB broth), and degradation rate reached 63.3～91.6%and 70.7～89.4%,in 6 h after each time of replacement.It was found that the colony forming units(CFUs) the total and A3 cells of biofilms formed by A3 with M22 or M9,were significantly higher than those of other three dual-species biofilms.The relatively high quantity of degrading strains in biofilm also demonstrated that strain A3 can be well maintained in these two dual-specises biofllms over time.Thus,strain M9 and M22 did enhance the degrading strain A3 fixed.Therefore,it seems feasible to use some specific biofilm-forming bacteria as an viable option for bioaugmentation to enhance immobilization of degrading bacteria in biofilm in an engineering setting,and the self-immobilization may also help maintain a durable and stable degradation capacity.Investigation of the effects of different nutrient status on the mixed inoculation of A3 with M9 or M22 showed that different amounts of LB broth added into 3,5-dinitrobenzoic acid mineral medium(DCMM) had no significant effects on the degradation rates of 3,5-DNBA,but the increase of LB broth from 0%to 5%,the nutriment enhanced the formation of biofilms greatly.The results from flow cytometer showed tnat the ratios of fluorescing bacteria in dual-specises biofilm of A3 with P.putida M9(pTR102gfp) and A3 with A.hydrophila M22(pTR102gfp) enhanced with the increase of LB broth.The P. putida M9(pTR102gfp) and the A.hydrophila M22(pTR102gfp) on the biofilms increased from 43.8%to 82.3%and from 39.2%to 79.6%,respectively.The distribution of the two bacteria within the biofilms and the three-dimensional differentiation observed by confocal laser scanning microscopy(CLSM) showed that A3 was homogeneously fixed in the biofilms;The overall evaluation indicated that 2%LB of addition to DCMM wastewater was the best to obtain a higher biofilm biomass and a higher percentage of degrading bacteria immobilized in the biofilms.The above investigation on the biofilm formation by the mixed culture of two functional bacteria carded out in the laboratory conditions was of significance in practical treatment of the wastewater by regulating the nutrient composition to promote immobilization of the degrading bacteria.If the bacteria with high biofilm-forming capability are able to enhance the self-immobilization of the degrading bacteria,this immobilization might make the degrading abilities run in a long term.The bioaugmentation of derading bacterium A3 and biofilm-forming bacteria M9 was detected in an 8L bioreactors.The results show that 3.5-DNBA can be quickly degraded in the reactor 1 with A3 and M9 and the reactor 2 with only A3,and in the following 35 days,the degradation is well remained.The results from the culture of selecting agar plates and DGGE detection demonstrated the existence of degrading bacterium A3 within the biofilm.In the reactor 3 with the only activated sludge from the municipal wastewater treatment factories,almost none of 3,5-DNBA has been degraded within first two weeks.However,after 15 days the degradation rate of 3,5-DNBA was greatly increased.And after 21 days the degrading rate from reactor 3 was no more different from the ones from reactor 1 and reactor 2.DGGE profles of the biofilm of reactor 3 are lack of the band similar to Strain A3,demonstrating that the aboriginal microorganisms can degrade 3,5-DNBA if they had been cultured for 2～3 weeks.On the 35th day,on the DGGE pattern of biofilms in those two reactors into which M9 had not been added,bands that were similar to Strain M9 were found.And this showed that M9 might exist in inoculated sludge or environment and it had quite high capability to form biofilm.