Download Gamma-Proteobacteria Identified in Commercial Bio

Gamma-Proteobacteria Identified in Commercial Bio-aids
Used in Wastewater Treatment Industrial
Y-W Chen, P-L Lee, C-Y Huang, C-N Chang, H-W D. Kuo
Department of Environmental Science and Engineering,
Tung-Hai University, Taichung, Taiwan
ABSTRACT
Three commercial bio-aids (i.e., TF1-CMB, TF4-MB, and TF5-POB) had showed good
trouble-shooting capabilities during several biological wastewaters processing. However, the
microbial contents in the bio-aids were still unclear. In this study, we analyzed their
eubacteria contains based on 16S rDNA sequence comparisons. Results showed that the
major functional microbes for C/N removal in activated sludge systems such as Klebsiella
pneumoniae, Pantoea agglomerans, P. ananatis, P. vagans, and Pectobacterium cypripedii
belonging to γ sub-division of Proteobacteria (purple bacteria) and Rummeliibacillus
stabekisii of Gram-positive bacteria (4 colonies) were identified in the these bio-aids. This
research provided further microbial information for the three studied bio-aids to support
insight reasons toward successful trouble-shooting cases during previous applications; and,
would be helpful for future selection of suitable bio-aids for various troubles during
biological wastewater processing.
Keywords: wastewater biological treatment, bio-aid, 16S rDNA
INTRODUCTION
Bio-augmentation with addition of bio-aids has been practiced for years in attempts to
improve treatment performances of wastewater biological processes (Rittmann and
Whiteman, 1994; Gentry et al., 2004; Foster and Whiteman, 2007). This technique has few
typical functions including (1) shortening start-up time, (2) enhancing BOD/COD removal
efficiency, (3) increasing capability to overcome shock loading, (4) improving sludge settling
conditions, (5) decreasing sludge production, (6) preventing odor problems, etc (Buckley,
1992; Stephenson and Stephenson, 1992). Several previous studies have shown that the
treatment performance could be improved by adding some bio-aids while no significant
enhancement was noticed from other study cases (Wilderer et al., 1991; Bouchez et al., 2000;
Boon et al., 2003; Dabert et al., 2005; Parker and Wanner, 2007; Tayà et al., 2011). To appropriately
select and successfully apply particular bio-aids under various conditions depends on
sufficient information about microbial contents in the bio-aids. However, this information for
many applied bio-aids is somewhat deficient comparing with that about their applicability
and chemical components. The major objective of this study is to analyze the microbial
contents for three commercialized bio-aids by according to 16S rDNA sequence comparisons.
MATERIAL AND METHODS
Microbes in the three analyzed bio-aids (i.e., TF1-CMB, TF4-MB, and TF5-POB)
were cultured with autoclaved LB medium for four days. The microbial growth and activity
were monitored daily using microscope observation and oxygen uptake rate (OUR, mg
O2/L/min) measurement, respectively. Genomic DNAs of the 4-day cultured microbes (600 μl)
were extracted by using the Tissue & Cell Genomic DNA Purification Kit (GeneMark,
Taiwan). Target 16S rDNAs of eubacteria were PCR amplified with a primer set (i.e.,
27f/1522r). The PCR products (~1,500 bps) were cloned into yT&A vector (Yeastern Biotech,
Taiwan) and further transformed into E. coli cells (ECOS 101, Yeastern Biotech, Taiwan). A
total of 96 colonies were picked (32 for each bio-aid sample) and checked with colony PCR
using the M13F/R primer set. The positive clones were sent out for sequencing; and, the
revealed 16S rDNA sequences were compared to those from the NCBI database to identify
microbes presented in the bio-aids samples. The 16S rDNA sequences from both database
and bio-aid samples were subsequently aligned by using the Clustal X (Thompson et al., 1997)
for further constructing a phylogenetic tree that could be displayed with TreeView (Page,
1996).
RESULTS AND CONCLUSIONS
Results showed that significant microbial growth (according to microscope
observations, not showed) and activities (measured as oxygen uptake rate, Fig. 1) were
noticed after 2-4 days incubation in LB medium. Within 96 selected colonies (3 × 32), 54
were positive clones; and, among which, 27 clones were sequenced. Based on 16S rDNA
sequence comparison with NCBI databases, the bio-aid of TF1-CMB mainly included
Klebsiella pneumoniae belonging to γ sub-division of Proteobacteria (purple bacteria) (Fig.
2). In TF4-MB, Pantoea agglomerans, Pantoea ananatis, and Pectobacterium cypripedii of
γ-Proteobacteria were found. In addition, not only Pantoea agglomerans and Pantoea
vagans of γ-Proteobacteria but also Rummeliibacillus stabekisii of Gram-positive bacteria (4
colonies) were identified in the TF5-POB. The Klebsiella sp., Pantoea sp., and
Pectobacterium sp. had been recognized as the major functional microbes for C/N removal
(Wagner et al., 2002; Ding et al., 2011); thus would be practical for improvement of activated
sludge systems. In summary, this research provided further information about microbial
contents for the three studied bio-aids to support insight reasons toward successful troubleshooting cases during previous applications; and, would be helpful for future selection of
suitable bio-aids for various troubles during biological wastewater processing.
Figure 1. Activity of the microbes from the bio-aids increased as incubation time was
increased during the 4-day cultivation period.
Figure 2. The phylogenetic tree showed that most of the microbes in the analyzed bio-aids
belong to the γ sub-division of the Proteobacteria.
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