## 2 Materials and Methods
### Sampling, Analyses and Calculations  
Samples of influent and effluent from the VFF and HFF units were collected once a week and analyzed for pH, EC, Tot-N, NH\({}_{4}\)-N, NO\({}_{3}\)-N, COD, _Salmonella_ spp. and _E. coli_. The incoming wastewater and the filter effluents were analyzed using Spectroquant(r) cell kits number 14772-14773 \begin{table}
\begin{tabular}{c c c c c c c} \hline \multirow{2}{*}{**ID**} & **Duration** & **HLR** & **OLR (g COD** & **NLR (mg Tot-N** & & \\  & **(Weeks)** & **(L m\({}^{-2}\) day\({}^{-1}\))** & **m\({}^{-2}\) day\({}^{-1}\))** & **Parameters Investigated** \\ \hline \multirow{2}{*}{HLR-23 [1]} & \multirow{2}{*}{31} & \multirow{2}{*}{23} & \multirow{2}{*}{11.0 \(\pm\) 4.8} & \multirow{2}{*}{878 \(\pm\) 284} & Tot-N, NH\({}_{4}\)-N, NO\({}_{3}\)-N, COD, pH, & \\  & & & & & & _Salmonella_ spp. and _E. coli_ \\ \hline \multirow{2}{*}{HLR31-1} & \multirow{2}{*}{12} & \multirow{2}{*}{31} & \multirow{2}{*}{14.4 \(\pm\) 8.3} & \multirow{2}{*}{2323 \(\pm\) 187} & Tot-N, NH\({}_{4}\)-N, NO\({}_{3}\)-N, COD, & \\  & & & & & & & \\  & & & & & & & \\ \hline \multirow{2}{*}{HLR-39} & \multirow{2}{*}{12} & \multirow{2}{*}{39} & \multirow{2}{*}{18.9 \(\pm\) 5.9} & \multirow{2}{*}{1403 \(\pm\) 203} & Tot-N, NH\({}_{4}\)-N, NO\({}_{3}\)-N, COD, pH, & \\  & & & & & & & _E. coli_ \\ \hline \multirow{2}{*}{HLR31-2} & \multirow{2}{*}{46} & \multirow{2}{*}{31} & \multirow{2}{*}{15.5 \(\pm\) 4.2} & \multirow{2}{*}{1206 \(\pm\) 269} & \multirow{2}{*}{} & \multirow{2}{*}{} \\  & & & & & & & \\ \end{tabular}  
* This loading period included the start-up and restart of the filter after reaching steady state.  
\end{table} 
Table 1: Loading conditions in the experiment investigating the effects of changes in hydraulic loading rate (HLR). OLR = organic loading rate, COD = chemical oxygen demand, NLR = nitrogen loading rate. Values shown are mean \(\pm\) SD. for determination of COD, 00683 for NH\({}_{4}\), 09713 for NO\({}_{3}\) and 14963 for Tot-N (Merck KGaA, Darmstadt, Germany). Concentrations were determined colorimetrically using a Nova 60 photometer (Merck KGaA). The pH was measured using WTW pH/ion 340i meter and the electrical conductivity (EC) was measured using a Condi 330i conductivity meter (WTW, Weilheim Germany). The cultivation of _Salmonella_ spp. was performed by incubating a single colony of _Salmonella_ spp. in unselective microbial growth medium (nutrient broth, Swedish Veterinary Institute, Uppsala, Sweden) for 24 \(\pm\) 7 h at 37 \({}^{\circ}\)C under continuous mixing of the bacteria and nutrient broth. For analysis of _E. coli_ and _Salmonella_ spp., 0.1 mL samples were taken from sterile tubes containing series of dilutions of influent and effluents, and added to Petri dishes with Chromocult agar (VWR International, Helsinki, Finland) for _E. coli_ or XRD for _Salmonella_ spp. The dishes were incubated at 37 \({}^{\circ}\)C for 24 \(\pm\) 5 h and visible colonies of _E. coli_ and _Salmonella_ spp. were counted. Removal efficiency (_E_) was calculated as the difference in concentration between influent and effluent of the filters:  
\[E=100\ \frac{[C_{in}]-[C_{out}]}{[C_{in}]} \tag{1}\]  
where \(E\) is the removal efficiency (%), [\(C_{in}\)] is the concentration in influent (mg L\({}^{-1}\)) and [\(C_{out}\)] is the concentration in effluent (mg L\({}^{-1}\)). Log\({}_{10}\) reduction in _E. coli_ and _Salmonella_ spp. was calculated as:  
\[\text{Log}_{10}\ \text{reduction}=\text{Log}_{10}\ [\text{C}_{\text{in}}]- \text{Log}_{10}\ [\text{C}_{\text{out}}] \tag{2}\]  
where [C\({}_{\text{in}}\)] is the concentration of the microorganism in influent and [C\({}_{\text{out}}\)] is the concentration in effluent (CFU mL\({}^{-1}\)). For _Salmonella_ spp., [C\({}_{\text{out}}\)] was the concentration in the effluent collected 24-48 h after feeding the filters with the wastewater spiked with _Salmonella_ spp.