Phylogenetic Identification and Evaluation of Biosafety of Selected Strains

Full 16S rRNA gene sequences were obtained for microbial strains B and F, respectively. The sequences of strain B was 99.4% identical to Klebsiella pneumoniae [Gene bank access number is AY292866.1], and strain F was 99.9% identical to Pseudomonas veronii [Gene bank access number is AY512619.1]. The sequences were of 1338 and 1408 bases in length, respectively. Both isolates belong to subclass of y-Proteobacteria.

A disadvantage of wastewater treatment with microbial granules in comparison with the conventional activated sludge system is the long start-up period. To accelerate the granulation start-up and to prevent the growth of filamentous bacteria, high COD loading, 8.5 g COD L-1 day-1, was used in this study because there was no negative effect on granulation when COD increased even up to 15 kg m-3day-1(Moy et al., 2002). Another approach was selection of cells with high aggregation ability. Cells of selected strains Klebsiella pneumoniae strains B and Pseudomonas veronii F could either form aggregates by themselves or coaggregated with other microbial strains of activated sludge. Such ability of these strains could be the main reasons why microbial granules can be formed faster in experiment with addition of these strains to activated sludge than in control with activate sludge as inoculum.

Cultivation of cells from the dispersed granules on solid medium showed that 12 ± 3% of colonies from the granules formed for 10 days of cultivation in experimental reactor (R2) were colonies of Klebsiella

Fig. 10.8. SEM of aerobic granule in experimental reactor (R2). (a) x 70 magnification; (b) x 5000 magnification.

pneumonia strain B and 40 ± 6% colonies were colonies of Pseudomonas veronii strain F. The concentrations of Klebsiella pneumoniae and Pseudomonas veronii were both 36 mg L-1 at the beginning of cultivation. After 10 days of cultivation, these concentrations increased to 350 and 1400 mg L-1, respectively. It indicated that the added strains B and F were dominant cultures in formed aerobic granules.

Bioaugmentation of activated sludge systems with specialized bacterial strains (microbial seeds), has been practiced since the 1960s. Their application in wastewater treatment was originally the efforts to solve operational problems such as shock loading in treatment plant (Limbergen et al., 1998). Bioaugmentation could be a powerful tool and cost-effective method to improve several aspects in the wastewater treatment process such as improved flocculation and degradation of recalcitrant compounds (Limbergen et al., 1998). A stable enhanced biological phosphorus removal (EBPR) in a bioreactor was installed within 15 days using bioaugmentation of sludge by the phosphorus-accumulating organism (Dabert et al., 2005). In our experiments, environmental conditions and process parameters in both control and experimental reactors were the same and only the difference between the reactors was an addition of two selected strains with high aggregation ability into experimental reactor. Experimental data demonstrated that this addition significantly reduced the time of granule formation and facilitated formation of dense granule with low SVI.

Both isolates, Klebsiella pneumoni strain B and Pseudomonas veronii strain F, belong to y-Proteobacteria, which in agreement with previous studies that Proteobacteria constitute a largest fraction of the microbial granules (Jiang et al., 2004b). The authors isolated seven strains and one from them, Comamonas sp. D22, exhibited strong flocculation activity and could form auto-aggregates with high extra-polysaccharide content, which might play an important role in the formation and maintenance of the phenol-degrading aerobic granules (Jiang et al., 2004a,b).

However, the species K. pneumoniae is a urinary tract pathogen and could be considered as opportunistic human pathogen. The release of the strain of this species into environmental engineering system might potentially cause health problems for human and animals. Therefore, K. pneumoniae strain B was considered as not suitable for environmental engineering application because of biosafety issue. Strain of Pseudomonas veronii might be considered as suitable for the treatment of wastewater because until now there were no published data on its pathogenicity. Therefore, Pseudomonas veronii strain F was selected for further large-scale trials as a starter culture for facilitated formation of microbial granules in aerobic wastewater treatment.

However, survival and stable activity of introduced strain in the system is a common problem in bioaugmentation applications. Therefore, selected strain of Pseudomonas veronii cannot be considered as universal strain for all cases, where microbial granulation is required. The feasibility of microbial granulation start-up with this strain must be tested for every specific process. Another problem of bioaugmentation of microbial granules with selected strain is that not only duration of granulation, but also the specific activities of granules such as nitrification, spectra of degradation of natural organic compounds and xenobiotics, accumulation of phosphate, cell survivability, and other properties are important in wastewater treatment. Therefore, enhancement of specific activities of microbial granules by incorporation of other microbial strains into the granules must also be studied.

Bacterial cultures of Klebsiella pneumoniae strain B and Pseudomonas veronii strain F, with self-aggregation index of 65 and 51%, respectively, and coaggregation index of 58%, were isolated from enrichment culture. The mixture of these strains with activated sludge was used as inoculate in an experimental sequencing batch reactor to start-up aerobic granulation process. Aerobic granules with mean diameter of 446 ± 76 |xm have been formed in experiment after 8 days of cultivation but the microbial granules were absent in control. Considering biosafety issues, Klebsiella pneumoniae strain B will be excluded from further studies, but Pseudomonas veronii strain F was selected for larger scale testing. Time of granule formation from the flocs of activated sludge was from 8 to 14 days but can be reduced to 2 days if selected bacterial strains with enhanced self-aggregation ability will be used instead of activated sludge.

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