# Neutralization of Toxic Malodorous Gases from Cattle Slurry
## 2 Materials and Methods
### Microorganisms  
In this study, three _Bacillus_ and three _Pseudomonas_ strains were investigated. They were selected based on their enzymatic activity, from among 27 strains isolated from soil, compost, pig, or cattle slurry in our previous studies [42]. The nucleotide sequences of the 16S rDNA gene and the MALDI-TOF (Matrix Assisted Laser Desorption/Ionization Time of Flight) mass spectrometer IDplus Confidence(SHIMADZU, Japan), were used in the species identification of various groups of bacteria. The methodological details of microbial profiling were presented in our previous publication [42]. The lipolytic, proteolytic, amyloidytic, cellulolytic, and chitinolytic activities were studied using the plate methods [42]:  
1. lipolytic activity: opaque zone around colonies on medium with Tween 80 after 2 days of incubation at 37 \({}^{\circ}\)C,
2. proteolytic activity: clear zone around the colonies on Milk Plate Count Agar after 2 days of incubation at 37 \({}^{\circ}\)C,
3. cellulolytic activity: translucent zone around colonies on medium with carboxymethylcellulose and trypan blue after 2 days of incubation at 37 \({}^{\circ}\)C,
4. amyloidytic activity: clear zone around the colonies on Starch Agar after 2 days of incubation at 37 \({}^{\circ}\)C,
5. chitinolytic activity: purple zone around colonies on medium with colloidal chitin after 2 days of incubation at 37 \({}^{\circ}\)C.  
The characteristics of the microorganisms used in this research are presented in Table 2.  
Eleven identified _Bacillus_ and three _Pseudomonas_ strains were assigned GenBank accession numbers and were included in [PERSON] [PERSON] Institute of Agricultural and Food Biotechnology--State Research Institute's Collection of Industrial Microorganisms (pol. KKP). The Protect Microorganism Preservation System--Protect Multipurpose kit TS/80-MX (Technical Service Consultants Ltd., Heywood, UK) was used for long-term storage of bacteria (\(-20\)\({}^{\circ}\)C). The liquid cultures were grown in Luria Broth (LB, BTL, Lodz, Poland) in a shaking incubator (Innova 4000, Brunswick Scientific, Edison, NJ, USA, 140 rpm) for 22 h. The inoculum was added to the cattle slurry in the amount of 12.5 cm\({}^{3}\)/250 cm\({}^{3}\) of the CS in 500 cm\({}^{3}\) Schott bottles. The concentration of viable cells was performed by tenfold serial dilutions in Tryptone Salt broth (Biokar Diagnostics, Beauvais, France) of samples and spread plate on TSA (30 \({}^{\circ}\)C for _Bacillus_ species and 25 \({}^{\circ}\)C for _Pseudomonas_ species). The results obtained after 22 h of incubation were expressed as the logarithm of the number of colony-forming units (CFU). The concentration of tested strains ranged from 6.7 to 8.4 log CFU/cm\({}^{3}\). The bacterial \begin{table}
\begin{tabular}{c c c c c c c c} \hline \multirow{3}{*}{**Microorganisms**} & **KKP** & **GenBank** & \multicolumn{6}{c}{**Enzymatic Activity**} \\  & **Accession** & **Accession** & **Liquid** & **Proteolytic** & **Cellularolytic** & **Amyloidytic** & **Chitinolytic** \\ \hline _Bacillus_ & 3899 & PP069822 & + & + & +++ & +++ & + \\ _subtilis_ 1 & & & & & & & \\ _Bacillus_ & 3901 & PP069832 & ++ & ++ & ++ & + & \(-\) \\ _subtilis_ 1 & & & & & & & \\ _Bacillus_ & 3903 & PP069947 & + & ++ & +++ & ++ & + \\ _Pseudomonas_ & 3909 & PQ483896 & \(-\) & \(-\) & \(-\) & \(+\) & + \\ _fluorescens_ 1 & & & & & & & \\ _Pseudomonas_ & 3910 & PQ483894 & \(-\) & \(-\) & \(-\) & \(-\) & + \\ _fluorescens_ 1 & & & & & & & \\ _Pseudomonas_ & 3911 & PQ483898 & \(-\) & \(-\) & \(-\) & \(-\) & \(-\) \\ sp. 1 & & & & & & & \\ \hline \end{tabular}  
* Notes: Origin: 1—cattle slurry, 2—pig slurry. Lipolytic, proteolytic, and chitinolytic activity: ++ zone diameter \(\geq\) 2 cm, ++ zone diameter \(\geq\) 1 cm and <2 cm, + zone diameter less than 1 cm, \(-\) lack of zone; cell-ludolytic and amyloidytic activity: +++ zone diameter \(\geq\) 3 cm, ++ zone diameter \(\geq\) 2 cm and <3 cm, + zone diameter less than 2 cm, \(-\) lack of zone.  
\end{table} 
Table 2: Characteristics of microorganisms used in the research.