Evaluation of fungal lactic acid accumulation using glycerol as the sole carbon sourceby Xiaoqing Wang, Zhenhua Ruan, Webster Guan, Robert Kraemer, Yuan Zhong, Yan Liu

Biotechnology and Bioprocess Engineering


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Biotechnology and Bioprocess Engineering 20: 389-395 (2015)

DOI 10.1007/s12257-014-0799-5

Evaluation of Fungal Lactic Acid Accumulation Using Glycerol as the

Sole Carbon Source

Xiaoqing Wang, Zhenhua Ruan, Webster Guan, Robert Kraemer, Yuan Zhong, and Yan Liu

Received: 16 November 2014 / Revised: 28 March 2015 / Accepted: 1 April 2015 © The Korean Society for Biotechnology and Bioengineering and Springer 2015

Abstract This study investigated the glycerol utilization for lactic acid accumulation by the filamentous fungus

Rhizopus oryzae 9363 and compared it with the conventional carbon source glucose. No lactic acid accumulated in the glycerol media at 30oC, in contrast to the glucose media.

Increasing the temperature from 30 to 37oC led to a 63% decrease in the average growth rate of R. oryzae in glycerol media and a 61% increase in the average cel mass yield, and the cultures on glycerol media at 37oC were able to generate 0.6 g/L lactic acid. While, raising temperature significantly inhibited lactic acid production on glucose media.

Moreover, supplementing cultures with sodium pyruvate significantly improved the lactic acid synthesis of R. oryzae on glycerol media, with lactic acid concentrations reaching 1.33 g/L at 37oC and 0.67 g/L at 30oC, respectively. Our results indicate that glycerol utilization for lactic acid accumulation by Rhizopus sp. is limited by the availability of intracelular pyruvate, and controling pyruvate flow is a key to enhancing the lactic acid accumulation.

Keywords: Rhizopus oryzae, lactic acid, glycerol, pyruvate 1. Introduction

Glycerol has recently gained significant atention as an atractive feedstock for value-added chemical production due to its abundance and relatively cheap price resulting from the rapid growth of the biodiesel industry [1,2].

Compared to the chemical and thermal processes of glycerol utilization, microbial fermentation provides an environmentaly friendly approach to convert glycerol into valueadded products. Many microorganisms are able to metabolize glycerol as part of fine chemical production processes, such as 1,3-propanediol production from Clostridium Butyricum and Klebsiela Pneumoniae, dihydroxyacetone from Gluconobacter oxydans, and Klebsiela pneumoniae, succinic acid from Anaerobiospirilum succiniciproducens, hydrogen from Enterobacter Aerogenes, and D-lactic acid from

Escherichia coli [3]. However, limited information is available about the glycerol utilization of filamentous fungi [4-6]. The filamentous fungus Rhizopus oryzae was selected to investigate fungal glycerol utilization for lactic acid accumulation.

Lactic acid has been widely used in the food industry as an acidulant, flavoring, and preservative [7]. Over the past ten years, ecological concerns have driven its use as a substrate in the production of the biodegradable plastic poly-lactic acid, which requires a highly purified, preferably

L-(+)-lactic acid anhydrous monomer [8]. Compared to the bacterial synthesis of lactic acid isomers, the fungus

R.oryzae is able to synthesize opticaly pure L-(+)-lactic acid in relatively simple media [9]. Various carbon sources such as starch and pentose have been studied for their efects on lactic acid production by R. oryzae [10]. However, limited information is available regarding glycerol utilization by R. oryzae for lactic acid production.

Therefore, the objectives of this study were to investigate the glycerol conversion to lactic acid by R. oryzae and to evaluate the main factors that influence the eficiency of lactic acid accumulation.

Xiaoqing Wang, Zhenhua Ruan, Robert Kraemer, Yuan Zhong, Yan Liu*

Biosystems and Agricultural Engineering, Michigan State University, East

Lansing, MI 48824, USA

Tel: +517-432-7387; Fax: +517-432-2892

E-mail: liuyan6@msu.edu

Webster Guan

Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

RESEARCH PAPER 390 Biotechnology and Bioprocess Engineering 20: 389-395 (2015) 2. Materials and Methods 2.1. Fungal strain and spore inoculum preparation

The Strain Rhizopus oryzae NRRL 395 (ATCC 9363), obtained from American Type Culture Colection (Manassas,

VA, USA), was grown on potato dextrose agar at 30oC for two weeks. Spores were washed as described previously [11], and the spore solution was stored at a concentration of 1×108 spores/mL at 4oC. 2.2. Cultivation

The salts used for batch culture include: KH2PO4 (1 g/L) (Malinckrodt Bakker); MgCl2·6H2O (0.5 g/L) (Malinckrodt

Bakker); ZnSO4·7H2O (1.4 mg/L) (Sigma); MnSO4·H2O (1.6 mg/L) (Sigma); CoCl2·6H2O (3.6 mg/L) (Sigma);

FeSO4·7H2O (2.7 mg/L) (Sigma). The sole nitrogen source for al experiments was 1 g/L NH4Cl (Sigma). Glucose (Sigma) and glycerol were used as carbon sources, with initial concentrations of 10, 30, 50, and 70 g/L. To initiate the culture, 0.5 mL spore solution was inoculated into 250 mL Erlenmeyer flasks filed with 100 mL of growth medium and grown on a rotary shaker (Thermo Scientific) with shaking at 180 rpm at 30 or 37°C. During the cultivation, CaCO3 was added to the broth to maintain the pH at approximately 4 ~ 5. 2.3. Analytical methods

Mycelia cel mass was colected by filtration, washed with 6 mol/L HCl to neutralize the excess CaCO3 in the cel mass, and then washed with distiled water. The washed cel mass was dried at 92.5 ± 0.5°C overnight until a constant weight was achieved. Glucose, glycerol, ethanol, lactic acid and other organic acids were detected by HPLC equipped with a Bio-rad Aminex HPX-87H organic acid column and a refractive index detector [12]. The mobile phase was 5mmol/L sulfuric acid at a flow rate of 0.6 mL/min.

The column temperature was set to 65°C. 2.4. Reductive lactic acid dehydrogenase activity assay

Lactic dehydrogenase (LDH) activity is traditionaly assayed at an absorbance of 340 nm to monitor the decrease in the reduced nicotinamide adenine dinucleotide (NADH) concentration during the reaction [13]. One unit (U) of enzymatic activity was defined as the amount of enzyme necessary to convert 1 µmol of NADH to NAD+ per min [13]. However, this method is not suitable for analyzing the activity of crude enzyme extracts, as other oxido-reductases such as pyruvate decarboxylase and alcohol dehydrogenase in the extracts may also afect the NADH concentration and interfere with the spectrophotometric readings [13].