Sporulation Test

Sporulation Test
1. To determine the progression of sporulation in cultures of C. beijerinckii_M1GS^DisA, _M1GSC^cpA, or _pMTL500E, all three strains were grown in glucose + arabinose medium in triplicate (as described by Ujor et al., 2021)* in loosely capped culture bottles in the anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI, USA) with a modified atmosphere of approximately 82% N₂, 15% CO₂, and 3% H₂).
2. Samples were taken from each culture every 12 h. In the anaerobic chamber, the culture bottles were tightly closed, and samples (1 ml) were taken immediately after gently inverting each culture bottle three times (be careful due to gas production), to ensure even distribution of spores and vegetative cells. The samples were placed in 2 ml tubes and then diluted 1:100 in a total volume of 1 ml.
3. For the dilutions, each sample in a 2 ml tube was inverted three times and then, 100 μl was pipetted immediately afterwards and placed in a sterile 1.5 ml tube pre-filled with 900 μl of sterile distilled water to make a 1:10 dilution.
4. Then, 100 μl of the 1:10 diluted sample was drawn from the tube immediately after inverting the tube three times and transferred into a fresh sterile 1.5 ml tube containing 900 μl of sterile distilled water to achieve a 1:100 dilution, cumulatively. For all inversions, culture bottles or tubes were tightly capped before inverting to avoid spillage.
5. Subsequently, a portion of the sample (500 μl) was drawn after inverting the tube three times, transferred into a fresh sterile 1.5 ml tube, heat-shocked at 75°C for 8 min, cooled on ice for 2 min and then, plated on TGY agar (0.5%, w/v) to determine the number of spores per sample. The remainder of each sample (500 μl) was plated out on TGY agar (0.5%, w/v) without heat treatment to determine the number of vegetative cells in the samples relative to the spores (heat-shocked portion). All plates contained erythromycin (25 μg/mL) and were incubated in the anaerobic chamber for 24 h at 35 °C.
6. Subsequently, the colonies were counted for both the heated and unheated samples.
7. Except for heat shocking, all experiments were conducted in the anaerobic chamber.
8. To heat-shock the spores, 500 μl of each sample was placed in a sterile 1.5 ml tube, capped tightly and then moved out of the anaerobic chamber following standard protocols for operating an anaerobic chamber.
9. Each sample was placed on a heating block (Fisher brand dry bath), preset at 75°C and incubated for 8 min. The sample holes in the heating block were pre-filled with sterile sand to enhance contact and heat transfer.
10. After heating, the samples were cooled on ice as earlier stated and then, transferred back to the anaerobic chamber following standard protocols for operating an anaerobic chamber.
11. After 24 hours, the colonies on each plate were counted.
12. Based on the number of colonies on each plate (500 μl of plated sample), the total number of vegetative cells and spores in each culture (100 ml) were proportionally estimated, taking the 1:100 dilution into account. Further, standard protocol for percentage calculation was used to calculate the percentage spores in each culture (colonies on plates containing the heat-shocked samples_spores only) relative to the total colony counts on the culture plates containing unheated samples (spores + vegetative cells).

*Ujor VC, Lai LB, Okonkwo CC, Gopalan V, Ezeji TC. Ribozyme-Mediated Downregulation Uncovers DNA Integrity Scanning Protein A (DisA) as a Solventogenesis Determinant in Clostridium beijerinckii. Front Bioeng Biotechnol. 2021 Jun 8;9:669462.