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Effect of Rare Earth Elements on Vitamin C Fermentation by Mixed Cultures.

Byline: Shuxia Lyu Zhiyong Guo Jian Pan Yu Yang Weichao Yang Hongquan Chen and Zhongze Zhang

Abstract

In recent years the research on rare earth metals has expanded to the field of microbiology. In China the production of Vitamin C is through mixed culture fermentation which transforms L-sorbose to 2-keto-L-gulonic acid (2-KGA) the precursor of Vitamin C. In this study we compared the yields of 2-KGA by an acid-producing strain of Ketogulonigenium vulgare that was co-cultured with different companion bacterial strains in the presence of four light rare earth elements lanthanum cerium neodymium and samarium at selected concentrations. We found that all of the tested rare earth elements had a promoting effect on 2-KGA production at concentration up to 5 mM. At or above 10 mM the yield of 2-KGA was reduced. When cultured separately with 5 mM of given rare earth elements the growth of two companion bacterial strains (Bacillus megaterium 25-B and B. subtilis A9) were both significantly reduced while that of the K. vulgare strain was enhanced. Copyright 2014 Friends Science Publishers

Keywords: 2-keto-L-gulonic acid; Ketogulonigenium vulgare; Companion strain; Mixed cultures; Rare earth elements

Introduction

The mixed culture fermentation of L-sorbose for producing 2-keto-L-gulonic acid (2-KGA) the precursor of Vitamin C (Vc) is the main method of industrial Vc production in China (Zhang et al. 2008 2010; Ai et al. 2013). The mixed culture fermentation is realized by the cooperation of two microorganisms: Ketogulonigenium vulgare and Bacillus megaterium. As a Gram-negative bacterium the strain of K. vulgare is usually referred to as the acid-producing strain which grows poorly and its production of 2-KGA is low under K. vulgare mono-culture system; As a Gram-positive bacterium the strain of B. megaterium is the companion strain (or co-culture helper) which promotes the growth and 2-KGA production of K. vulgare in mixed culture fermentation of vitamin C (Feng at al. 1998).

Rare earth elements (REEs) also known as lanthanides are a group of 15 metals with similar properties which are used for a broad spectrum of applications in industry agriculture and medicine. REEs also affect the growth of microorganisms. Generally low concentrations of REEs promote and high concentrations of REEs inhibit the growth of microorganisms (Tang et al. 2001; Wang et al. 2005; Jiang et al. 2008).

In this study an acid-producing strain of K. vulgare was cultured with two different companion bacterial strains B. megaterium 25-B and B. subtilis A9. Four light REEs lanthanum cerium neodymium and samarium were added at selected concentrations to the mixed cultures. Our results demonstrated that these light REEs could increase the yield of 2-KGA significantly. These results of the experiments could provide critical information that can be referenced for large-scale production of vitamin C.

Materials and Methods

Materials

The acid producing strain of K. vulgare and the companion strain of B. megaterium 25-B used in this study for vitamin C fermentation were provided by Northeast Pharmaceutical General Factory of Vitamin C Company (Shenyang China). The B. subtilis A9 strain was from Shenyang Agriculture University collection. The slant agar in g/L consisted of: L-sorbose 5; peptone 10; corn steep liquor 5; yeast extract 3; beef extract 3; MgSO4 0.2; agar 25. The isolation medium in g/L consisted of: L-sorbose 20; peptone 10; corn steep liquor 3; yeast extract 3; beef extract 3; urea 1; MgSO4 0.2; KH2PO4 1; CaCO3 1; agar

25. The seed medium in g/L consisted of: L-sorbose 20; glucose 2; corn steep liquor 5; urea 1; CaCO3 1. The companion strain medium in g/L consisted of: glucose 2; corn steep liquor 5; urea 1. The fermentation medium in g/L consisted of: L-sorbose 90; corn steep liquor 10; urea 12; KH2PO4 1; MgSO4 0.2; CaCO3 1.

Methods

Fermentation conditions used in this study consisted of 5 mL of fermentation medium mixed with 10% (v/v) of seed culture in a 50 mL flask containing different concentrations of the four rare earth elements. The inoculated mixed culture was incubated at 29oC for 24~72 h with orbital shaking aeration at 180 rpm. The initial culture acidity was adjusted to pH 6.8~7.0 (Gao et al. 2012). The 2-KGA concentration was determined using the iodometric method as previously described (Chen and Yan 1981) with three independent repetitions. The conversion rate of 2-KGA was calculated using the formula:

Equation

Where: 1.0776 is the relative molecular mass ratio of 2-KGA and L-sorbose.

Statistical Analysis

Design of the experiments was completely randomized with three to five replications. Statistical analyses for all experiments were performed using the Proc Mixed model (general linear model GLM) of SAS 9.3 (SAS Inst. Inc. Cary NC.). When effect(s) were statistically significant mean comparisons were performed with Sidak adjusted P values to maintain experiment-wise error (a) at 0.05.

Results

Effect of REEs on the Yield of 2-KGA with megaterium 25-B as Companion Strain

For each of the light REEs concentrations of 1 5 10 and 15 mM were tested for the effect on the yields of 2KGA by K. vulgare cultured with companion strain of B. megaterium 25-B for three independent repetitions. Similar effects on the yields of 2-KGA were observed for all of the four tested REEs (Fig. 1). At concentrations up to 5 mM the yields of 2-KGA increased comparably for all four REEs. The average increases of 2-KGA yields were 13.6 and 25.6% in the presence of 1 and 5 mM of REEs respectively. The highest yield of 2-KGA was obtained with the addition of 5 mM of La3+ which reached 73.4 g/L. In comparison to the control (54.4 g/L) this represented a 34.9% increase of 2-KGA yield. The calculated conversion rate (from L-sorbose to 2-KGA) increased from 63.1 to 87.8%. At or above concentrations of 10 mM each of the four REEs resulted in a sharp decrease in 2-KGA yields.

Addition of each of the light REEs at 10 mM resulted in a drop of 2-KGA yields to approximately 25% of that of the control.

Yield of 2-KGA by K. vulgare stimulated by 5 mM of La3+ with B. subtilis A9

With the concentration of each of the REEs from 0~5 mM the curves of the yield of 2-KGA are similar to Fig. 1. With three independent repetitions the yield of 2-KGA increased with the addition of up to 5 mM of each of the REEs. When the concentration of REEs was 5 mM the yield of 2-KGA was maximum. When REE was added at concentration at or above 10 mM the amount of 2-KGA decreased significantly (Fig. 2). The yield of 2-KGA with the addition 5 mM of La3+ reached 83.5 g/L in contrast to the control which yielded 72.1 g/L and resulted in the conversion rate (from L-sorbose to 2-KGA) increasing from

86.3 to 99.98%. Of interest the yield of 2-KGA by K. vulgare companion with B. subtilis A9 increased 13.8% than that of K. vulgare cultured with companion strain of B. megaterium 25-B. This suggested that the companion strain B. subtilis A9 plays more important role in stimulating K. vulgare to produce 2-KGA than that of B. megaterium 25-B.

Effect of Rare Earth Elements on the Growth of K. vulgare and Companion Bacterial Strains

The effect of the REEs was further examined by cultivating K. vulgare and the companion strains of B. megaterium 25B and B. subtilis A9 individually in the presence or absence of 5 mM of the REEs respectively. In five independent repetitions the growth of the K. vulgare strain was all significantly stimulated by the addition of REEs in the seed medium (Fig. 3) but that of both B. megaterium 25-B and B. subtilis A9 were strongly inhibited (Fig. 4 and 5). In the absence of a companion strain 2-KGA production by K. vulgare in the fermentation medium is less than 10% of that in the presence of B. megaterium 25-B or B. subtilis A9.

Although adding 5 mM of La to K. vulgare culture stimulated 2-KGA production the yield of 2-KGA was much higher in the mixed cultures of K. vulgare and companion strain of 25-B (or A 9) under the same condition (Fig. 6).

Discussion

Three bacterial strains used in this study K. vulgare B. megaterium 25-B and B. subtilis A9 appeared to have different capacities of accumulating REEs (Tsuruta 2006). With the concentrations of La3+ at 5 mM the growth of K. vulgare was promoted while that of B. megaterium 25-B and B. subtilis A9 strongly inhibited. However it was previously shown that the growth of B. megaterium 25-B and B. subtilis A9 was promoted at much lower concentrations of La3+ (Yang et al. 2008). Therefore it can be concluded that 5 mM of La3+ exceeded the tolerance limits of these strains.

Acknowledgements

The authors wish to thank Dr. R.E. Levin (University of Massachusetts Amherst MA USA) for critical reading and linguistic revision of the manuscript. The authors are also grateful to the Northeast Pharmaceutical Group General Factory of Vitamin C Company (Shenyang P. R. China) for providing bacterial strains. Funding was provided by the National Natural Science Foundation of China (31370077).

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Article Details
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Author:Shuxia Lyu; Zhiyong Guo; Jian Pan; Yu Yang; Weichao Yang; Hongquan Chen; Zhongze Zhang
Publication:International Journal of Agriculture and Biology
Article Type:Report
Geographic Code:9CHIN
Date:Dec 31, 2014
Words:2096
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