Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

Abstract

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.

Introduction

Lactic acid is a simple compound containing both hydroxyl and carboxylic acid groups, and it has been produced commercially by fermentation since 1883. Recently, there has been an increasing interest in its polymer polylactic acid as a raw material for biodegradable and renewable plastics, which will play an important role in reducing the dependence on petroleum-based plastics. However, lactic acid production suffers from the too high cost relative to that of petroleum-based plastics. Hence, recent research on the fermentative production of lactic acid has been focused on reduction of material cost and improvement of fermentation performance. To reduce the material cost, a large number of carbohydrate materials have been used, tested or proposed for lactic acid production. However, one should note the fact that nutrient cost, such as the cost of yeast extract (YE), is much higher than that of a carbohydrate material, and 90% of lactic acid is produced by lactic acid bacteria, which need specific minerals, B-vitamins, and several amino acids to ensure their optimum growth. The use of low-cost nutrients, such as whey protein hydrolyzate [1], [2] and soy protein hydrolyzate [3], could reduce the nutrient cost, however, supplementation of very large amounts of them causes an increase in the concentration of impurities, with a corresponding increase in separation cost and the decrease in lactic acid recovery [4], [5].

As is well known, yeasts hardly produce lactic acid because they have no lactate dehydrogenase (LDH) gene. In our previous study, we have developed a lactic acid producing yeast by introducing six copies of the bovine L-LDH gene into the genome of Saccharomyces cerevisiae [6], [7]. This transgenic yeast can produce lactic acid efficiently in a poor medium and the lactic acid produced is of high optical purity, i.e., above 99.9%, indicating its potential to replace common lactic acid bacteria. Until now, however, only basic investigations on this transgenic yeast have been conducted, involving a batch mode fermentation, to confirm the possibility of lactic acid production. For that reason, further investigation is needed to improve the fermentation performance.

Cell immobilization is an approach for improving the fermentation performance because immobilized cells exhibit many advantages over free cell, such as easier separation of the product and operation of the bioreactor, higher productivity and the reuse of cells for long-term lactic acid production. Commonly, cell immobilization is achieved by cell entrapment [8], [9], [10], [11], cell attachment [12], [13] or the formation of cell pellets [14], [15]. Entrapment in Ca-alginate gel is most widely used procedure for cell immobilization [10], [11].

Photo-crosslinkable resin generally has a main chain of polyethyleneglycol or polypropyleneglycol, which has photo-crosslinkable ethylenic double bonds at its ends. Three dimensionally crosslinked gels are formed by irradiating actinic ray to the ethylenic double bonds [16], [17]. The resin gels have a chemically, mechanically and thermo-stable matrix. Moreover, their sizes can be changed by changing the length of the chain between the double bond bases. Although there have been some studies on ethanol production with a photo-crosslinkable resin based immobilized cells, there have not been any investigations on lactic acid production with the immobilization method. Therefore, we selected photo-crosslinkable resin gels for immobilization of the engineered yeast. Since the engineered yeast can efficiently produce high optical purity lactic acid in a nutrient-poor medium, e.g., a molasses medium, low-cost production of high optical purity lactic acid would be more possible when the productivity of the engineered yeast is enhanced. Therefore, the main objective of this study was to investigate the possibility of using the engineered yeast immobilized in photo-crosslinkable resin gels for lactic acid production by testing the operational stability of immobilized cells and discussing the performance of immobilized cells in lactic acid production.