Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source

Abstract

Efficient utilization of crude glycerol, a by-product from biodiesel production, could bring significant economic and environmental benefits. In this work, a low-grade glycerol was used as the sole carbon source in phytase production with recombinant Pichia pastoris possessing a pGAP-based constitutive expression vector. In batch and fed-batch modes, the effects of important cultivation conditions were investigated using both analytical and biodiesel glycerols in batch and fed-batch fermentations. The following factors were analyzed: initial glycerol concentration, dissolved oxygen level, and the effect of feeding strategy on cell growth biomass and protein production. Significant cell growth inhibition was observed in batch fermentation when the initial crude glycerol concentration was as high as 70 g/L. However, such inhibition was overcome in fed-batch mode by starting the cultivation with a lower crude glycerol level. Finally, cell densities and phytase activity levels of as high as 146 g (dry cell weight)/L broth and 1125 U/mL supernatant were achieved in the fed-batch fermentation with crude biodiesel glycerol as the sole carbon source. The study has proven the potential of using crude glycerol from biodiesel production as the carbon source for industrial scale phytase mass production in high cell density fermentations with recombinant P. pastoris.

Introduction

The surging price of fossil hydrocarbons and increasing concerns about the environment have led to the rapid growth of biodiesel production worldwide. It is estimated that the annual production capacity of biodiesel in the US alone will reach 2.24 billion gallons in 2008 [1]. Glycerol is the main, by-product of the conversion of vegetable oils into biodiesel, comprising approximately 10% by mass of the vegetable oil fed to the system [2]. The increased production of biodiesel has caused a sudden increase in production of glycerol creating a glut in the glycerol market [3]. The price of high purity glycerol in the United States plunged from US$1.00/lb in 1995 to less than US$0.40/lb in 2005 [4] and it is suggested that the price of crude glycerol might become stable at as low as $0.05/lb [5]. It is believed that refining crude glycerol to high purities is too costly and energy-intensive; therefore, it is urgent to discover innovative utilizations for crude glycerol that will make biodiesel production more profitable and sustainable. Researchers around the world are currently looking at the thermal, chemical, and biological conversion of crude glycerol to a variety of value-added products. One application that has been evaluated is the potential of using crude glycerol from biodiesel in animal feeds [6]. Another application for crude glycerol that is currently being investigated is the fermentation of glycerol to 1,3-propanediol, an intermediate compound for the synthesis of polymers used in cosmetics, foods, lubricants, and medicines [7], [8].

Glycerol has long been used as a major carbon source in culture medium for the cultivation of microorganisms in industrial fermentation. Less expensive carbon sources, such as glucose, have limited the use of glycerol in fermentations. Now that glycerol has decreased in price, as a result of a dramatic increase in biodiesel production, its use as a carbon source needs to be re-evaluated. If feasible, large-scale cultivation in industry will create a substantial demand for glycerol.

Phytase is an important industrial enzyme and is used as an animal feed additive in diets largely for swine and poultry, and to some extent for fish [9]. The whole market volume of phytase was estimated to be in the range of 150 million£ [10]. Adding phytase to the diets of monogastric animals can improve the uptake of phosphate from phytate, therefore effectively improving nutrient digestion and a reduced need for the supplementation of external phosphate in their diets. Furthermore, the breakdown of phytate liberates chelated minerals such as calcium, magnesium, iron, and zinc. Meanwhile, the release of phosphate from phytate will reduce the pollution caused by undigested phytate remaining in animal feces. The promising application of phytase in conjunction with environmental concerns has led to studies concerning phytase technology and phytase production.

Pichia pastoris has become a popular host for the expression and mass production of industrial enzymes, including phytase. Traditionally, P. pastoris cultivation is performed in fed-batch fermentation using the methanol-inducible system, an AOX1-based expression system. In this system, excessive accumulation of methanol suppresses cell growth, making process control very difficult [11]. Another strong expression system, pGAPZ – based system, is reported to produce protein at a comparative level to the AOX1 – based system, although the level appeared to vary depending on the protein being expressed and the carbon source used for cell growth [12]. For the pGAPZ-based system, foreign protein was expressed constitutively without induction using methanol, which is costly and hazardous to handle in large volumes [13].

This research will study the feasibility of using crude glycerol as the sole carbon source for P. pastoris cultivation using a pGAPZ-based expression vector. The goal is to propose a new way to use crude glycerol from biodiesel, a waste product, to produce economically useful products on a large-scale.