Highly Efficient Erythritol Recovery from Waste Erythritol Mother Liquor

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Highly Efficient Erythritol Recovery from Waste Erythritol Mother Liquor by a Yeast-mediated Biorefinery Process Siqi Wang, Hengwei Wang, Jiyang Lv, Zixin Deng, and Hairong Cheng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04112 • Publication Date (Web): 08 Dec 2017 Downloaded from http://pubs.acs.org on December 10, 2017

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Journal of Agricultural and Food Chemistry

Highly Efficient Erythritol Recovery from Waste Erythritol Mother Liquor by a Yeast-mediated Biorefinery Process Siqi Wang1, Hengwei Wang2, Jiyang Lv1, Zixin Deng1, Hairong Cheng1* 1

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China 2 Innovation and Application Institute (IAI), Zhejiang Ocean University, Zhoushan 316022, China 1

1

ACS Paragon Plus Environment


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ABSTRACT

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Erythritol, a natural sugar alcohol, is produced industrially by fermentation and

4

crystallization, but this process leaves a large amount of waste erythritol mother liquor

5

(WEML) which contains more than 200 g/L erythritol as well as other polyol

6

by-products. These impurities make it very difficult to crystallize more of erythritol.

7

In our study, an efficient process for the recovery of erythritol from the WEML is

8

described. The polyol impurities were first identified by HPLC and GC-MS, and a

9

yeast strain Candida maltose CGMCC 7323 was then isolated to metabolize those

10

impurities so as to purify erythritol. Our results demonstrated that the process could

11

remarkably improve the purity of erythritol, and thus made the subsequent

12

crystallization easier. This newly developed strategy is expected to have advantages in

13

the WEML treatment and provide helpful information with regards to green cell

14

factories and zero-waste processing.

15 16 17

KEYWORDS: KEYWORDS Erythritol, waste mother liquor, Candida maltosa, Yarrowia lipolytica, green cell factories.

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INTRODUCTION

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Sugar polyols are a class of polyhydroxyl compounds which lack the reducing

21

groups required for cell growth, carbon storage and fixation in many microorganisms.

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Some sugar polyols are currently used as food additives and sweeteners because they

23

are non-cariogenic and safe for people with diabetes. Among these sugar polyols,

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erythritol has been produced industrially by microbial fermentation of glucose for

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more than half a century.

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Erythritol, a simple polyol with four carbons each with a hydroxyl group, is

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approximately 75% as sweet as sucrose and exists naturally in many fruits, fermented

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foods, seaweed and mushrooms.1 Over 90% of erythritol absorbed in the human

29

gastrointestinal tract is excreted from the body directly without being metabolized.

30

Erythritol tastes sweet and leaves no bitter aftertaste and has a lower energy content

31

than table sugar (0.2 vs. 4 kcal/g), thus it may be used in conjunction with other

32

intense sweeteners, such as aspartame, a chemical sweetener with a bitter aftertaste.2

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Due to the above advantages, erythritol has been widely used as an ingredient in foods,

34

beverages and pharmaceuticals.3-5 Furthermore, erythritol can be used as an

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intermediate in the synthesis of the anti-aging ingredient mannosylerythritol lipid that

36

is commonly used in cosmetics and quasi-drugs.6

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Though erythritol can be secreted by various yeasts and bacteria,7 commercially

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it is only produced by fermentation using strains with the highest yields, such as

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Aureobasidium (44%),8 Torula corallina (48.9%),9 Torula sp. (48%),10 Candida

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magnoliae (43%),11,12 Moniliella sp. (39.4%),13 Pseudozyma tsukubaensis (61%),14

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Trichosporonoides megachiliensis (47%)15 and Yarrowia lipolytica (44% for glycerol

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and 60% for glucose).16,17 During erythritol production, some by-products such as

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glycerol, ribitol, mannitol, D-arabitol, fumarate and citrate are produced, depending

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on different strains.13,18 After fermentation, erythritol is purified by crystallization

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from the concentrated fermentation supernatants. This process leaves a large amount

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of waste erythritol mother liquor (WEML), a viscous and reddish-brown liquor also

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known as waste molasses, which contains multiple organic components. The WEML 3



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contains erythritol (30~ 40% of the total soluble solids) as well as other waste polyols

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and is of low cost and difficult to deal with. In China, one of the main

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erythritol-producing countries, approximately 10,000 tons of erythritol and more than

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2,000 metric tons of WEML were produced in 2016, which leads to environmental

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concerns associated with its disposal.

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Attempts have been made to increase the value of this waste mother liquor by

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individually separating erythritol and other polyols by simulated moving bed

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chromatography (SMBC). However, this method has not been readily adopted due to

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its high equipment investment and running costs and poor separation efficiency.

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Currently, the difficulties in WEML treatment still remain a challenge.

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In recent years, biological removal (or bioremoval) and biotransformation have

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become attractive approaches due to their high efficiency and specificity, especially,

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in the environmental remediation and recovery of high-value compounds from crude

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sugar feedstocks.19,20 During the past six years, our group has developed various

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strategies for the recovery of L-arabinose and L-arabitol from waste xylose mother

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liquor (WXML) and xylitol mother liquor, and for the production of xylitol from

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WXML by one-pot process. 21-24

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In the present study, we aimed to develop an efficient procedure to purify

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erythritol from WEML. To achieve this purpose, we first identified the polyol

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impurities in WEML, then a yeast strain, Candida maltose SJTU828 (also CGMCCC

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7323) was isolated to biologically enrich erythritol in WEML by depleting the polyol

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impurities. We also studied our newly developed bioremoval strategy in shake flasks

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and fermentors, and discussed its advantages in the WEML treatment and green cell

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factories with regards to zero-waste economy.

72 73

MATERIALS AND METHODS

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Preparation of WEML in laboratory

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The WEML samples were prepared through two rounds of crystallization of

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erythritol from fermentation supernatants. The fermentation medium contained 6 g/L 4


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yeast extract, 4 g/L peptone, 2.5 g/L (NH4)2HPO4, 2 g/L KH2PO4, and 0.2 g/L

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MgSO4·7H2O supplemented with 260 g/L glucose (pH 3.5). The fermentation was

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carried out using Y. lipolytica BLC13 at 30 °C, 350 rpm and 1.5 vvm aeration in a 5-L

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fermentor. After fermentation, the yeast cells were removed by centrifugation, and the

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supernatant was decolorized by mixing with activated carbon at 80 °C. The obtained

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colorless fermentation broth was then clarified by removal of the activated carbon via

83

filtration and deionized using ion-exchange resin columns. The success of ion

84

removal was verified by a conductivity test (≤ 100 µs/cm). The solution was then

85

concentrated until 70% (w/v) soluble solids and cooled gradually from 80 °C to 4 °C

86

to allow erythritol crystals to precipitate. The erythritol crystals were then removed by

87

centrifugation. The subsequent clear supernatant was further concentrated to 70%

88

(w/v) soluble solids and cooled gradually from 80 °C to 4 °C to allow another round

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of erythritol crystallization. After two rounds of crystallization, the concentration of

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erythritol in the solution decreased remarkably, the content of impurities increased,

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and erythritol crystals could no longer formed in the solution when cooling. The

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solution was designated as WEML, and it was found to have approximately 520 g/L

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total soluble solids and a pH of 3.0~ 3.5.

94 95

Identification of Polyol Ingredients

96

The concentrations of sugar and sugar alcohols were analyzed by high

97

performance liquid chromatography (HPLC) system equipped with a Shodex RI 101

98

refractive index detector. HPLC analysis was performed on a Shodex SP0810 sugar

99

column (8 × 30 mm, Pb2+ cation exchange column) using distilled water as the mobile

100

phase at a flow rate of 1.0 mL/min at 70 °C. The fractions with the same retention

101

times as the standards erythritol and D-arabitol (13.9 and 18.7 min, respectively) were

102

collected, lyophilized and also analyzed by thin-layer chromatography (TLC) and gas

103

chromatography-mass spectrometer (GC-MS).

104

High-performance TLC on silica gel plates was used to compare the Rf values of

105

the HPLC fractions with those of the standards. The silica plates were developed in a

106

chamber using a solution of pyridine:ethyl acetate:acetate:water (5:5:3:1, v/v). Then, 5



107

the plates were dried and sprayed with 1% sodium periodate and a color developing

108

agent (1% benzidine in 95% ethanol).

109

To conduct GC-MS analysis, the lyophilized samples were dissolved in 100 µL

110

anhydrous pyridine in 2-mL brown tubes; an equal amount of derivatizing agent,

111

N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA), was added and the tubes were

112

heated at 80 °C for 45 min. Then, aliquots of the derivatized samples (1 µL) were

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subjected to GC-MS analysis. All GC-MS experiments were performed on an Agilent

114

gas chromatograph interfaced with an Agilent ion trap mass spectrometer (Agilent

115

6850/5975C). The analytical column was a HP5-MS column (30 m×0.32 mm i.d.,

116

0.25 µm) with highly pure helium as the carrier gas at a flow of 1 mL/min. After

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separation on the column, the ionized samples were analyzed by a mass selective

118

detector (MSD). The temperature program was as follows: initially, 60 °C for 1 min;

119

increased to 280 °C at 60 °C/min and held for 5 min; and then increased to 30 °C at

120

20 °C/min and held for 2.5 min. Peaks were identified by comparing their retention

121

times with those of internal standards in the selected ion monitoring (SIM) mode. The

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most intense ions were at m/z 217 for D-arabitol and m/z 61 for xylitol with a dwell

123

time of 50 ms per pixel.

124 125

Screening of Yeast Strains to Purify Erythritol in the WEML

126

After identification of the polyol components in the WEML, we screened yeast

127

strains that could utilize the impurities (ribitol, glycerol, D-arabitol, and mannitol) but

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not the target erythritol. Those strains could therefore be used to improve the relative

129

content of erythritol and facilitate its crystallization. The solid medium used for the

130

screening contained 6.7 g/L yeast nitrogen base medium (Difco, USA) and 15 g/L

131

agar (YNB), supplemented with 10 g/L of either glucose (YNG), glycerol (YNGy),

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erythritol (YNE), ribitol (YNR), D-arabitol (YNA), or mannitol (YNM).

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Approximately 400 yeast strains from our laboratory stock were screened by

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inoculating each strain on the above solid media and cultured at 30 °C for 3~ 5 days.

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The cultures that could grow on other media but not on YNE were then inoculated 6


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into solid YNB medium supplemented with 40% (v/v) WEML at pH 5.0 and cultured

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at 30 °C for another 7 days. The culture that showed the highest growth rate based on

138

its colony size was selected. The strain was then grew on a solid medium containing

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40% (v/v) WEML at pH 5.0, 10 g/L yeast extract, 10 g/L tryptone and 20 g/L agar

140

(W40YT) for 30 rounds to further improve its adaptability to the WEML.

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For the taxonomic identification of this yeast strain, genomic DNA was extracted

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according to the simple phenol lysis method to amplify partial sequences of its 18S

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rDNA and internal transcribed space (ITS)1-ITS4 DNA 25. The primers used for the

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amplification of 18S rDNA were 5’-ATC CTG CCA GTA GTC ATA TGC TTG TCT

145

C-3’ and 5’-GAG GCC TCA CTA AGC CAT TCA ATC GGT A-3’, while those for

146

the ITS1-ITS4 (partial 18S-5.8S-partial 28S sequence) were 5’-TCC GTA GGT GAA

147

CCT GCG G-3’ and 5’-TCC TCC GCT TAT TGA TAT GC-3’. The PCR conditions

148

were as follows: 95 °C for 3 min, 30 cycles of denaturation at 94 °C for 35 s,

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annealing at 56 °C for 45 s, extension at 72 °C for 60 s, and a final extension at 72 °C

150

for 10 min. Each PCR product was independently ligated into T-vector and sequenced.

151

A homology search was performed using the basic local alignment tool (BLAST)

152

available

153

(https://www.ncbi.nlm.nih.gov/).

from

the

National

Center

for

Biotechnology

Information

154 155

Staining of Polyol Dehydrogenases

156

The above identified yeast strain was cultured in a liquid medium containing 10

157

g/L yeast extract and 5 g/L tryptone supplemented with either 20 g/L glucose,

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glycerol, ribitol, D-arabitol, sorbitol, or mannitol at 30 °C and 200 rpm for 2 days.

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The cells were harvested by centrifugation at 5000 g for 10 min, washed twice with

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100 mM Tris-HCl buffer (pH 8.5) and then disrupted by ultrasonication. The samples

161

were then clarified by centrifugation at 10,000 g to remove the insoluble cell debris.

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The sample protein contents were quantified with a Lowry assay kit (Takara, Dalian,

163

China) using bovine serum albumin (BSA) as standard. To stain polyol

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dehydrogenases, 100 µg of protein was subjected to native polyacrylamide gel 7



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electrophoresis (PAGE). After electrophoresis, the gels were incubated in an assay

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mixture containing either glycerol, erythritol, ribitol, D-arabitol, sorbitol, or mannitol,

167

until appearance of blue bands. The staining was according to the method described

168

by Birken and Pisano (1976) with some modifications.26 The staining mixture

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consisted of 50 mL of 100 mM Tris-HCl buffer (pH 8.5), 25 mg of nitroblue

170

tetrazolium (NBT), 3 mg of phenazine methosulfate (PMS), 30 mg of NAD, and 100

171

mg of polyol as substrate.

172 173

Fermentation by Candida sp. on Different Carbon Sources

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To determine the products by the obtained yeast strain Candida sp., it was

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cultivated in shake flasks containing liquid YT medium (10 g/L yeast extract, 5 g/L

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tryptone, pH 5.0) supplement with 200 g/L of different carbon sources (either glucose,

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glycerol, ribitol, D-arabitol, or mannitol) under 32 °C and 200 rpm. Aliquots were

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periodically withdrawn from the fermentation broth. The supernatants were first

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analyzed with HPLC, and the fractions with the same retention time as erythritol were

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collected and lyophilized for GC-MS analysis.

181 182

Fermentation of Candida sp. using WEML in 500-mL Shake Flasks

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The test tube medium contained 10 g/L yeast extract, 5 g/L tryptone and 10 g/L

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dextrose (natural pH). The W25Y medium used in flask fermention contained 25%

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(v/v) WEML and 10 g/L yeast extract (pH 5.0). The Candida sp. cells were first

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incubated in 50-mL test tubes at 32 °C and 200 rpm for 24 h. To start the fermentation,

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the cells were collected by centrifugation and transferred into 100 mL of W25Y

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medium in 500-mL baffled shake flasks. The cells were cultivated at 32 °C and 200

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rpm. Samples were periodically withdrawn at 12-h intervals and analyzed by HPLC.

190 191

Fermentation of Candida sp. using WEML in a 150-L Fermentor

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In factory, the yeast Candida sp. was first grown on slants at 30 °C for 3~ 5 days.

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The slant medium contained 25% (v/v) WEML, 5 g/L yeast extract, 5 g/L corn steep

194

powder and 20 g/L agar and was sterilized in eggplant-shaped bottles at 108 °C for 30 8


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min. The W30YC medium used in the 150-L fermentor contained 30% (v/v) WEML,

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5 g/L yeast extract, 5 g/L corn steep powder and was autoclaved at 108 °C for 30 min.

197

To prepare the inoculum, a total volume of 800 mL of sterilized glucose solution (50

198

g/L) was added into these slants to collect the yeast cells. The cells were then

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inoculated into the fermentor containing 120 L of W30YC medium at an initial pH 5.0.

200

The cultivation was carried out at 32 °C, 250 rpm and an aeration of 1.5 vvm.

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Samples were withdrawn at 4-h intervals, and the concentrations of erythritol,

202

mannitol and D-arabitol were determined by HPLC.

203 204

Fermentation of Candida sp. using WEML in a 120-m3 Fermentor

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The first step was the cultivation and transfer of the Candida sp. cells from 150 L

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to 30,000-L fermentors. The yeast cells grown on the slants were collected using

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glucose solution and transferred to a 150-L fermentor containing 120 L of W30YC

208

medium, and cultivated at 32 °C, 250 rpm and an aeration of 1.5 vvm until the cell

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density at OD600 reached 8.0 (approximately 22 h). Then, the 120 L of yeast cells was

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transferred sequentially to a 2,000-L fermentor and a 30-m3 fermentor and cultivated

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until OD600 8.0. In the next step, to conduct the erythritol enrichment, approximately

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13 m3 of the cells was transferred into a 120-m3 fermentor containing 80 m3 of

213

W30YC medium, and cultivated under the same conditions. When mannitol and

214

D-arabitol were depleted, 40 m3 of the fermentation broth was transferred to a 120-m3

215

storage tank and 40 m3 of WEML containing 3 g/L yeast extract and 2 g/L corn steep

216

powder (pH 5.0) was then supplemented into the 120-m3 fermentor. The cultivation

217

was continued under the same conditions until mannitol and D-arabitol were depleted

218

again. This fed-batch process could be repeated for three times.

219 220

Crystallization of Erythritol from the Yeast Biopurification Broth

221

Colorless, highly concentrated erythritol syrup was obtained through

222

ultrafiltration (removal of yeast cells), nanofiltration (removal of macromolecules),

223

decolorization, ion-exchange and concentration, and it contained approximately 650

224

g/L erythritol. To do the crystallization, erythritol seed crystals were added to the 9



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above syrup at 75 °C to further supersaturate it, and the syrup was then transferred to

226

a crystallizer and stirred in a linear cooling process from 75 °C to 15 °C. The

227

erythritol crystals formed were separated by centrifugation. The resulting mother

228

liquor was further concentrated by evaporation at 75 °C and was then subjected to

229

crystallization under the same conditions. Generally, erythritol can be crystallized

230

three times from the above colorless syrup. If necessary, the obtained erythritol can be

231

re-crystallized to further improve its purity.

232 233

Determination of Crude Single Cell Protein Content of Dry Yeast Cells

234

The yeast cell density was determined at an absorption wavelength of 600 nm

235

(OD600). To obtain dry cells, the yeast cells were washed using 9 g/L NaCl solution

236

twice, collected by centrifugation and dried at 105 °C to a constant weight. The total

237

nitrogen content was determined by the modified semi-micro Kjeldahl method as

238

described by AOAC and the American Public Health Association (1992). 27, 28 K2SO4

239

was used instead of KCl to extract the inorganic nitrogen from the yeast cells with an

240

automatic Kjeldahl apparatus (type KND-1, Shanghai Leizi Co., Ltd)

241

protein values were obtained by multiplying the total nitrogen content by 6.25.

29

. The crude

242 243

RESULTS

244

Identification of the Polyol Components in the WEML by HPLC, TLC, and

245

GC-MS

246

The yeast strain Y. lipolytica BLC13 was from our laboratory and our previous

247

results showed that it could produce erythritol from glucose at a yield of 50%. 16 The

248

purity of erythritol peak at 14.1 min was approximately 85% in the fermentation broth

249

(Figure 1A), including some co-eluted impurities. After two rounds of erythritol

250

crystallization, the WEML obtained was analyzed by HPLC (Figure 1B). The

251

fractions at 14.1 min and 18.5 min were collected, lyophilized, and analyzed by TLC.

252

The TLC spots show that the fraction at 14.1 min contained erythritol and other

253

co-eluted compounds. The fraction at 18.5 min also contained at least two compounds 10


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(Figure 1B).

255

GC-MS was performed to further identify the components in the fractions at 14.1

256

min and 18.5 min. The results of GC analysis show that the fraction with the HPLC

257

retention time of 14.1 min contained three compounds, peak a, b and c (at 7.002,

258

12.030 and 16.984 min in Figure 2A), same as those of BSTFA-derivatized glycerol,

259

erythritol and ribitol, which were further identified by their mass spectrum results

260

(Figure 2B, 2C and 2D). The GC results also demonstrate that in the WEML, the total

261

concentration ratio of the two co-eluted polyols to the main product erythritol was

262

approximately 1: 20, and it was less than 1: 40 in the fermentation broth, indicating

263

that we could evaluate the concentration of erythritol in the fermentation broth using

264

HPLC system in spite of the presence of the impurities (Figure 1A and 2A). The

265

fraction with the HPLC retention time of 18.5 min contained two compounds, peak d

266

and e (16.879 and 20.166 min in Figure 3A), same as those of BSTFA-derivatized

267

D-arabitol and mannitol, which were identified by their mass spectrum results (Figure

268

3B and 3C).

269

Our results demonstrate that some polyol by-products could be produced in the

270

erythritol fermentation when using glucose and Y. lipolytica BLC13. The relative

271

concentrations of these by-products to erythritol increased after the crystallization and

272

recovery of erythritol, from approximately 1: 9 to (1~2): 1 (Figure 1A and 1B). In

273

addition, the typical polyol impurities in the WEML mainly included ribitol, glycerol,

274

D-arabitol, mannitol, and sometimes residual glucose, and the total concentration of

275

such polyol components in the WEML varied to some extent, depending on different

276

factories and sources. To purify erythritol from the WEML, we expected to screen

277

yeast strains which could metabolize other polyols but not erythritol.

278 279

Screening, Selection and Identification of the Yeast Candida sp.

280

Among the 400 yeast strains screened, only 26 strains could utilize all carbon

281

sources (glucose, glycerol, erythritol, ribitol, D-arabitol and mannitol), and 6 strains

282

could utilize the other five carbon sources but not erythritol. Among the 6 strains, one 11



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strain grew faster on the solid YNB medium supplemented with 40% (v/v) WEML

284

than the other five strains. After 30 cycles of adaptation to the WEML on the solid

285

W40YT medium, the strain was found to grow faster (colony size 1.0 ~ 1.5 mm) than

286

its initial one (colony size 0.2 ~ 0.5 mm) in a 5-day cultivation. This strain was

287

designated SJTU828 and was selected for the subsequent biopurification of erythritol

288

from the WEML.

289

We then identified the screened yeast strain SJTU828 based on its partial

290

sequences of 18S rDNA and ITS1/ITS2. Partial DNA (1.5 kb and 0.8 kb) were

291

amplified using 18S and ITS primers and were partially sequenced. The two DNA

292

sequences were analyzed by the BLAST search. The sequenced 18S rDNA fragment

293

(GenBank accession No. HQ901201) from yeast SJTU828

294

Candida yeasts such as Candida maltosa (EF120588.1), Candida aquaetextoris

295

(GU142861.1), Candida viswanathii (EU589205.1), Candida tropicalis (EF428133.1)

296

and Candida parapsilosis (FJ153126.1). The 0.8 kb fragment of ITS1/ITS2 (GenBank

297

accession No. HQ901202) was 99% identical to a homologous sequence from

298

Candida maltose CBS5611 (KJ722417.1) and Candida albicans 21A (JN159659.1).

299

Thus, we identified the screened yeast as a kind of Candida maltosa, and it has been

300

deposited in CGMCC (accession No. 7323).

was 99% identical to

301 302

Activities of Polyol Dehydrogenase in Candida maltosa CGMCC 7323

303

To investigate why the yeast Candida maltosa CGMCC 7323 lost its ability to

304

grow on erythritol, we detected the activities of various polyol dehydrogenases in its

305

crude cell extracts. The strain was grown in a liquid medium supplemented with either

306

glucose, glycerol, ribitol, D-arabitol, sorbitol, or mannitol as carbon source. Figure 4A

307

shows that at least two bands of dehydrogenase formed in the yeast cells grown on

308

glucose when using glycerol, ribitol, D-arabitol, sorbitol, mannitol and erythritol as

309

polyol substrate in the dehydrogenase staining. Figure 4B demonstrates that the

310

erythritol dehydrogenase activity would always exist, being independent of the polyol

311

carbon sources used in the cultivation. Our results indicate that the loss of ability to 12


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grow on erythritol was not due to its deficiency in erythritol dehydrogenase activity.

313 314

Metabolism of Carbon Sources in the Cultivation of Candida maltosa CGMCC

315

7323

316

Interestingly, the yeast Candida maltosa CGMCC 7323 could produce some kind

317

of polyol from mannitol and glucose, which has the same HPLC retention time as

318

erythritol. But there were no polyols produced when using glycerol, D-arabitol or

319

ribitol as carbon source (data not shown). Figure 5 shows the HPLC profile of the

320

polyol produced from mannitol (peak I at 90 h) and its MS results. The mass spectra

321

of the BSTFA-derivatized polyol show the same characteristic fragmentation pattern

322

as the standard erythritol (Figure 5 C), indicating that Candida maltosa CGMCC 7323

323

could produce erythritol. In the YT medium containing 200 g/L mannitol, erythritol

324

reached 11.6 g/L in 90 h, which corresponded to a yield of 4.5%. And when using 200

325

g/L glucose, erythritol reached 9.0 g/L in 90 h.

326 327

Enrichment of Erythritol in WEML Using Candida maltosa CGMCC 7323 on an

328

Industrial Scale

329

The yeast Candida maltosa CGMCC 7323 could grow on carbon sources such as

330

glucose, glycerol, ribitol, D-arabitol, sorbitol and mannitol, but hardly on erythritol,

331

suggesting that it could be used in the biopurification of erythritol by depleting other

332

polyols. In the scale-up experiments from shake flasks to fermentors, the WEML that

333

we used was from factory wastes. Our results show that the depletion of polyol

334

by-products was completed quickly in fermentors. It took 48 h to deplete the

335

by-products in shake flasks but only 18 h in the 120-m3 fermentor (Figure 6A, 6B and

336

6C). The improvement was mainly due to the increase in the biomass, especially, the

337

yeast cell density could reach 40 OD600 in 18 h in the 120-m3 fermentor, in contrast to

338

approximately 25 OD600 at 48 h in shake flasks. The purity of erythritol in the broth

339

increased significantly from approximately 34% to 65% at the end of fermentation

340

(Figure 6A, 6B and 6C). The fed-batch of WEML in the 120-m3 fermentor started

341

only when mannitol and D-arabitol were depleted and it was repeated for three rounds. 13



342

This process took approximately 22 h. The final concentration of erythritol increased

343

to approximately 155 g/L and 65% in purity in 40 h (Figure 6D). The removal of yeast

344

cells by traditional filtration and removal of the macromolecules in the broth by

345

nanofiltration further increased the purity of erythritol to 87% (Figure 6E). Then, the

346

broth was concentrated up to more than 600 g/L soluble solids and subjected to

347

crystallization. The purity of the erythritol crystals was up to 99.9% by HPLC (Figure

348

6F). The total recovery rate of erythritol from the WEML could reach 90%. Based on

349

our technology, approximately 1.0 ton of pure erythritol crystals is expected to be

350

produced from 5.5 tons of WEML.

351 352

DISCUSSION

353

The WEML is hard to process by some conventional technologies and causes a

354

high chemical oxygen demand (COD) and biological oxygen demand (BOD) when it

355

is discharged improperly into the environment. Our biorefinery technology developed

356

in this study allows for the easy recovery of the target erythritol from its waste mother

357

liquor with a high efficiency, and thus can decrease the environmental burden greatly.

358

Our approach for enriching erythritol was based on the elimination of by-products

359

from this mother liquor. Hence, we aimed to select a yeast strain which could rapidly

360

deplete the by-products and could adapt to high concentrations of erythritol mother

361

liquor. The yeast strain Candida maltose CGMCC 7323 met these requirements and

362

was used as a green degradation factory to purify erythritol from the WEML.

363

Dehydrogenases in the Yeast Candida maltose CGMCC 7323 and its Growth on

364

Erythritol

365

In our study, the yeast cells could not grow on solid plates when using erythritol

366

as the sole carbon source. But they showed dehydrogenase activty which could

367

oxidize erythritol with NAD+ as a coenzyme, and such enzymes were observed to be

368

constitutively expressed when using glucose, glycerol, D-arabitol, ribitol or mannitol

369

(Figure 4). In some yeast cells, it is common to contain polyol dehydrogenases in

370

cytoplasm but they cannot assimilate these polyols, or it takes a long adaptation 14


Page 14 of 31

Page 15 of 31


371

period for their genomes to express active polyol dehydregenases.30,31

372

We suggest that the growth retardation may be due to the lack of erythritol

373

transporter (erythritol/H+ symporter) that specifically allows erythritol entry into cells,

374

or the transporter gene may have been silenced due to its localization in subtelomeric

375

regions.32 The existence of polyol transporters has been detected in several yeasts. The

376

yeast Debaryomyces hansenii is a halotolerant yeast which produces and assimilates a

377

wide variety of polyols. Five polyol/ H+ symporters have been identified and

378

characterized, with different specificities and affinities for some polyols including

379

glycerol, D-galactitol and D-sorbitol. 33

380

Erythritol Production by the Yeast Candida maltosa CGMCC 7323

381

An advantage of our strategy is that the yeast strain Candida maltosa CGMCC

382

7323 can produce a small amount of erythritol, not other by-product polyols, from

383

glucose and mannitol. Mirończuk and her colleagues summarized the principal

384

metabolic pathways for erythritol synthesis from glycerol and glucose in a strain Y.

385

lipolytica,18 suggesting that there might be a similar pathway in our yeast strain. In

386

regard to the erythritol synthesis from mannitol, it is reported that mannitol was first

387

oxidized to fructose by dehydrogenases, and converted to fructose-6-phosphate (F6P)

388

by kinases and then transformed to erythritol by the non-oxidative phase of the

389

pentose phosphate (PP) pathway.18,34 Howerver, in our experiments, no erythritol was

390

detected when using glycerol or D-arabitol as carbon source (data not shown).

391

Theoretically, according to the non-oxidative PP pathway, erythritol can be produced

392

from glycerol and D-arabitol.18,34,35 Tomaszewska et al. reported that addition of

393

glycerol resulted in efficient erythritol production of 201.2 g/L in the media

394

containing only yeast extract and crude glycerol. 35 The reason why there was none of

395

erythritol observed in our experiments when using glycerol or D-arabitol is currently

396

under evaluation. And they also provided a valuable suggestion that the yeast strain

397

would not utilize erythritol in the presence of other carbon sources and this feature

398

could be used to increase the product purity. 35

399

Green Factories Using Yeast Cells for Biodegradation 15



400

There is a substantial demand for new yeast cell factories which can synthesize

401

target compounds more efficiently and in higher yields than chemical synthesis.

402

Engineered microbial cell factories, especially those of yeast cells, are economically

403

feasible and sustainable for the production of various fine chemicals and functional

404

sugars.36-41

405

Green factories using yeast cells for biodegradation have received much attention

406

in recent years, especially in regard to down-stream processing. 42,43 Typically, the

407

yeast Y. lipolytica is used not only in synthesis but also in biodegradation, especially,

408

bioremediation of aquatic environments, due to its plentiful enzymes such as esterases

409

and lipases.44 Above all, this yeast strain is considered safe and friendly to

410

environments.

411

Another potential advantage of our strategy is that the yeast strain Candida

412

maltose CGMCC 7323 is expected to be considered safe, and with a total crude

413

protein content of 32%, the biomass produced industrially, up to approximately 9

414

gram protein per litter in the 120-m3 fermentor, may be used as animal feed.

415

Considering that most of the organic compounds other than erythritol in the waste

416

mother liquor were depleted by the yeast cells, our strategy meets circular and

417

zero-waste economy.

418

In a word, we demonstrated in our study that a waste material, WMEL, could be

419

used as an excellent source for producing value-added product erythritol without

420

producing other wastes. The procedure developed did not require costly SMBC, and

421

erythritol crystals could be obtained easily from the enriched fermentation broth by

422

common purification procedures. This newly developed strategy is expected to have

423

excellent economic benefits, and to be environmental friendly. To the best of our

424

knowledge, this is the first report to employ yeast cells in the recovery of erythritol

425

from its mother liquor, a waste produced on a large scale in the erythritol industry.

426

Our technical strategy may be helpful in the production of other valuable chemicals

427

from industrial wastes.

428

16


Page 16 of 31

Page 17 of 31


429 430

AUTHOR INFORMATION Corresponding Author

431

*Hairong Cheng, Phone: +86-21-34206722. Fax: +86 21-34206722.

432

E-mail: [email protected].

433

Authorship

434

Siqi Wang and Jiyang Lv performed this study; Hairong Cheng and Hengwei Wang

435

designed the study and wrote the paper; Hengwei Wang and Zixin Deng discussed the

436

results.

437 438

FUNDING

439

We acknowledge financial support through Grants from the Research Projects of

440

Public-Welfare Technology, Zhejiang Province (No. LGG18E040002) and the

441

National Basic Research Program of China (No. 2013CB733901).

442 443

ABBREVIATIONS

444

BOD,

445

trifluoroacetamide); CGMCCC, China General Microbiological Culture Collection

446

Center; COD, chemical oxygen demand; GC-MS, gas chromatography mass; HPLC,

447

high performance liquid chromatography; MSD, mass selective detector; NAD,

448

nicotinamide adenine dinucleotide; NBT, nitroblue tetrazolium; OD600, optical density

449

at 600 nm; PMS, phenazine methosulfate; rpm, revolutions per min; SCP, single cell

450

protein; SMBC, simulated moving bed chromatography; TLC, thin layer

451

chromatography.

biological

oxygen

demand;

BSTFA,

N,O-bis

(trimethylsilyl)

452 453

REFERENCES

454

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455

1994, 1/2, 27-33.

456

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457

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from Cephalosporium chrysogenus. J. Bacteriol. 1976, 125, 225-232.

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chemists (15th edn.). Arlington: Association of Official Analytical Chemists Inc. 1990,

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nitrogen analysis by the Kjeldahl method. Communications in Soil Science and Plant

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Analysis. 1992, 23, 1805-1813.

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Saccharomyces cerevisiae. J. Gen. Microbiol. 1987, 133, 1675–1684.

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gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae. Gene. 1994,

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study on glycerol metabolism to erythritol and citric acid in Yarrowia lipolytica yeast

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cells. FEMS Yeast Res. 2014, 14, 966-976.

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production from glycerol by Yarrowia lipolytica. Biomass & Bioenergy. 2014, 64,

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(36) Kavšček, M.; Stražar, M.; Curk, T.; Natter, K.; Petrovič, U. Yeast as a cell factory:

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current state and perspectives. Microb. Cell Fact. 2015, 14, 94.

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(37) Lin, B.; Tao, Y. Whole-cell biocatalysts by design. Microb. Cell Fact. 2017, 16,

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(38) Zhang, L.; An, J.; Li, L.; Wang, H.; Liu, D.; Li, N.; Cheng, H. R.; Deng, Z.

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Highly efficient fructooligosaccharides production by an erythritol-producing yeast

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Yarrowia lipolytica displaying fructosyltransferase. J. Agric. Food Chem. 2016, 64,

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3828-3837.

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(39) An, J.; Zhang, L.; Li, L.; Liu, D.; Cheng, H.; Wang, H.; Nawaz, M. Z.; Cheng, H.

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R.; Deng, Z. An alternative approach to synthesizing galactooligosaccharides by

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cell-surface display of β-galactosidase on Yarrowia lipolytica. J. Agric. Food Chem.

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approach to producing high-purity trehalose from maltose by the yeast Yarrowia

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lipolytica displaying trehalose synthase (TreS) on the cell surface. J. Agric. Food

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display of sucrose isomerase from Pantoea dispersa on Yarrowia lipolytica. J. Funct.

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Engineered yeast whole-cell biocatalyst for direct degradation of alginate from

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macroalgae and production of non-commercialized useful monosaccharide from

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applications of Yarrowia lipolytica. Appl. Microbiol. Biotechnol. 2009, 84, 847-865.

583

22


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Page 23 of 31


584

FIGURE CAPTIONS

585

Figure 1. HPLC and TLC analysis of the polyol components in the fermentation broth

586

of Yarrowia lipolytica BLC13 and in the WEML.

587

(A) HPLC analysis of the fermentation broth; (B) HPLC and TLC analysis of the

588

WEML prepared in laboratory.

589 590

Figure 2. GC-MS analysis of polyols in the fraction of 14.1 min isolated by HPLC

591

(A) GC analysis of the fraction at 14.1 min. Peaks a, b and c has the same retention

592

time as those of BSTFA-derivatized glycerol, erythritol and ribitol standards; (B), (C)

593

and (D), comparison of mass spectrum of the polyols in peak a, b and c with glycerol,

594

erythritol and ribitol standards. Blue vertical lines: peak polyols; Red vertical lines:

595

standard polyols.

596 597

Figure 3. GC-MS analysis of polyols in the fraction of 18.5 min isolated by HPLC

598

(A) GC analysis of the fraction at 18.5 min. Peaks d and e has the same retention time

599

as those of BSTFA-derivatized D-arabitol and mannitol standards; (B) and (C),

600

comparison of mass spectrum of the polyols in peak d and e with D-arabitol and

601

mannitol standards. Blue vertical lines: peak polyols; Red vertical lines: standard

602

polyols.

603 604

Figure 4. Zymogram analysis of polyol dehydrogenases in the Candida maltose cell

605

extracts by non-denaturing PAGE.

606

(A) Staining of polyol dehydrogenases by various polyols. The yeast cells were grown

607

in a liquid YT medium containing glucose. The polyol substrates used for the staining

608

included glycerol, ribitol, D-arabitol, sorbitol, mannitol and erythritol; (B), Staining of

609

polyol dehydrogenases using erythritol. The yeast cells were grown in liquid YT

610

medium containing various polyols including glycerol, ribitol, D-arabitol, sorbitol or

611

mannitol.

612 613

Figure 5. HPLC and GC-MS analysis of polyol products in the cultivation of Candida 23



614

maltosa using mannitol. (A) and (B) HPLC results of the fermentation broth at 0 h

615

and 90 h; (C) comparison of mass spectrum of the polyol in peak I with erythritol

616

standard. Blue vertical lines: peak polyols; Red vertical lines: standard polyols.

617 618

Figure 6. Erythritol enrichment experiments from 500-mL flasks to 120-m3

619

fermentors. HPLC results of samples from the 500-mL flasks (A), a 150-L fermentor

620

(B) and the 120-m3 fermentor (C) are shown; (D) HPLC results of the WEML

621

fed-batch in the 120-m3 fermentor; (E) and (F) HPLC results of the broth

622

nanofiltration and erythritol crystallization.

623

24


Page 24 of 31

Page 25 of 31

mA


A

min

mA

B Erythritol

min

Figure 1



Page 26 of 31

A Peak b

Peak c Peak a

100

B

73

147

Peak a vs. glycerol 50

117

103

0

32 36

C

79

101

85 89 93

Scan 2462 (9.534 min): glycerol.D\ data.ms

129

111

150

140

191

177

163

130

120

110

100

90

80

70

60

50

40

30

133

45 41 47 52 55 59 63 66

190

180

170

160

200 m/z

Trimethylsilyl ether of glycerol

Side by Side MF=801 RMF=813

Peak b vs. ribitol

m/z 100

D

73

147

50

0

217

Peak c vs. ribitol 205

103

26 20

45 40

319 117

59

79 89

60

80

129 157

100

120

Scan 283 (7.304 min): 20110720-1.D\ data.ms

307

189

140

243

175

160

180

200

220

253 265

240

277

260


Figure 2 ACS Paragon Plus Environment

280

332

291 300

320

340

395 407 360

380

400

Ribitol, 1,2,3,4,5-pentakis-O-(trimethylsilyl)-

422 420

m/z

Page 27 of 31


Peak d

A

Peak e

%

B

Peak d vs. D-arabitol

73

100

147

103

50

117 0

45 53 59 66 40

60

205

129 157

100

140

120

%

C

243

175

160

180

200

Scan 283 (7.304 min): 20110720-2.D\ data.ms

100

307

189

89 80

217

220

263

240

260

280

332

291 300

320


Peak e vs. mannitol

73

277

319

395 407

351 340

360

380

400

422 420

m/z

d-(+)-Arabitol, pentakis(trimethylsilyl)ether

319

205 217

147

50 103

0

45 54 40

60

117

89 80

100

307 129

120

157 140

169

160

Scan 333 (8.049 min): 20110720-2.D\ data.ms

189 180

229 200

220

243 255 240

277 291

260

280

300

331 345 320

340


Figure 3


393 405 421 434 360

380

400

420

440

524 460

480

500

520

D-Mannitol, 1,2,3,4,5,6-hexakis-O-(trimethylsilyl)-

m/z


A

Yeast cells grown on glucose

B

Yeast cells grown on other polyols

Erythritol

Substrates used for dehydrogenase staining

Figure 4


Page 28 of 31

Page 29 of 31

mA


A

0h Mannitol

mA

min

B

90 h Peak I

min

%

C

Peak I vs. erythritol

m/z

Figure 5



A

Flasks

B

150-L Fermentor

48 h

Page 30 of 31

C

28 h

120-m3 Fermentor

18 h

Fed-batch fermentation

D E

Nanofiltration

Figure 6 ACS Paragon Plus Environment

F

Crystallization

Page 31 of 31


34% Nanofiltration & Decolorization

Erythritol crystals Biopurification

99.9%

65%

Crystallization

TOC


Highly Efficient Erythritol Recovery from Waste Erythritol Mother Liquor

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