## 3 Result and Discussion  
### Generation of Thermo-Tolerant Strains of L. bulgaricus  
Starting from the isolated pure culture of _L. bulgaricus_ grown at 42 \({}^{\circ}\)C, temperature elevation was used as an environmental stimulus to induce ALE. Over the course of 20 days, sub-culturing of bacteria, which survived the elevation of temperature, was performed, and at every temperature point, the bacterial cells were examined for morphology under a light microscope. As shown in Figure 2a, initially, the control, _L. bulgaricus_ grown at 42 \({}^{\circ}\)C had short, rod-shaped cells, with 7.62 log CFU/mL on average when grown on LMRS. We were able to increase the temperature to 52 \({}^{\circ}\)C and achieve 4.66 log CFU/mL. The survival rate of _L. bulgaricus_ is known to decrease due to the loss of its polysaccharide production ability starting from 47 \({}^{\circ}\)C. Hence supplementation with lactose at 2% to MRS has helped with development of _thermo-tolerant strain_. Studies have shown that adding lactose has increased the growth of _L. bulgaricus_ and provided effective protection to _L. bulgaricus_ in extreme temperature [31; 32]. Lactose can replace structural water molecules in membranes after dehydration at extreme temperature, preventing protein unfolding and aggregation by hydrogen bonding with protein polar groups [33]. As shown in Figure 2b, the thermo-tolerant strain showed change in bacterial morphology to elongated cells compared to the short rod shapes at 42 \({}^{\circ}\)C, as a response to environmental stimuli, as explained by [34]. The enhanced capability of bacteria to survive after exposure to a lethal temperature has been associated with the production of heat-shock proteins [35; 36]. Filamenting temperature-sensitive mutant Z (FtsZ) is the first tubulin-like protein to respond during cell division. FtsZ recruits other synthesis proteins that produce Z-ring for cell division and form filaments with smooth morphology as elongation [37]. As the colony count observed in this study was much less than expected, it was assumed that the thermo-tolerant bacteria is lacking FtsZ, resulted in no cell division but elongation into filaments [38]. Figure 2: Microscope images (\(\times\)1000 magnification) of Gram-stained _L. bulgaricus_. (**a**) Pure _L. bulgaricus_ strain incubated at 42 \({}^{\circ}\)C, showing short and rod shape. (**b**) Thermo-tolerant strain of _L. bulgaricus_ incubated at 52 \({}^{\circ}\)C, showing elongated shape. (**c**) First incubation at 52 \({}^{\circ}\)C after storing at \(-80\)\({}^{\circ}\)C for 3 weeks, showing truncation in elongated cells and formation of cocci. (**d**) Post to three rounds of sub-culturing at 52 \({}^{\circ}\)C, showing further transformation of truncated elongated rod-shaped cells (from (**c**)) into cocci. (**e**) Colony A and B are examples of _L. bulgaricus_ showing irregular edges at 52 \({}^{\circ}\)C.  
When both the control and the thermo-tolerant strains were frozen at \(-80\)\({}^{\circ}\)C in glycerol for three weeks to test for freeze-thaw stability, another change in morphology was detected but only in the thermo-tolerant strain. This strain was re-incubated and sub-cultured on LMRS agar for 48 h. As shown in Figure 2c, morphology has changed to shorter chins and cocc on the first sub-culture. After three rounds of sub-culturing (48 h per sub-culture), more of the rod-shaped cells were converted into cocci (Figure 2d), with irregular edges (circled in Figure 2e). The observations may be explained as adaptation mechanism of _L. bulgarcus_ to the heat stress by taking appropriate molecular responses to reduce the extreme effects and restore and survive [39]. The observed change in morphology may be caused by mutation occurring in MreB, which is an actin-like cytoskeletal protein found in most rod-shaped bacteria. Previously, transition from growing as a rod-shaped cell to coccus has been observed in _E. coli_, _Arthobacter_, _Acinetobacter_, and _Rhodococcus equi_[40].