Sugaring-out extraction of 2,3-butanediol from fermentation broths

Accepted Manuscript Title: Sugaring-out extraction of 2,3-butanediol from fermentation broths Author: Jian-Ying Dai Chun-Jiao Liu Zhi-Long Xiu PII: DOI: Reference:

S1359-5113(15)30058-1 http://dx.doi.org/doi:10.1016/j.procbio.2015.08.004 PRBI 10493

To appear in:

Process Biochemistry

Received date: Revised date: Accepted date:

20-4-2015 3-8-2015 3-8-2015

Please cite this article as: Dai Jian-Ying, Liu Chun-Jiao, Xiu Zhi-Long.Sugaringout extraction of 2,3-butanediol from fermentation broths.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2015.08.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Sugaring-out extraction of 2,3-butanediol from fermentation broths

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Jian-Ying Dai Chun-Jiao Liu Zhi-Long Xiu*

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School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, P. R. China

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*Corresponding author.

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Zhi-Long Xiu

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e-mail : [email protected]

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Tel/Fax: 86-411-84706369

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Graphical abstract

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Highlights

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1 Sugaring-out extraction of 2,3-butanediol from fermentation broth was studied.

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Most of 2,3-butanediol was distributed to top phase and glucose to bottom phase.

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The glucose-rich bottom phase was reused for fermentation of 2,3-butanediol.

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The phenomenon in the bottom phase-based medium was similar to that in the normal.

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Abstract 1

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Sugaring-out is a novel phase separation method which was found firstly in the separation of acetonitrile.

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Nowadays sugaring-out extraction was tried in the recovery of metal ions, biomolecules, drugs and proteins,

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while its application in separation of hydrophilic primary metabolites from fermentation broths was unknown. In

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this work sugaring-out extraction of 2,3-butanediol was explored. The effects of sugars and solvents on phase

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separation and 2,3-butanediol distribution were investigated to obtain an appropriate sugaring-out extraction

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system. The system consisting of t-butanol/glucose/water was chosen to obtain the operation conditions

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according to 2,3-butanediol distribution and recovery, including glucose addition, 2,3-butanediol concentration

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and (NH4)2HPO4 concentration. The glucose-rich bottom phase was reused for fermentation of 2,3-butanediol

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after removing the solvent. When the fermentation broth (60.3 g/L 2,3-butanediol) was mixed with 30% (w/v)

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glucose，1.8% (w/v) (NH4)2HPO4 and equal volume of t-butanol, 76.3% 2,3-butanediol was distributed into the

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top phase and 80.4% glucose into the bottom phase, and 78.0% soluble proteins and 86.8% lactic acid were

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separated from 2,3-butanediol. When the bottom phase was diluted to prepare fermentation medium, glucose

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consumption and 2,3-butanediol production were similar to those using freshly prepared normal fermentation

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medium. Sugaring-out extraction is a novel alternative separation for bio-based chemicals.

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Keywords: 2,3-butanediol; sugaring-out extraction; fermentation; separation

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1. Introduction

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2,3-Butanediol (2,3-BD) is a platform chemical with wide applications in chemicals, pharmaceuticals,

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foods, fuels and aerospace [1, 2]. One of the key problems for large-scale production of 2,3-BD is the

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downstream processing. The general procedure of 2,3-BD downstream processing includes solid-liquid

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separation, primary recovery and final purification, and many methods have been tried in the primary recovery

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[3], such as membrane filtration [3, 4], alcohol precipitation [5], liquid-liquid extraction [3, 6], reactive

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extraction [7, 8] and salting-out extraction [9]. Among these methods, liquid-liquid extraction is prospective in

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industrialization with the advantages of easy scale-up and low energy consumption. However, it is hard to extract

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2,3-BD from fermentation broths efficiently if a solvent is solely used due to the high hydrophilicity of 2,3-BD.

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Therefore, solvent extraction is not applicable in 2,3-BD recovery. In order to improve the extraction efficiency,

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other chemicals were added to assist the extraction. The most effective method is salting-out extraction, which

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could recover 90~99% 2,3-BD from fermentation broth and remove most of soluble proteins and part of organic

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acids by one-step extraction [9]. By mixing a certain amount of hydrophilic solvent and inorganic salt with

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fermentation broth, two aqueous phases were formed and 2,3-BD was distributed into the solvent-rich top phase.

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Because a large amount of inorganic salt was distributed in the bottom phase, the recovery and reuse of inorganic

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salt became the key problem for this method. Some efforts have been made on the recovery of inorganic salts.

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For example, the phosphate could be recovered at a yield about 94% by pH adjustment and solvent precipitation

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(ethanol or acetone) [10, 11], ammonium sulfate at a yield of 97% by methanol precipitation [12], and carbonate

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could be recovered at a yield of 94% by absorbing CO2 [13]. Although the recovered salts from the bottom phase

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could be reused, the recovery of target product showed a slow descending tendency [11]. To avoid this problem,

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carbohydrates were used to replace inorganic salts in this study because sugar is a substrate in most of the

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fermentations and can be reused by bacteria after extraction.

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The phase separation method “sugaring-out” was first observed by Wang et al in 2008, who introduced a

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monomeric sugar or disaccharide into an acetonitrile-water solution and found acetonitrile was distributed to the

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top phase and sugar to the bottom phase [14, 15]. Until now, the studied sugaring-out extraction systems have

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included acetonitrile-sugar [14-20], ethanol-sugar [21] and propylene glycol-sugar [21], and were tried in the

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recovery of metal ions [18], biomolecules [14, 19, 21], drugs [16, 20] and proteins [17]. High efficiency was

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shown in the separation of chemicals with low hydrophilicity, such as syringic acid [14], para-coumaric acid

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[14], ferulic acid [14], phenolic compounds [21], while its application in the recovery of bio-based chemicals

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with high hydrophilicity was unknown.

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In this study, the feasibility of employing sugaring-out extraction as a primary recovery method to extract

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2,3-BD from fermentation broths was investigated. The systems consisting of different solvents and sugars were

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screened and the effects of glucose addition, 2,3-BD concentration and inorganic salt addition were explored.

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Finally the reuse of glucose-rich bottom phase for 2,3-BD fermentation was tried. This work could act as a

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typical example of sugaring-out extraction of bio-based chemicals from fermentation broths.

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2. Material and methods

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2.1. Chemicals and materials

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2,3-BD and lactic acid standards were purchased from Sigma. The glucose standard was purchased from

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Shandong Academy of Sciences, China. Bovine serum albumin (BSA) was obtained from Shanghai Institute of

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Bio-products, Ministry of Health of China. Coomassie Brilliant Blue G250 was purchased from Shanghai Boao

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Biotechnology Corp in China. Glucose, t-butanol, (NH4)2HPO4 and other chemicals were purchased from

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Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

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2.2. Preparation of fermentation broths

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The strain used was Enterobacter cloacae CGMCC 6053. The medium for seed culture was prepared

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according to the published paper [22]. The medium for fed-batch fermentation was the same as the seed culture

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medium except the concentrations of glucose and (NH4)2HPO4, which were135 g/L and 18.0 g/L, respectively.

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The fermentation broth was prepared by a fed-batch process in a stirred bioreactor controlled at pH5.8, 250 rpm

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with an aeration rate of 0.1 vvm. The concentration of 2,3-BD and residual glucose in the fermentation broth was

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60-90 g/L and 10-15 g/L, respectively. The cells in fermentation broth were removed by centrifugation at 8000

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rpm for 15min under 4 C, and the supernatant was used for the following sugaring-out extraction experiments.

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2.3. Sugaring-out extraction of 2,3-butanediol from fermentation broths

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All the extraction experiments were carried out at ambient temperature (about 15 ~ 25C). The sugar was

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dissolved in 2,3-BD fermentation broths to obtain sugar mixture. Then, organic solvent with equal volume of

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fermentation broths was added into the sugar mixture and mixed thoroughly by a vortex mixer. The obtained

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mixture was stood overnight. Each experiment was carried out in triplicate.

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The parameters including phase ratio (R), distribution coefficient (K) and recovery (Y) were calculated as the followed:

R

VT VB

(1)

where VT and VB were the volumes of top phase and bottom phase, respectively.

K

CT CB

(2)

where CT and CB were the concentrations of 2,3-BD in the top phase and bottom phase, respectively.

Y (%) 

CT  VT  100 CO  VO

(3)

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1

Where VT andV0 were the volumes of top phase and fermentation broth, respectively; CT andC0 were the

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concentrations of 2,3-BD in the top phase and fermentation broth, respectively.

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2.4. Reuse of the glucose-rich bottom phase

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Glucose (90 g) and (NH4)2HPO4 (5.4 g) were mixed with fermentation broth (300 mL), then t-butanol (300

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mL) was mixed. The mixture was stood overnight to obtain the bottom phase. Part of the organic solvent in the

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bottom phase was removed by vacuum distillation with votary evaporation instrument. The bath temperature and

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the vacuum pressure of the vacuum distillation were 30~40C and -0.08~-0.1 MPa, respectively. The final

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concentration of the t-butanol was less than 0.5%. Water was added into the residual solution (water/solution:

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1~2/1 (v/v)) in order to dilute the glucose concentration to 110-130 g/L. The cells (10% v/v) prepared from seed

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culture medium were inoculated into the diluted solution and cultured at 37C, 200 rpm for 70-90h by

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shaking-flask. Each experiment was carried out in triplicate.

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2.5. Analytical methods

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The concentrations of 2,3-BD and t-butanol were analyzed by gas chromatography (SHIMADZU GC-2010,

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Japan) equipped with a FID and a column packed with Chromosorb 101 (2 m × Φ5 mm) and operated with N2 as

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carrier gas at a flow rate of 50 mL/min. The column temperature was 180 C, and the temperatures for injector

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and detector were 220 C. The concentration of lactic acid was analyzed by HPLC (Agilent 1100) equipped with

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a C18 column (Sino Chrom ODS-BP, 5 m 4.6 × 250 mm) and an ultraviolet detector (214 nm), eluted by a

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mobile phase composed of 2‰ phosphoric acid and acetonitrile (96.5:3.5, v/v) at a rate of 1 mL/min. Glucose

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was assayed by a glucose analyzer (Biosensor SBA-50, Shandong Academy of Sciences, China). The Bradford

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method was used to detect the concentration of protein with BSA as a standard protein. The biomass was

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measured by the optical density at 620 nm using a spectrophotometer. The concentration of (NH4)2HPO4 was

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calculated from a calibration equation (Y = 0.00096X - 0.931), where Y is the concentration of (NH4)2HPO4, X is

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the value of conductivity (2800 < X < 12000).

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3. Results and discussion

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3.1. Screening of sugaring-out extraction systems

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Sugaring-out extraction of 2,3-BD has not been reported before, so a suitable solvent-sugar system has to

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be screened out for the separation of 2,3-BD. In the study of salting-out extraction, the systems formed by short

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chain alcohols, especially methanol and ethanol, showed strong ability in the separation of 2,3-BD from

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fermentation broth [9], but the high solubility of methanol and ethanol in water caused a high residue in bottom

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phase which would inhibit the cell growth. So the screening of sugars was explored with propanol and butanol.

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Dimethyl carbonate has been considered as a green reagent for its non-toxicity and biodegradability [23], so it

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was also used for the sugar screening experiments.

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Six sugars were used to explore the phase separation and 2,3-BD distribution (Table 1). Glucose is the

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common carbon source for fermentation. Fructose is the main unit of inulin which is rich in Jerusalem artichoke

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tuber and is a good substrate for 2,3-BD production [24, 25]. Xylose and arabinose are two important

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components in the hemicelluose hydrolysate, and sucrose is the main ingredient of sugarcane molasses which is

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a cheap carbon source for 2,3-BD production [26, 27]. As shown in Table 1, the recovery of 2,3-BD (Y) obtained

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from n-propanol system was always higher than that from i-butanol and dimethyl carbonate systems when the

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same sugar was used. The xylose system demonstrated a higher efficiency than other sugars, which was related

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with its ability to form a two-phase system. For example, only 15 g/L xylose was required to induce the phase

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separation of ACN-water solution, while the concentration for arabinose, glucose or fructose was 25 g/L, and

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sucrose 30 g/L at 1 C [15]. The difference of 2,3-BD recovery among these sugars was within 6% except

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n-propanol-inulin system when the same solvent was used. Considering the common usage of glucose in

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fermentation, glucose was selected for the following experiments.

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In order to study the effect of solvents on phase separation and 2,3-BD partition, a series of solvents was

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screened at two concentration levels of glucose. Due to the high hydrophilicity, homogeneous phase was

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observed when using methanol and ethanol systems at present concentrations of glucose. As shown in Table 2,

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two phases were observed when the solvent was mixed with the fermentation broth in which 30% (w/v) glucose

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was added, while homogeneous phase was kept when i-propanol or acetone was mixed with the fermentation

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broth in which 15% (w/v) glucose was added. Glucose was mainly partitioned to the bottom phase and the

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recovery varied from 27% to 99%. The recovery of 2,3-BD (Y) in the top phase varied from 1.35% to 83.1%,

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distribution coefficient (K) varied from 0.01 to 0.99, and phased ratio (R) varied from 1 to 12. The difference of

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these parameters among the solvents was larger than that among the sugars, so solvent played a more important

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roles in the separation of 2,3-BD.

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The data in Table 2 clearly showed that the recovery in short chain alcohol-glucose system was generally

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higher than that in ester-glucose system. This phenomenon was similar to that in salting-out extraction which

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was mainly resulted from the solubility of water and 2,3-BD in the solvent [12]. High recovery of 2,3-BD was

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obtained with i-propanol- and acetone-glucose systems, but the large phase ratio hindered its application. Water

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has a high specific heat capacity, while the large phase ratio indicated a high water content in the top phase.

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Finally, the energy consumption of top phase concentration would be greatly increased. So these two systems

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were not considered for the following experiments. The recovery obtained from t-butanol system was a little

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lower, but the small phase ratio was favored. On the other hand, gram-negative bacteria showed a higher

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metabolic activity in the solvent with higher logP values according to the experiments of Anvar i and K hayati

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[6]. Based on the data provided by ChemBioDraw, it could be concluded that the solubility and cell toxicity of

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t-butanol was smaller than propanol which was beneficial for the reuse of bottom phase, so t-butanol-glucose

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system was selected to separate 2,3-BD from fermentation broths.

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3.2. Partition behavior of 2,3-butanediol in the t-butanol-glucose system

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3.2.1. Effect of glucose addition on the phase separation and 2,3-butanediol distribution

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The effect of glucose addition on phase separation and 2,3-BD distribution was explored in order to find out

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an appropriate separation condition. For operation convenience the experiments were carried out at a fixed

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volume ratio (1:1) of fermentation broth to t-butanol at ambient temperature. The volume of top phase decreased

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when adding more glucose into the fermentation broth, while the volume of bottom phase increased, which

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indicated more water was distributed into the bottom phase. Thus, the phase ratio (R) decreased with increasing

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glucose concentration as shown in Fig. 1. Similar phenomenon was also observed by Tubimdee and Shotipruk in

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the extraction of phenolics with the ethanol-glucose system [21]. The distribution coefficient of 2,3-BD (K)

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increased to a highest point at a range of glucose addition between 30~35%, then decreased when increasing

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glucose concentration. The recovery (Y) is a combination of K and R, so it showed a slow tendency of decrease

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in Fig. 1. The addition of 30% glucose was selected for the following experiments.

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3.2.2. Effect of 2,3-butanediol concentration on the phase separation and 2,3-butnediol distribution

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2,3-BD can be produced by various types of bacteria and the concentration of 2,3-BD is usually in the

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range of 30-150 g/L in the fermentation broths [2]. Due to the production ability of strain used in this study, the

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concentration of 2,3-BD in fermentation could not reach 150 g/L. Therefore, the water-solution of 2,3-BD was

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used in this experiment. As shown in Fig. 2, the existence of 2,3-BD would affect the phase separation and the

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recovery of 2,3-BD. The volume of top phase increased with the increasing concentration of 2,3-BD, while the

2

volume of bottom decreased. Consequently, the phase ratio increased with increasing 2,3-BD concentration. The

3

increasing phase ratio indicated more 2,3-BD was extracted from bottom phase. So the distribution coefficient

4

and recovery increased, too. Therefore, high concentration of 2,3-BD in fermentation broths was favorable for

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the recovery of 2,3-BD.

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3.2.3. Effect of (NH4)2HPO4 addition on the phase separation and 2,3-butanediol distribution

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The above results showed that the recovery of 2,3-BD by sugaring-out extraction was lower than those by

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salting-out extraction [9], so inorganic salt was considered to assist the extraction. Both salt and sugar compete

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water molecules with 2,3-BD, thus push more 2,3-BD to the top phase. The salt (NH4)2HPO4 was an ingredient

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of fermentation medium and the amount was the largest among the inorganic salts added. In the separation of

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1,3-propandiol the addition of (NH4)2HPO4 increased the distribution coefficient of 1,3-propandiol due to the

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salting-out effect [28]. In this work a certain amount of (NH4)2HPO4 was added to the sugaring-out extraction

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system to increase the recovery of 2,3-BD and act as a complement of phosphate and nitrogen sources for the

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reuse of bottom phase. Due to the limitation of (NH4)2HPO4 concentration in fermentation medium, the amount

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of (NH4)2HPO4 was much lower than the salt used in the salting-out extraction [9]. It was observed that the top

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phase volume reduced and bottom phase volume increased gradually with increasing addition of (NH4)2 HPO4.

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As a result, the phase ratio decreased (Fig. 3). The increasing distribution coefficient and recovery of 2,3-BD

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indicated more 2,3-BD was pushed from bottom into top phase due to salting-out effect of (NH4)2HPO4. In fact,

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(NH4)2HPO4 was not exhausted in the fermentation broth used. There was 16 g/L salt existed in the fermentation

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broth according to the conductivity. When the concentration of (NH4)2HPO4 was increased from16 to 106 g/L,

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the recovery of 2,3-BD from fermentation broth (60.3 g/L 2,3-BD) increased from 75.6% to 82.7%. These data

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clearly showed that the salt with low concentration did work although the change of parameters was not large.

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Because glucose added into the fermentation broth was about two-fold of that in fermentation medium,

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additional 18 g/L (NH4)2HPO4 was added into the fermentation broth during the extraction experiments to

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control a similar ratio as that of glucose. At this point, the concentration of by-product lactic acid was 1.87 g/L in

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the top phase and 9.54 g/L in the bottom phase. The removal of proteins and lactic acid from the top phase was

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78.0% and 86.8%, respectively, and the recovery of glucose and (NH4)2HPO4 in the bottom phase was 80.4%

7

and 97.3%, respectively. Because 19.6% glucose was existed in the top phase, glucose and 2,3-BD must be

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separated after the removal of solvent. One probable method is sugaring-out. Two phases might form at an

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appropriate concentration after removing part of water by vacuum distillation. The top phase is 2,3-BD and the

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bottom phase contains glucose which can be reused in fermentation. Further work will be done later to develop

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the method.

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3.3. Reuse of the bottom phase

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3.3.1. Effect of t-butanol concentration on 2,3-butanediol fermentation

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In the t-butanol-glucose system part of t-butanol was partitioned into the bottom phase which would affect

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the growth of bacteria. To explore the effect of t-butanol on 2,3-BD production, t-butanol was added into the

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fermentation medium at the beginning of culture and the results were shown in Fig. 4. With the increasing

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concentration of t-butanol the bacterial growth and 2,3-BD production were gradually inhibited. When the

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concentration of t-butanol was 5.33 g/L, the existence of t-butanol has little impact on 2,3-BD fermentation.

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When t-butanol concentration was greater than 16.1 g/L, the cell growth was ceased at first 18 hours and trace

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glucose was consumed. It seemed that almost all the consumed glucose was used to maintain the survival of

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bacteria, not the production of 2,3-BD. The recovery of cell growth in the late phase mainly resulted from

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1

acclimatization of bacteria and the reduced concentration of t-butanol. These data indicated that the

2

concentration of residual t-butanol had to be controlled lower than 0.5% in the culture medium when the bottom

3

phase was reused.

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3.3.2. Production of 2,3-butanediol with medium prepared from bottom phase

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Most of t-butanol in the bottom phase was removed by vacuum distillation, and the treated bottom phase

7

was diluted as fermentation medium in which the concentration of t-butanol was less than 0.5%. The seed was

8

freshly prepared using the seed culture medium and the inoculation was 10% (v/v). As shown in Fig. 5, the

9

glucose consumption and 2,3-BD production showed a similar trend as that using the normal fermentation

10

medium. After 90 h incubation 98.8 g/L glucose was consumed and 38.3 g/L 2,3-BD was produced, and the

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productivity was 0.43 g/(Lh) which was same as that from normal fermentation medium. The value of OD620

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was 4.8 and 5.8 for bottom phase-based medium and fermentation medium, respectively. The result indicted the

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feasibility of reuse of bottom phase.

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4. Conclusion

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This paper provided a systematic work of sugaring-out extraction of 2,3-BD from fermentation broths.

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After the screening of sugaring-out systems formed by different sugars and solvents, t-butanol-glucose system

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was selected to study the extraction of 2,3-BD from fermentation broths. When 1.2 g glucose and 4 ml t-butanol

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were mixed with 4 ml fermentation broth containing 60.3 g/L 2,3-BD, two phases were appeared. 75.6% 2,3-BD

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was distributed into the top phase, and 80.4% glucose into the bottom phase. The bottom phase could be diluted

21

to prepare fermentation medium after removing part of t-butanol. At appropriate conditions, glucose

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consumption and 2,3-BD production were similar to those using normal fermentation medium. This work

12

1

provided a typical example for the sugaring-out extraction of bio-based chemicals.

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Acknowledgement This work was supported by the National Natural Science Foundation of China (Grant No. 21476024).

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extraction for the determination of sulfonamides in honey by high-performance liquid chromatography with fluorescence detection. J Chromatogr A 2010;1217:7812-7815. [17] Dhamole PB, Mahajan P, Feng H. Sugaring out: A new method for removal of acetonitrile from preparative RP-HPLC eluent for protein purification. Process Biochem 2010;45:1672-1676. [18] Zhang C, Huang K, Yu P, Liu H. Sugaring-out three-liquid-phase extraction and one-step separation of Pt(IV), Pd(II) and Rh(III). Sep Purif Technol 2012;87:127-134. [19] Cardoso GdB, Mourão T, Pereira FM, Freire MG, Fricks AT, Soares CMF, Lima ÁS. Aqueous two-phase systems based on acetonitrile and carbohydrates and their application to the extraction of vanillin. Sep Purif Technol 2013;104:106-113. [20] Zhang J, Myasein F, Wu H, El-Shourbagy TA. Sugaring-out assisted liquid/liquid extraction with acetonitrile for bioanalysis using liquid chromatography–mass spectrometry. Microchem J 2013;108:198-202. [21] Tubtimdee C, Shotipruk A. Extraction of phenolics from Terminalia chebula Retz with water–ethanol and water-propylene glycol and sugaring-out concentration of extracts. Sep Purif Technol 2011;77:339-346. [22] Qin J, Xiao Z, Ma C, Xie N, Liu P, Xu P. Production of 2,3-butanediol by Klebsiella Pneumoniae using glucose and ammonium phosphate. Chinese J Chem Eng 2006;14:132-136. [23] Tundo P, Selva M. The chemistry of dimethyl carbonate. Acc Chem Res 2002;35:706-716. [24] Li D, Dai J-Y, Xiu Z-L. A novel strategy for integrated utilization of Jerusalem artichoke stalk and tuber for production of 2,3-butanediol by Klebsiella pneumoniae. Bioresour Technol 2010;101:8342-8347. [25] Sun L-H, Wang X-D, Dai J-Y, Xiu Z-L. Microbial production of 2,3-butanediol from Jerusalem artichoke tubers by Klebsiella pneumoniae. Appl Microbiol Biotechnol 2009;82:847-852. [26] Jung M-Y, Park B-S, Lee J, Oh M-K. Engineered Enterobacter aerogenes for efficient utilization of sugarcane molasses in 2,3-butanediol production. Bioresour Technol 2013;139:21-27. [27] Dai J-Y, Zhao P, Cheng X-L, Xiu Z-L. Enhanced production of 2,3-butanediol from sugarcane molasses. Appl Biochem Biotechnol 2015;DOI 10.1007/s12010-015-1481-x. [28] Wu SH, Wang YJ. Salting-out effect on recovery of 1,3-propanediol from fermentation broth. Ind Eng Chem Res 2012;51:10930-10935.

14

1

Figure legends:

2

Fig. 1 Effect of glucose addition on the phase separation and 2,3-butanediol distribution with t-butanol as

3

solvent. The volume ratio of fermentation broth to organic solvent was 1:1, the concentration of 2,3-BD in

4

fermentation broth was 60.3 g/L.

5

Fig. 2 Effect of 2,3-butanediol concentration on the phase separation and 2,3-butanediol distribution in the

6

t-butanol-glucose system. The volume ratio of 2,3-BD solution to organic solvent was 1:1, glucose addition

7

was 30% of 2,3-BD solution (w/v), (NH4)2HPO4 concentration in 2,3-BD solution was 18 g/L.

8

Fig. 3 Effect of (NH4)2HPO4 on phase separation and 2,3-butanediol partition in the t-butanol-glucose

9

system. The volume ratio of fermentation broth to organic solvent was 1:1, glucose addition was 30% of

10

fermentation broth (w/v), the concentration of 2,3-BD in fermentation broth was 60.3 g/L.

11

Fig. 4 Effect of t-butanol concentration on bacterial growth and 2,3-butanediol production. A, time course of

12

bacterial growth in shaking-flask culture at 37 C and 200 rpm with different initial t-butanol concentrations

13

in fermentation media; B. glucose consumption and 2,3-BD production after 28 h culture.

14

Fig. 5 Time course of glucose consumption and 2,3-BD production in shaking-flask culture at 37C and 200

15

rpm.

16 17

15

1

Table 1 Effect of sugars on the partition behavior of 2,3-butanediol. n-Propanola

i-Butanola

Dimethyl carbonatea

R

K

Y (%)

R

K

Y (%)

R

K

Y (%)

Xylose

2.56±0.01

1.57±0.04

80.9±1.4

1.65±0.05

0.835±0.007

56.5±1.7

1.04±0.02

0.433±0.025

31.0±1.4

Arabinose

1.98±0.02

1.54±0.03

76.2±1.9

1.26±0.07

0.947±0.032

54.5±1.9

1.07±0.03

0.423±0.040

31.1±1.5

Glucose

1.97±0.01

1.38±0.13

75.4±1.4

1.20±0.08

0.833±0.025

49.9±0.6

1.22±0.02

0.330±0.014

28.5±1.2

Fructose

2.00±0.06

1.87±0.18

76.0±2.0

1.49±0.03

0.873±0.025

56.2±1.1

1.13±0.01

0.410±0.085

28.4±0.1

Sucrose

1.99±0.02

1.78±0.04

76.6±2.3

1.21±0.03

0.843±0.024

50.5±0.3

1.18±0.02

0.307±0.012

26.4±1.0

Inulin

1.84±0.04

1.43±0.02

70.3±1.9

1.20±0.03

0.897±0.040

51.8±1.1

1.26±0.02

0.327±0.060

26.9±2.1

2

a

3

concentration of 2,3-BD in fermentation broth was 85.5 g/L.

30% sugar was added into the fermentation broth (w/v), volume ratio of fermentation broth to solvent was 1:1; the

4

16

1

Table 2 Effect of different solvents on the partition behavior of 2,3-butanediol. Glucose 15%a

R

i-Propanol

K

-

Glucose 30%a

YGb (%)

Y (%)

-

-

R

K

Y (%)

YGb (%)

9.00±0.01

0.530±0.001

83.1±0.9

26.7±0.4

n-Propanol

4.33±0.01

0.750±0.014

75.8±1.4

50.0±0.9

2.59±0.01

0.990±0.014

72.7±1.9

68.8±1.6

t-Butanol

3.00±0.01

0.990±0.056

73.6±2.2

64.3±1.2

2.20±0.01

0.913±0.040

66.7±0.9

76.4±0.8

n-Butanol

1.67±0.01

0.810±0.001

56.7±1.5

87.2±1.1

1.50±0.01

0.720±0.056

54.7±1.9

94.3±1.1

i-Butanol

1.50±0.01

0.590±0.001

53.0±0.9

77.9±0.1

1.45±0.05

0.645±0.064

48.6±1.4

96.9±1.4

Pentanol

1.29±0.01

0.580±0.026

44.4±1.5

94.1±1.5

1.29±0.01

0.553±0.011

41.5±0.5

99.9±1.2

Oleyl alcohol

1.22±0.01

0.070±0.001

7.24±0.84

88.5±1.3

1.22±0.01

0.070±0.001

7.75±0.52

92.1±0.1

Acetone

-

-

12.0±0.01

0.315±0.021

78.1±1.7

17.9±1.3

Tetrahydrofuran

1.60±0.01

0.750±0.026

54.5±1.0

73.2±1.8

1.16±0.01

0.545±0.021

39.7±1.7

90.4±2.2

Ethyl acetate

1.03±0.03

0.090±0.001

8.26±0.25

93.8±1.7

1.02±0.03

0.103±0.006

9.37±0.33

99.8±3.3

Butyl acetate

1.04±0.04

0.010±0.001

1.35±0.17

94.9±0.1

1.11±0.01

0.040±0.001

3.94±0.12

98.0±1.3

-

2

a

3

2,3-BD in fermentation broth was 69.7 g/L; “-”, homogeneous phase. bRecovery of glucose in the bottom phase.

Glucose added into the fermentation broth (w/v), volume ratio of fermentation broth to solvent was 1:1; the concentration of

4

17

1

2 3

Fig. 1.

4

18

1

2 3 4

Fig. 2.

5

19

1 2

Fig. 3.

3

20

1 2

3 4

Fig. 4.

5

21

1

2 3

Fig. 5.

22