# Bioaugmentation Treatment of a PAH-Polluted Soil in a Slurry Bioreactor
## Materials and Methods
### Slurry bioreactor and Experimental Design  
The aerobic SB used (Figure 1) was a 40-l stainless-steel recipient designed for pilot-scale experiments. The reactor had a stainless-steel central shaker with speed control, operated in this set of experiments at 150 rpm. A compressor was used to provide oxygenation through an air diffuser locatedat the bottom of the reactor. This setup facilitates a regular distribution of air throughout the slurry, thereby keeping solids in suspension. Dissolved oxygen in the slurry was continuously controlled and adjusted initially to 8 mg/L. A heating system (casing) achieved a continuous operational temperature of 28 \({}^{\circ}\)C. Oxygen and pH, and temperature probes were continuously monitored by means of several monitoring probes. Also, a pre-treatment tank was used for slurry preparation, which was achieved by blending 4 kg of soil with 20 L of distilled water (1:5 ratio for the solid:liquid relation in the final pulp). After thorough mixing, the slurry was pumped into the SB. The slurry samples were obtained using a sampling valve, and a second pumping system was also available to recover the slurry-load in an outflow tank connected to a chamber filter press. The system was designed to incorporate additional reactors connected in parallel.  
Three experiments were performed to study PAH biodegradation in the SB. The first (Treatment A) was based on biostimulation of the autochthonous microbial population by adding nutrients, specifically NH4 NO3 (two different sources of N) and NaH2 PO4 (P source and buffer effect), adjusted to a C:N:P ratio of 100:10:1 on the basis of the amount of hydrocarbons present at the beginning of the experiments. A commercial biodegradable surfactant, Bioversal HC (Bioversal International, IEP Europe), was also added at dosages selected on the basis of previous experiences in other studies (including field sites) and on the manufacturer's recommendations [41]. The two other experiments were based on biostimulation of the autochthonous microbial population combined with bioaugmentation with _Rhodococcus erythropolis_ (Treatment B) and _Pseudomonas stutzeri_ (Treatment C). Both bacteria had previously been isolated from the polluted soil used in this study during a  
Figure 1: The 40-l slurry bioreactor and auxiliary devices.  
full-scale biopile treatment at the same site, and showed a high capacity to degrade naphthalene and fluorene [27]. A concentrated inoculum of each bacterium (around 10\({}^{8}\) UFC/mL final bioreactor concentration) was added to the SB at the beginning of each experiment (time 0), and the samples were removed at different times over 15 days to analyze the parameters mentioned below. Samples were transferred to dark bottles, sealed and stored at 4 \({}^{\circ}\)C, and analyzed within a week. A summary of the operating conditions and experimental variants measured in the three treatments are shown in Table 1. Furthermore, continuous monitoring of the slurry phase pH, temperature, and dissolved oxygen (DO) was carried out using a Multi 340i apparatus (WTW).