| Waste activated sludge(WAS) is the by-product of wastewater treatment plants, 30 million tons was produced in China in 2011, and its production is still increasing with urbanization and population growth, which will rise up to 60 million tones in 2020(80% moisture content). It is well known that the predominant components of WAS are organic matters(protein, carbohydrate and lipid, etc.). This obviously suggests that it has a great potential to be utilized as renewable resource. Currently, as anaerobic digestion technology can simultaneously achieve resource recovery and WAS treatment, it is widely used for WAS treatment all over the world. However, most of organic matters in WAS are either enclosed in the microbial cell or enmeshed in the extracellular polymeric matrix, leading to lower digestion rate and resource efficiency. Thus, enhancing the hydrolysis of WAS is one of the key factors to improve its biodegradation and further enhance resource recovery. Both mesophilic and psychrophilic bacteria would either be dead or lose their enzymatic activities at 60 °C, but thermophiles can maintain stable extracellular enzyme activities even at higher temperatures. Based on this, a study was carried out by successfully isolating thermophiles from WAS by thermophilic acclimation, which can hydrolyze WAS. The lytic performances of WAS by thermophiles were investigated, meanwhile, combined pretreatment methods for further enhancement of lytic activities of WAS and short-chain fatty acids(SCFAs) accumulation were established, which offer the technical and theoretical guidance for WAS treatment.Three strains of thermophiles(G1, G2 and G3) were isolated from WAS at 60 °C, which had protease activities. In that, strains G1 and G2 were members of the Geobacillus, named Geobacillus sp. G1(Accession No. JX522538), and Geobacillus sp. G2(Accession No. KJ190160), and G3 was a member of the Aneurinibacillus, named Aneurinibacillus sp. G3(Accession No. KJ190161). Among the three strains of thermophiles, Geobacillus sp. G1 showed the best performance in lysis of E.coli, which was selected as the experimental strain in this study. Uniform design and response surface methodlogy(UD-RSM), was employed to optimize the conditions of Geobacillus sp. G1 for enhancing WAS hydrolysis. The results showed that the maximum lysis rate of E.coli was 50.1% at 4 h treatment period, and it increased by 16.2% compared with the control. The optimized dosage ratio of Geobacillus sp. G1 to WAS was 35%:65%(VG1:VWAS), soluble protein was increased to 695 mg COD/L, which was 5.0-fold higher than that obtained in the control.The maximal protease activity(403.4 ± 4 μg FDA/(m L·h), was 1.5-fold of that obtained in the control). This was achieved under the following conditions: Geobacillus sp. G1 dosage was 10%(V/V) and the pretreatment time was 6 h. The corresponding performances of WAS solubilization were as follows: SCOD 4130 ± 170 mg/L, soluble protein 1063 ± 15 mg COD/L, and carbohydrate 213 ± 6 mg COD/L, which were 1.5-fold, 1.7-fold and 1.1-fold high of that obtained in the control, respectively. Meanwhile, the production of SCFAs was also improved to 3529 ±150 mg COD/L in the Geobacillus sp. G1 pretreatment, which was 2.0-fold of the control. The successive process of microbial community analysis revealed that, the dominant lytic bacteria in Geobacillus sp. G1 treatment at the hydrolysis stage was Calormator, which belonged to Clostridia. During the fermentation stage, the proportion of Calormator was increased from 9% ~ 25% to 66% ~ 74% compared with hydrolysis stage, the strains had shifted from thermophilic hydrolysis bacteria to mesophilic hydrolysis and acidification bacteria, and the content of protease genes decreased. Further study on the effects of Geobacillus sp. G1 on WAS hydrolysis combined with three typical pretreatments(alkaline, ultrasonic, and freezing/thawing methods) were carried out to verify whether Geobacillus sp. G1 can further enhance WAS hydrolysis. The results demonstrated that lytic performances greatly improved under combined pretreatments, compared with the corresponding single pretreatments. In the hydrolysis stage, the concentrations of protein in supernatants pretreated by Geobacillus sp. G1 & alkaline(AG), Geobacillus sp. G1 & ultrasonic(UG), Geobacillus sp. G1 & freezing/thawing(FG) were 2.0-fold, 1.7-fold, and 1.4-fold higher than the control, respectively. And in acidification stage, the SCFAs accumulation was 2.7-fold(AG), 2.5-fold(UG), and 2.0-fold(FG) higher than that obtained in the control treatment, respectively. The successive process of microbial community structures showed that the combined pretreatment methods had significant influence on the microbial community structures and the species of functional microorganisms in the acidification stage remained consistent under different pretreatments. Clostridia and Bacteroidia were the predominating functional microorganisms for the WAS hydrolysis and further acidification, γ-Proteobacteria and Actinobacteria were the two dominant microorganisms in anaerobic digestion process of WAS, which were closely related to WAS hydrolysis.The biological pretreatment techniques proposed in this research study has opened up a new approach to enhance lytic performances of WAS, and further provided some fundamentals for resource recovery from WAS, especially as a carbon source. |
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