2. material and methods

2.1 description of the TAnD digester designed for this study

The semi full-scale one stage TAnD digester was installed in Molina de Segura WWTP (Murcia, Spain) and consisted of a 15m3 continuously stirred tank reactor (CSTR) covered by a heat resistant, 10 cm trick polyurethane layer, for its thermal insulation and cladding. The heating system consisted of a coil heat exchanger of 75.5 kW in which the circulation of the sludge and temperature controlled water from the primary cooling circuit occurred. The biogas output from the TAnD digester was passed through a stainless steel pipe of 32mm diameter and was burnt off in a torch. An over pressure relief valve was installed to release gas whenever the pressure exceeded 20 kPa. The foam production was controlled with the installed of a polyester rack of a 25 mm reticulum. The sludge stream was operated in batch mode. The temperature of incoming and outgoing sludge, pH, electrical conductivity (EC), and oxidation reduction potential (ORP) were measured continuously with set of probes installed in the digester and registered with a SCADA(supervisory control and data acquisition). Fig 1 shows a schematic diagram of the TAnD digaster.

2.2 mesophilic anaerobic digestion

Molina de segura WWTP consists of a double stage biological treatment followed by a coagulation flocculation tertiary system, lamellar settling, filtering, and ultraviolet radiation disinfection, with an average flow of 25.000 m3 d-1. Primary and secondary sludge produced in the WWTP are first thickened separately and then mixed in the introduction into the mesophilic anaerobic digester. The mesophilic anaerobic digester has an effective volume of 7612 m3 and serves a population equivalent of 290.000 p.e. the required temperature of the process (around 35oC) is maintained by the Heatamix system by using hot water from the refrigerating circuit of the motogenerator GUASCOR SFGLD 480/55 of 760 kW. The biogas generated during the digestion process in stored in a double membrane gasometer of 1410 m3 volume to be utilized by the motogenerator. If the storage capacity of the gasometer is surpassed, the excess of biogas will burnt off in an automatic ignition torch.

2.3 Physical and chemical analysis

Analysis of total solids (TS), volatile solids (VS), and alkalinity were performed daily according to the Standard Methods. Destruction of Volatile Solids (VDS) was calculated as the fraction of volatile solids destroyed by the constant ash method. The biogas flow rate was measured with a T-Mass AT-70 Thermal Mass Flow meter (Endress + Hauser, IN,USA). The biogas composition and volatile fatty acid (VFA) were analyzed by gas chromatography. Total organic carbon (TOC) was determined by oxidation with K2Cr2O7 in an acid medium (H2SO4) evaluating the excess of dichromate with (NH4)2Fe(SO4)2. Total nitrogen (N) was determined by

the Kjeldahl method modified by Bremner and Mulvaney. Total phosphorus (P), total potassium (K), and heavy metals were determined by colorimetry following the Murphy and Riley method and K and heavy metals by atomic absorption spectrometry.

2.4 Dewaterability

The evaluated the dewatering characteristics of the sludges, two laboratory tests were performed: a dynamic jar test and a drainage test. The dynamic jar test was used for the determination of the optimum type and polymer done (OPS) for an accurate dehydration. This test consisted of the mixing of the sludge and the polymer by a determined number of sludge transfers made from one jar to another. Then, the sludge was mixed with its appropriate type and amount of polymer and the number of necessary transfers to determine the following parameters was calculated; number of transfers needed to achieve flocculation (TF), floc breakage (TR), re-flocculation (TFR) and breakage of re-flocculated floc (TRR). The drainage test was used to determine the volume and rate of water released from sludge.

2.5 cultivation and molecular of pathogens

Raw sludge (mixed primary secondary sludge), sludge produced after mesophilic anaerobic digestion (MAD Sludge) and sludge produced after thermophilic anaerobic digestion (TAnD sludge) were sampled weekly during the stable operational periods of the thermophilic digester. Total coliforms, E.coli, C. Perfringens spores, and salmonella spp. Were cultivated and pathogenity genes invA and cpa PCR amplified as described by Lloret at al.

2.6 statistical analysis

For the analysis of chemical parameters, one way ANOVA was performed with type of sludge as factor followed by Tukey,s HSD as a post hoc test (p <0.05) to control for multiple testing. For the analysis of pathogenic microorganisms the data (log transformed) were submitted to a non-parametric test for several independent samples (Kruskal-wallis test). Pair-wise comparisons between different types of sludge were performed using the Mann-Whitney U test (p < 0.05). The statistical software SPSS 20.0 was used for the analysis (SPSS inc).

3. Result and Discussion

3.1 TAnD digester start up and operational periods

The start-up of this type of bioreactor is a challenging task that can be accomplished in two different ways: by using an inoculum from another thermophilic anaerobic reactor, or by using a mesophilic inoculum. In this study, due to the absence of a thermophilic inoculum, the second option was chosen. In this case, the conversion strategy from mesophilic to thermophilic temperatures may be performed either: (a) by a sequential and gradual

temperature rish, or (b) by a rapid and unique change from mesophilic to thermophilic temperatures. The conversion strategy chosen in this work was to rapidly increase the temperature. In this respect, previous contradictory observations have been reported describing this strategy as not successful for mesophilic-thermophilic temperature conversions or as the favorable option to first establish a thermophilic culture when converting from mesophilic operation. Consequently, the TAnD digester was filled with mesophilic sludge from Molina de Segura WWTP, sealed, the temperature was increased up to 55oC and the reactor was maintained without any feed during the following 15 days. Then, the digester was fed with raw sludge with an OLR of 0.7 kg VS m-3 d-1 that was progressively increased up to 1.0 kg VS m-3

d-1 within the following 40 days, with the aim of controlling the production of VFA. Fig.2 shows the evolution of the concentration of VFA in the digester during the start up stage. It can be observed how the concentration of VFA started to increase during the first days of opartion. An initial accumulation of VFA, typical of hydrolysis and fermentation of the easily degradable substrates, is common during start-up process, as the activity of methanogens is exceeded.

After approximately 50 days, the concentration of VFA began to decrease, which led to an increase of the OLR up to 1.2 kg VS m-3 d-1. Nevertheless, as the concentration of VFA started to increase again, suggesting that the system was overloaded, the OLR was lowered to 1.0 kg VS m-3 d-1 once again and it was not increased until the stabilization of the digestion process occurred. It should be pointed out that the population of methanogenic microorganisms capable of adjusting from a mesophilic to a thermophilic condition is relatively small and needs a long period of time to acclimatize. At the same time, this new thermophilic anaerobic consortia established in the digester will be determinant for its stable and efficient operation.

After 250 days, the VFA concentration decreased and stabilized in a range between 2000 and 3000 mg/l, considering this period as the start-up phase. These result are in accordance to other studies that have described the experimental start-up period for a stable thermophilic operation as long even up one year before gas production reaches an acceptable level. Nevertheless, result from iranpour et al. that also chose rapid heating as the start-up strategy, show that a shorter process stabilization could be achieved, being their feeding plan proportionally lower.

3.2. TAnD digester operational parameters