Lactic acid production from renewable carbon sources by genetic engineered bacillus subtilis and mixed culture with lactobacillus rhamnosus

Abstract

Lactic acid has been produced commercially by fermentation since 1881. The fermentative production of this product accounts for half of the world production. The remainder is produced synthetically from e.g. lactonitrile. The main users of lactic acid are the food and pharmaceutical industries, but there are technical applications as well, e.g. production of poly lactic acid (PLA). PLA can be used as cell microcarriers, bone tissue engineering etc. Batch fermentation experiments were conducted to establish optimum operating conditions for the simultaneous saccharification and fermentation (SSF) of potato starch to lactic acid using Lactobacillus rhamnosus (ATCC 10863) with immobilized Aspergillus niger (ATCC 13496). Starch was first liquefied by bacterial alpha amylase. The alpha amylase solution (59 kunit/ml) was obtained from Bacillus subtilis 105 MU331 with alpha amylase gene inserted, after 6 hours thermoinduction. Immobilized Aspergillus niger alginate beads were cultivated in a sucrose medium followed by glucoamylase induction in a starch solution. The beads and Lactobacillus rhamnosus inoculum were mixed together with fermentation broth. All SSF were kept at pH 5.0 – 5.5 adjusted by feeding calcium carbonate at 43 C. The yield of lactate based on total sugar was about 75%. Lactate productivity was 1.354 g/l/h for SSF conducted under optimum conditions with 100 g/1 potato starch; 1.389 g/l/h with initial 150 g/1 potato starch. The productivities and yields did not change with the addition of yeast extract in SSF. Bacillus subtilis is an atypical strain for lactic acid production and it is rarely used as a lactic acid producer. In its' metabolism, glucose was metabolized through glycolysis, Krebs cycle and electron receptors yielding ATP. Lactic acid was produced only when there is a lack of oxygen. The aerobic condition showed a positive influence on growth and a negative effect on lactate production. Based on this, a 2-stage fed batch fermentation was developed, the initial aerobic culture with aeration rate of 21/1/min was changed to a micro-aerobic culture with aeration rate of 0.5 1/1/min. The biomass and lactic acid concentration produced were 6.2 and 10.0 g/1, respectively. To improve the strain, UV mutagenesis was performed. Mutants were isolated by mutagenizing the parent strain Bacillus subtilis 105 MU331. After fermentation, the concentration of biomass and lactic acid of the mutants were approximately 6.4 and 21 g/1, respectively. The productivity of lactic acid is high (2.625 g/l/h), in comparison with mesophilic lactic acid bacteria. The major metabolites produced was analyzed and identified by HPLC.