Effects of carbon concentration, oxygen, and controlled pH on the engineering strain Lactiplantibacillus casei E1 in the production of bioethanol from sugarcane molasses

Abstract

Sugarcane molasses are considered a potential source for bioethanol's commercial production because of its availability and low market price. It contains high concentrations of fermentable sugars that can be directly metabolized by microbial fermentation. Heterofermentative lactic acid bacteria, especially Lactiplantibacillus casei, have a high potential to be a biocatalyst in ethanol production that they are characterized by strong abilities of carbohydrate metabolism, ethanol synthesis, and high alcohol tolerance. This study aimed to evaluate the feasibility of producing ethanol by Lactiplantibacillus casei used the ethanologen engineering strain L. casei E1 as a starter culture and cane molasses as substrate medium. The effects of environmental factors on the metabolism of L. casei E1 were analyzed by high-performance liquid chromatography (HPLC) system, and the gene expression of key enzymes in carbon source metabolism was detected using quantitative real-time PCR (RT-qPCR). Results showed that the strain could grow well, ferment sugar quickly 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%. RT-qPCR results verified the strain preferentially ferment glucose and fructose of molasses to ethanol at the molecular level. In addition, the metabolism of sugars, especially fructose, would be inhibited by elevating acidity. Our findings support the theoretical basis for exploring Lactic acid bacteria as a starter culture for converting sugarcane molasses into ethanol.

Keywords: Bioethanol; Fermentation; Lactiplantibacillus casei; Sugarcane molasses.

Fig 1

Growth of strain in different concentrations of cane molasses

Fig. 2

Carbohydrate utilization in different concentrations of cane molasses. Carbohydrates tested: Sucrose , Glucose , Fructose . a 5 °BX; b 7.5 °BX; c 10 °BX; d 20 °BX

Fig. 3

Products in different concentrations of cane molasses. Products tested: Lactate , Acetate , Ethanol . a 5 °BX; b 7.5 °BX; c 10 °BX; d 20 °BX

Fig. 4

The products in aerobic (a) and anaerobic (b) fermentation. Products tested: Lactate , Acetate , Ethanol

Fig 5

Effect of pH control and non-pH control on the growth of strain. pH changes under non-pH control

Fig. 6

Effect of pH control (a) and non-pH control (b) on carbohydrate utilization. Carbohydrates tested: Sucrose , Glucose , Fructose

Fig 7

Effect of pH control and non-pH control on key enzyme gene expression of carbon source metabolism. a GK, glucokinase; b INV, invertase; c PFK, phosphofructokinase

Fig 8

RT-qPCR analysis of key enzyme gene expression under pH control and non-pH control . INV, invertase; GK, glucokinase; PFK, phosphofructokinase. a 4 h; b 8 h; c 12 h; d 16 h; e 24 h