## 3 Results and Discussion  
### Chemical Characterization of Raw Lipids and Optimization of Extraction Process  
Primary sludge and sewage scum were taken from several WWTPs in the Apulia region of southern Italy. After a preliminary determination of TS content, raw lipids were extracted from sewage sludge samples, quantified and chemically characterized (see Supplementary Materials of Section S.2). The results obtained are shown in Table 1.  
In primary sludge samples (E1-3), TS content was about 4%wt with a lipid content between 202.4 \(\pm\) 6.1 mg/gTS and 244.3 \(\pm\) 9.3 mg/gTS. Soaps were found to be the main component of the lipids isolated. In contrast, TS content in sewage scum samples (E4-9) ranged from 19.3 \(\pm\) 0.5%wt to 32.0 \(\pm\) 0.5%wt with a significantly higher lipid content of 350.1 \(\pm\) 8.4 mg/gTS-502.4 \(\pm\) 1.5 mg/gTS. Lipids consisted mainly of FFAs (47.5 \(\pm\) 0.8%-59.5 \(\pm\) 1.9%) and soaps (27.3 \(\pm\) 1.9%-34.5 \(\pm\) 1.0%). The different chemical nature of the lipids in primary sludge and sewage scum made it necessary to optimize the lipid recovery for each feedstock typology. The recovery of lipids from primary sludge, consisting mainly of calcium soaps (>70%), was achieved by extraction with hexane from acidified wet primary sludge with a recovery rate of 99% [53]. In the case of sewage scum, heating wet biomass at 80 \({}^{\circ}\)C and directly centrifuging it (4000 rpm, 3 min) allowed the extraction of lipid components with a recovery of 92-99%, without the addition of solvents or acids [54]. Subsequently, recovered lipids were activated by adding the stoichiometric amount of HCOOH to calcium soaps, thus obtaining their conversion into FFAs. The chemical composition of FFAs extracted from primary sludge and sewage scum is shown in Figure 2.  
\begin{table}
\begin{tabular}{c c c c c c c} \hline \hline
**E** & **WWTPs** & **PE** & \begin{tabular}{c} **TS** \\ **(\%wt)** \\ \end{tabular} & \
\begin{tabular}{c} **Lipids** \\ **(mg/gTS)** \\ \end{tabular} &
\begin{tabular}{c} **Lipid Composition (\%)** \\ **FFAs** \\ \end{tabular} \\ \hline \multicolumn{3}{c}{**Primary sludge**} & & & & \\ \hline
**1** & Bari Est & 360.000 & 4.4 \(\pm\) 0.3 & 202.4 \(\pm\) 6.1 & 9.5 \(\pm\) 0.3 & 71.3 \(\pm\) 1.5 \\
**2** & Bari West & 240.000 & 4.2 \(\pm\) 0.2 & 244.3 \(\pm\) 9.3 & 5.4 \(\pm\) 0.2 & 72.3 \(\pm\) 1.7 \\
**3** & Lecce & 120.000 & 4.3 \(\pm\) 0.1 & 211.5 \(\pm\) 7.4 & 4.3 \(\pm\) 0.2 & 81.6 \(\pm\) 2.1 \\ \hline \multicolumn{3}{c}{**Sewage scum**} & & & & & \\ \hline
**4** & Bari West & 240.000 & 22.4 \(\pm\) 0.3 & 502.4 \(\pm\) 1.5 & 51.7 \(\pm\) 1.6 & 30.4 \(\pm\) 0.8 \\
**5** & Andria & 100.000 & 17.4 \(\pm\) 0.3 & 350.1 \(\pm\) 8.4 & 47.5 \(\pm\) 0.8 & 34.5 \(\pm\) 1.0 \\
**6** & Bisceglie & 50.000 & 16.6 \(\pm\) 0.5 & 379.3 \(\pm\) 11.1 & 59.5 \(\pm\) 1.9 & 27.3 \(\pm\) 0.6 \\
**7** & Barletta & 90.000 & 22.9 \(\pm\) 0.4 & 410.4 \(\pm\) 12.3 & 53.3 \(\pm\) 1.7 & 32.9 \(\pm\) 1.2 \\
**8** & Polignano & 12.000 & 32.0 \(\pm\) 0.5 & 469.3 \(\pm\) 13.4 & 45.7 \(\pm\) 1.3 & 28.3 \(\pm\) 0.5 \\
**9** & Putignano & 12.000 & 19.3 \(\pm\) 0.5 & 435.4 \(\pm\) 11.3 & 52.0 \(\pm\) 0.9 & 34.0 \(\pm\) 0.7 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Chemical characterization of raw lipids extracted from primary sludge and sewage scum samples up-taken from WWTPs of Apulia region, southern Italy.