Effects of molasses concentration on the growth and metabolism of the strain
As the main component of molasses was sugar, molasses' concentration is directly related to the content of carbohydrates in the medium, the higher its concentration, the higher the osmotic pressure of the extracellular environment to cells. The increase in osmotic pressure would lead to the deterioration of the intracellular microenvironment. It can affect the growth and metabolism of the strain, causing the dehydration and inactivation of protein, which hinder cell cycle and inhibit cell growth (Radmaneshfar et al. 2013). Some studies have found that, with the increase of osmotic stress, the cell will actively regulate osmosis that causes intracellular water outflow, shrinking of cells, and even death (Djelal et al. 2017). Microorganisms would increase energy for maintaining the stability of the intracellular microenvironment (enhancing intracellular stress response and tolerance pathway). These change the metabolic network and regulatory pathways of cells, inhibit the fermentation rate, and change product accumulation (Xu 2010). It was found that the cell growth and pyruvate accumulation rate were inhibited by the increase in osmotic pressure in the pyruvate production (Kamzolova and Morgunov 2016). In arabitol production, when the osmotic pressure was increased, the product's yield would significantly decrease (Koganti et al. 2011). Therefore, combined with the results of growth and metabolism, four low concentrations of molasses, 5 °Bx, 7.5 °Bx, 10 °Bx, and 20 °Bx, were chosen to investigate the metabolism characteristics of the strain in molasses.
The utilization rate of carbohydrates in different molasses concentrations is also used as an index to select the optimal concentration medium. The results showed that there are enough carbohydrates in the 5 °Bx medium to ferment the strain. Moreover, the lower the concentration of substrate residual sugar, the higher the fermentation efficiency.
In conclusion, it is feasible to use the engineered strain L. casei E1 to produce ethanol with molasses as substrate conclusion of comparative genomics is verified (Wang et al. 2015). As the carbohydrates utilization rate and the ethanol production yield in 5 °Bx molasses are the highest, 5 °Bx is the optimum concentration of molasses for ethanol fermentation.
The sequence of carbohydrate metabolism of the strain
The metabolization of carbohydrates by strain E1 is in sequence by inference. This characteristic of strain was showed in all the four low concentrations molasses fermentation (Fig 2), and confirmed by the gene expression results (Fig 7). Agbogbo et al. (2006) used Pichia pastoris (P. stipitis CBS6054) to ferment a mixed medium of glucose and xylose. It was found that the consumption rate of glucose was higher than that of xylose. This indicated that there is a priority order in carbohydrate metabolism of microorganisms.
Effects of oxygen on the ethanol production
Lactobacillus casei is an aerobic anaerobe like other lactic acid bacteria. Under aerobic conditions, cells proliferated in large amounts and produced lactic acid (Maresca et al. 2019). Reactive Oxygen Species (ROS), present in the environment or metabolized by the strain, can seriously threaten the strain's survival. Some LAB can be subjected to aerobic growth, with a consequent change in the LAB's physiological metabolism, including the reduction of biomass and the change of fermentation product types. ROS can destroy cellular proteins, lipids, and nucleic acids, causing cell aging and death. Therefore, anaerobic culture is the optimum culture condition for ethanol production by the strain.
Effects of pH on the ethanol production
Based on the results of acidity control of fermentation broth, it was found that a change in pH has a significant effect on the growth and metabolism of the strain. This is mainly because fermentation's organic acid will enter the cytoplasm in the form of diffusion and release proton H+ after dissociation, subsequently reducing the intracellular environment's pH value. The continuous acidification of the intracellular environment destroys DNA structures, the denaturation of proteins, and enzymes' inactivation. Moreover, changes in pH cause the channel proteins, transporters, and signaling pathway proteins on the membrane to lose their normal function that maintains cell-selective permeability, disturbing the balance of sodium-potassium ions inside and outside cells. Ultimately, the physiological activities being affected results in low production efficiency and poor product quality. In the study of succinic acid production by E. coli, with the accumulation of succinic acid, the biomass was gradually reduced, and the vitality of the somatic cells was steadily decreased. If the succinic acid is removed in time, the yield of succinic acid can be increased by more than 60% (Andersson et al. 2010). Roa et al. (2011) found that the lower the pH value, the lesser the yield of fumaric acid in the Fermentation of Rhizopus oryzae (Roa Engel et al. 2011).
Effects of pH on the gene expression of key enzymes in carbon source metabolism
Multiple metabolic pathways and enzymes regulate the growth and metabolism of bacterial cells. Under different pH conditions, the strain's ability to metabolize a specific sugar is affected by the activity of key enzymes and the strain's growth. The amount of enzyme gene expression indicates the demand for this enzyme by the metabolism of the strain. Therefore, the expression level of key enzyme genes in metabolism can reflect the metabolic level of bacteria to a certain extent.
The metabolism of carbohydrates by the strain mainly depends on the catalysis of metabolic-related enzymes. Comparative genomic analysis results revealed that the wild-type strain L. casei 12A could degrade nine sugars in cells (Wang et al. 2015). The strain first used glucokinase (GK, EC 2.7.1.2) to degrade glucose to 6-phosphate glucose, which is the common intermediate product and intersection of various metabolic pathways, including glycolysis (EMP pathway), pentose phosphate pathway, and glycogen synthesis and decomposition pathway. Therefore, GK is one of the key enzymes for glucose metabolism. Second, the strain could use phosphofructokinase (PFK, EC 2.7.1.56) to convert fructose to 1, 6-fructose diphosphate (FDP) and then facilitate its entrance into the glycolysis pathway. PFK is one of the key enzymes in fructose metabolism. Third, the strain hydrolyzed sucrose to glucose and fructose using invertase (INV, EC 3.2.1.26). INV is one of the key enzymes for the metabolism of sucrose. Two conditions of pH control and non-pH control were designed to detect three key enzymes' expression levels. This enabled us to investigate the effect of pH control on carbon source metabolism.
An in-depth analysis of the three enzymes' gene expression characteristics under two conditions showed that, on the one hand, the expression levels of three enzyme genes in the fermentation broth with regulated pH were almost more significant than those under the non-pH control state. The amount indicates that the increase in acidity inhibits the synthesis of critical metabolic enzymes (Fig 7). The genes' high expression levels suggested that the number of sugar molecules transported into the cell is large, which is beneficial to cells' rapid activation of metabolic function. Therefore, Fermentation in the medium with a constant pH of 6.0 is more conducive to the metabolism of the three sugars by the strain; on the other hand, the trend of the gene expression in the two conditions is very similar, indicating that the change in pH does not change the trend of enzyme gene expression.
In conclusion, this present study proves that L. casei, as a potential tool, can be used for bio-ethanol production due to its good tolerance of lower alcohols (Elena, 2015). Through the characteristic analysis of cell growth and metabolism, L. casei EI can metabolize sucrose, glucose, and fructose of molasses and synthesize ethanol. In practice, the engineered L. casei E1 was grown in different fermentation conditions subjected to the effects of cane molasses concentrations, oxygen, and pH on ethanol production. The genetic strain E1 showed good growth and quick sugar fermentation and yielded high ethanol in cane molasses. By fermenting this bacterium anaerobically at 37°C for 36 h incubation in 5 °BX molasses when the fermenter's pH was controlled at 6.0, ethanol yield reached 13.77 g/L, and carbohydrate utilization percentage was 78.60%.
The expression differences from the GK, INV, and PFK genes between pH-control and non-pH control conditions were detected using RT-qPCR. It could be seen that the genetic strain E1 under pH control preferentially fermented glucose and fructose of molasses to ethanol. The non-pH control did not affect the three enzymes' gene expression trends, but an elevated acidity would significantly inhibit the genetically modified strain E1 from metabolizing fructose to ethanol.