**5.8 Test of adhesion**

This test has been carried out by the adoption of strain B1, as it constitutes most part of biomass isolated microorganisms. This test allows one to quantify attached mass through the measurement of the optical density of a mature biofilm on a plastic surface [33]. In this respect, Christensen's method has been applied based on the modifications described by Baldassarri et al. [34]. In this study, 7 dilutions of overnight cultures at 28 ̊C have been made in order to obtain, in addition to an evaluation of the strain attachment characteristics, also a relationship between cellular concentration and bioattachment, at the following dilutions:


*Experimental Investigation of Biomass Attachment to Wastewater Reactors DOI: http://dx.doi.org/10.5772/intechopen.94426*


5 ml have been withdrawn from each dilution, 1 ml of which has been used to prepare suspension-dilutions with the aim to determine the bacterial load corresponding to each sample. Portions left have undergone spectrophotometric readings (600 nm). All samples, except lowest dilutions, have been centrifuged for 10 minutes at 10,000 rpm and, further to supernatant elimination, cells have been resuspended in sterile broth. 2 ml have been collected from each dilution and distributed into 10 pits (200 μl each) of a 96-wells Enzyme-Linked Immunosorbent Assay (ELISA) plate with flat bottom. The plates, provided with 10 wells filled PCB sterile broth as blank sample, have been incubated at 28 C for 24 hours. The broth has been ̊ then gently removed from the wells by means of Pasteur pipettes and wells have been washed three times with sterile deionized water to remove unattached cells. This phase has been conducted very carefully as it often causes removal of attached cells also, resulting in false findings. Unlike proposed by Christensen et al. [33], sterile pipettes have been preferred to direct immersion of plates into clean water, in order to minimize any disturbances and handling. Attached cells have then been fixed by exposure to 60 C for 1 hour and colored by Hucker crystal violet. Plates have been rinsed until ̊ rinsing water has been completely colorless, then overturned and dried for 30 minutes at 37°C. Biofilm density has been assessed by a spectrophotometer for ELISA plates, after calibrating the blank sample of the instrument with the wells containing noninoculated sterile broth. Readings have been taken at 550 nm. According to the adopted protocol, mean of taken readings for each well has been interpreted as:


### **5.9 Observation of the growth-curve shown by a pure culture in presence of a complex substrate**

A study has been carried out with the aim to investigate the growth of a B1 strain with a substrate consisting of pure butanol only. In this respect, two tests have been performed:


The Inoculum has been prepared from a strain stored in a freezer on slant PCA. Sterile broth culture contained by a 20 ml tube has been inoculated with B1 strain, and incubated for 15 hours overnight at 28 ̊C. The culture obtained was the 10%
