## 3 Results
### Functional Predictions using PICRUSt  
On the basis of the annotation of functional genes using PICRUSt, 41 gene families were identified in the sample, but only families with an abundance \(>\) 1% are shown in Figure 7a. The sample was enriched for functions involved in membrane transport (12.217%), amino acid metabolism (10.067%), and carbohydrate metabolism (9.597%) (Figure 7a).  
A deep examination of the carbohydrate metabolism revealed that pyruvate, amino sugar and nucleotide sugar metabolisms, glycosis/glucoseogenesis, and butanoate metabolism were major categories (Figure 7b). Putative genes responsible for starch and sucrose metabolism were moderately accounted for 0.57% of the total reads. In addition, subgroup genes such as galactosidase metabolism (0.472%) and glycosis/glucoseogenesis (0.986%) were potentially involved in the starch degradation.  
Biological starch removal is one of the most important functions in the activated sludge. In the present study, several starch hydrolytic enzymes were detected in the SBR system such as starch phosphorylase (0.043%), \(\alpha\)-amylase (0.043%), glucoamylase (0.001%), pullulanase (0.017%), \(\alpha\)-galactosidase (0.036%), \(\beta\)-galactosidase (0.072%), \(\alpha\)-glucosidase (0.060%), \(\beta\)-glucosidase (0.113%), 1_A_-\(\alpha\)-glucan branching enzyme (0.043%), and oligo-1,6_-glucosidase (0.002%) (Table 4). By analysing Kyoto encyclopedia of genes and genomes (KEGG) metabolism maps, we discovered that all enzymes were involved in the glycolysis pathway to create pyruvate from starch (Figure 8). Starch was converted by the actions of \(\alpha\)-amylase and glucoamylase to form \(\alpha\)-d-glucose; and the actions of 1_A_-\(\alpha\)-glucan branching enzyme and starch phosphorylase to form \(\alpha\)-d-glucose-1-phosphate. The enzyme hexokinase catalyzed the conversion of \(\alpha\)-d-glucose to  
Figure 6: Bacterial composition in the granular sludge sample at phylum level (**a**) and family level (**b**). The families that accounted for greater than 1.5% of reads were shown in the figure.  
\begin{table}
\begin{tabular}{c c c c} \hline
**No.** & **Species** & **Frequency** & **Quantity** \\ \hline
1 & _Lactococcus chungangensis_ & 20.69120 & 3227 \\
2 & _Janthinobacterium lividum_ & 6.50167 & 1014 \\
3 & _Megasphereal cerevisiae_ & 3.63555 & 567 \\
4 & _Sultfurrospirtillum arsenophilum_ & 3.59066 & 560 \\
5 & _Pseudomonas flaescens_ & 2.34034 & 365 \\
6 & _Pseudomonas veronii_ & 2.0898 & 318 \\
7 & _Macallibacteroides fermentatus_ & 1.76327 & 275 \\
8 & _Soonwoaa buanensis_ & 1.10926 & 173 \\ \hline \end{tabular}
\end{table}
Table 3: List of the most frequently occurring species in the granular sludge sample.  
\(\alpha\)-D-glucose-6-phosphate. Other enzymes that were involved in the glycolytic pathway included phosphoglucomutase, glucose-6-phosphate isomerase, 6-phosphofructokinase, fructose-biphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxylase. Pyrruvate was converted into acetyl-CoA in the presence of pyruvate dehydrogenase in order to enter the citric acid cycle. Additionally, L-lactate dehydrogenase catalyzed the reversible conversion of pyruvate to L-lactate.  
Figure 7: Functional analysis based on the Kyoto encyclopedia of genes and genomes (KEGG) subsystem level II (**a**) and carbohydrate metabolism (**b**). At KEGG subsystem level II, only functions with percent read > 1% are shown.