Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis based on metabolic flux analysis

Accepted Manuscript Title: Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis based on metabolic flux analysis Authors: Ping Song, Chen Chen, Qianqian Tian, Ming Lin, He Huang, Shuang Li PII: DOI: Reference:

S1369-703X(12)00316-6 doi:10.1016/j.bej.2012.11.011 BEJ 5607

To appear in:

Biochemical Engineering Journal

Received date: Revised date: Accepted date:

23-7-2012 30-10-2012 18-11-2012

Please cite this article as: P. Song, C. Chen, Q. Tian, M. Lin, H. Huang, S. Li*, Two-stage oxygen supply strategy for enhanced lipase production by Bacillus subtilis based on metabolic flux analysis, Biochemical Engineering Journal (2010), doi:10.1016/j.bej.2012.11.011 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.

*Highlights (for review)

Highlights A detailed network model for B. subtilis metabolism was constructed.

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Metabolic flux analysis (MFA) was used to investigate lipase production.

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Lipase production needs: high flux of tributyrin, TCA, and ATP; less biomass

ip t

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TOS strategy was carried out with a 51% increase of lipase production based on

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MFA.

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flux.

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*Manuscript

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Two-stage oxygen supply strategy for enhanced

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lipase production by Bacillus subtilis based on

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metabolic flux analysis

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Ping Song, Chen Chen, Qianqian Tian, Ming Lin, He Huang*, Shuang Li*

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State Key Laboratory of Materials-Oriented Chemical Engineering, College of

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Biotechnology

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Technology, No. 5 Xinmofan Road, Nanjing 210009, People’s Republic of China

University of

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Nanjing

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Engineering,

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Pharmaceutical

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*Co-corresponding author. Tel./fax: +86 25 83172094. E-mail address: [email protected] (H. Huang)

*Co-corresponding author. Tel./fax: +86 25 83172094. E-mail address: [email protected] (S. Li) 1 Page 2 of 44

Abstract

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Lipase production by Bacillus subtilis CICC20034 was assessed by metabolic flux

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distribution analysis. Lipase production was tested under various oxygen supply

4

conditions in a synthetic medium to obtain the optimal oxygen supply profile.

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Based on the metabolic flux analysis, a two-stage oxygen supply strategy (TOS)

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that maintained high oxygen supply conditions during early fermentation phase,

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and then step-wisely reduced aeration to keep a stable, smooth, and adequate

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changing dissolved oxygen (DO) level profile throughout the production phases

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was carried out. With the proposed control strategy, the final lipase activity in

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batch fermentation significantly increased and reached a high level at 0.56 U/ml,

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corresponding to a 51% increase. The relevant metabolic flux analysis verified the

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effectiveness of the proposed control strategy. By applying TOS in composite

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medium, the final lipase activity reached 5.0 U/ml.

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Keywords: Bacillus subtilis; Metabolic flux analysis; Lipase; Dissolved oxygen;

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Aeration; Fermentation

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1. Introduction

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Oxygen is an indispensable raw material that must be supplied in large amounts

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during industrial aerobic fermentation. Different oxygen levels induce or repress

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transcription, synthesis of different enzymes, or both, thereby it affects cell

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metabolism and consequently, product yield and productivity [1]. Aerobic

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microorganisms generally require large amounts of oxygen to reoxidize NAD(P)H

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or FADH2 and generate effectively ATP for metabolism [2]. In submerged

9

fermentations, growth under anaerobic conditions often leads to the formation of

10

toxic by-products, such as acetic acid and ethanol, which in turn strongly inhibit

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cell growth [3,4]. On the other hand, very high dissolved oxygen (DO)

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concentrations can also hamper the production of the desired product because the

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oversupply of oxygen may suppress enzyme activities, essential in creating the

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desired product [5].

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Oxygen supply is known to have an important influence on the enzyme

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production of aerobic microorganisms [6-11]. In the particular case of lipase

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fermentation, lipase production depends more extensively on oxygen than on cell

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growth [12]. Vadehra and Harmon [13] found that the presence of air is essential

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for lipase production by Staphylococcus aureus. Alford and Smith [14] reported

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the beneficial effect of shaking on lipase production by S. aureus. Preliminary

22

experiments in the current work showed a sharp decrease of lipase activity after

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reaching its maximum. This phenomenon has also been found in other lipase

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production reports [15-18]. This sudden sharp decrease causes difficulties in

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collecting lipase in industrial production settings. A possible explanation may be 3 Page 4 of 44

decreased cell vitality or the production of proteases in the late lipase production

2

phase. Research work regarding physiological responses under different oxygen

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supply conditions, particularly for lipase-production strains, is very limited.

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Lipases from Bacillus sp. are of particular interest because they exhibit optimal

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activity and stability at extreme alkaline pH values (>9.5) [19, 20]. In addition, the

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genus Bacillus is one of the most important groups of industrial microorganisms

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and is used in several fermentation processes. However, a large part of Bacillus

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lipases described in the literature was produced by their host cells in very small

9

amounts [21-23]. Thus, the regulation of fermentation process is very important.

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Systematic methods, such as metabolic flux analysis, can provide useful

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information and knowledge on the metabolic mechanisms of the bioprocess to

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understand the physiological characteristics of lipase-production strains like B.

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subtilis under different oxygen supply conditions. Furthermore, available

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strategies could be applied in lipase production from laboratory scale to industrial

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scale.

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The present work applies metabolic flux analysis to batch fermentations with B.

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subtilis CICC20034 to determine the optimal oxygen supply strategy required to

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achieve maximal and stable lipase production. Based on metabolic flux analysis, a

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two-stage oxygen supply strategy was proposed and applied to enhance lipase

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production. The optimal oxygen supply strategy for lipase production was

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concluded from the information obtained. Further application of this two-stage

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oxygen supply strategy was carried out in a composite culture medium to

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investigate the feasibility of the strategy.

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2. Materials and methods

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2.1. Strain and media

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A laboratory strain of Bacillus subtilis CICC20034 (obtained from the Centre of

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Industrial Culture Collection of China) was maintained on Luria-Bertani (LB)

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medium agar slants. The synthetic fermentation medium initially contained the

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following per liter: 12 g glucose, 0.5 g MgSO4·7H2O, 15.2 g K2HPO4, 1.4 g

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(NH4)2SO4, and 15 g tributyrin.

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The composite fermentation medium contained the following per liter: 8 g sorbitol,

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64 g beef extract, 0.5 g arabic gum, 0.34 g MnSO4·H2O, 2.6 g K2HPO4 and 2%

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peanut oil.

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2.2. Culture condition

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The cells growing on newly prepared slants were transferred to 250 ml

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Erlenmeyer flasks containing 50 ml LB medium and were incubated at 37 °C on a

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rotary shaker (200 rpm) for 6 h. After harvesting by centrifugation at 8000 g for 5

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min at 4 °C, cells were washed twice with sterile water, and then resuspended in

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sterile water to obtain a final concentration of 4×109 cells/ml.

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Lipase production was carried out by batch fermentation in a 3.0 l Bioflo 110

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fermenter (New Brunswick Scientific Co., New Brunswich, NJ) with a working

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volume of 2.0 l fermentation medium inoculated with 5% (v/v) cell suspension. 5 Page 6 of 44

The fermenter was equipped with a glass pH electrode (Mettler Toledo Inc.,

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Columbus, OH) to monitor and control pH. Temperature, pH, and agitation speed

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were kept constant at 37 °C, 8.0, and 200 rpm, respectively. Three different

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oxygen supply methods determined by controlling the aeration rate at 0.5, 1.0, and

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1.5 vvm were classified as low oxygen supply (LOS) level, medium oxygen

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supply (MOS) level, and high oxygen supply (HOS) level, respectively. DO

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concentrations under different aeration conditions were expressed in terms of DO

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saturation level (%), whereas 100% DO concentration level corresponded to

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actual DO saturation at 37 °C, 1 atm.

2.3. Determination of KLa and OUR

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The volumetric mass transfer coefficient (KLa) and oxygen uptake rate (OUR)

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were determined from gas analysis. The inlet flow rate was measured using

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Tandem Gas Analyzers (Magellan Instrument Ltd, Limpenhoe, UK), resulting

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from the mass fractions of oxygen in the inlet and the exhaust gas.

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KLa was calculated from the following balances Eq. (1):

F inO2 - FOout2

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KLa=

(1)

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whereas Fin and Fout are the molar flow rates measured at bioreactor inlet and

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outlet gas, V is the bioreactor volume.

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OUR was calculated from the following balances Eq. (2):

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OUR=KLa·(C*-CL)

V  (C * -C L )

(2)

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whereas CL is the oxygen concentration in the liquid phase and C* is the

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saturation concentration of the oxygen in the liquid under the temperature and

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pressure conditions in the bioreactor.

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2.4. Extracellular metabolite analysis

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Dry cell weight (DCW) was determined from an average of 3 samples a 10 ml cell

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suspension harvested by centrifugation, washed with distilled water, and dried at

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80 °C for 24 h to a constant weight.

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Glucose concentration in the culture was measured using a biosensor with glucose

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oxidase electrode SBA-40C (Shandong Academy of Sciences, Shandong, China).

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Amino acid and organic acid concentrations were measured by HPLC (Dionex

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P680 pump, Chromeleon controller, and Dionex UVD 170U Detector; Dionex

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Corporation, CA, USA). Amino acid concentrations were measured with a Sepax

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AA column (250 mm×4.6 mm×5 μm; Sepax Technologies, DE, USA) using the

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Sepax method (isothiocyanate was used as the reagent for pre-column

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derivatization) and detection at 254 nm. Organic acid concentrations were

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determined through a Sepax HP-C18 column (250 mm×4.6 mm×5 μm; Sepax

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Technologies, DE, USA), and detection at 210 nm.

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Tributyrin was determined as described previously [24].

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2.5. Lipase assays 7 Page 8 of 44

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The lipase assay was performed by measuring the increase in absorbance at

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410 nm in a PerkinElmer Lambda 25 spectrophotometer (PerkinElmer Life and

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Analytical Sciences, Boston, MA). Increase in absorbance was observed due to

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the release of p-nitrophenol after the hydrolysis of p-nitrophenyl palmitate (pNPP)

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at 40 °C for 10 min at pH 8.0 [25]. One unit of lipase (U) was defined as the

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amount of enzyme that releases 1 μmol p-nitrophenol per min under the assay

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conditions described above. The calibration curve was prepared using p-

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nitrophenol as the standard.

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2.6. Metabolic flux analysis

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A detailed network model for B. subtilis metabolism was constructed. The

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previously described comprehensive isotopomer model of B. subtilis central

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carbon metabolism was extended to accommodate the situations arising with co-

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feeding of tributyrin as the inducer and carbon source [26]. The exact amino acid

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composition of the lipase enzyme produced by B. subtilis (LipA) [27] was used

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for formulating amino acid demand and energy requirement for protein synthesis

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assuming 4.306 μmol ATP/μmol amino acids [28]. The network model contains

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reactions for glycolysis, glyoxylate shunt, anaplerotic pathways, pentose

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phosphate pathway (PPP), TCA cycle, amino acid biosynthesis [29] and biomass

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synthesis reactions (30). (Appendix A). The model comprises of 52 reactions and

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47 balanced intracellular metabolites. 2 substrates (glucose and tributyrin), 4

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products (Lac, Ac, Biomass, and Lipase) and 20 excreted amino acids were

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measured and used for MFA. 8 Page 9 of 44

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Matlab 7.1 was used for metabolic flux balancing. The theory and practice of this

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method have been thoroughly described elsewhere [31-34]. Both glucose and

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tributyrin were carbon sources for cells; therefore, flux normalization was united

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by the carbon source’s uptake rate and tributyrin was expressed as equivalent to

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the glucose uptake rate. A list of these selected reactions and metabolite

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abbreviations are provided in Appendices A and B.

3. Results and Discussion

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3.1. Effect of aeration rate on lipase production

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To better understand the effect of DO levels on lipase production in batch

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fermentation, the fermentation was divided into four phases, each corresponding

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to a particular physiological state (Figs. 1A and 1B). Phase I (0 h to 18 h) is the

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lag phase. No nutrients are consumed and no lipase is produced in this phase.

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Phase II (18 h to 21 h) is the exponential phase. All the nutrients are consumed for

18

bacterial growth and almost no lipase is produced in this phase. In Phase III (21 h

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to 30 h), the rates of lipase secretion and glucose and tributyrin consumption

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begin to increase while cell growth tends to cease. In Phase IV (30 h to 45 h),

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lipase synthesis rate drops rapidly to a stable level after reaching a maximal level.

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The oxygen supply methods and thus, the DO changing patterns, have significant

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impacts on cell growth, glucose and tributyrin consumption, and lipase production

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during the fermentation by B. subtilis CICC20034. Glucose and tributyrin are 9 Page 10 of 44

used as carbon sources in lipase fermentation. The more the substrates are

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consumed, the faster is the expected lipase production (Fig. 1A, panel 3 and 4).

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Figure 1A panel 4 indicates that the highest final lipase activity (0.48 U/ml) was

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obtained under HOS condition in phase III. Lower lipase activity was observed

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under MOS and LOS conditions with 0.37 and 0.23 U/ml, respectively. However,

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after 27 h ( phase IV), lipase activity under MOS was more stable than under HOS.

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In fact, lipase activity under HOS condition dropped rapidly. This finding

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suggested that a high oxygen supply level in later fermentation phases was not

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beneficial to lipase production.

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The oxygen transfer characteristics of the lipase production process were analyzed.

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DO levels (Fig. 1B, panel 1) decreased considerably faster operating MOS and

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LOS, leading to microaerobic conditions that were maintained until the

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fermentation was completed. During Phase III, DO was consumed more slowly

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under LOS condition because the cell growth rate was much slower due to low

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aeration. DO levels under HOS never fell below 30% and OUR (Table 1) was

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higher than under other conditions, resulting in a higher specific growth rate. The

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correlation between higher OUR at increased µ max was observed previously by

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Rowe et al. [35], which is supported by our results. OUR was higher at the end of

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Phase III, indicating for a higher ATP demand through respiration to support

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lipase synthesis in the latter phase III. Increases in aeration rate also increased the

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kLa values.

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The by-metabolites, organic acids and other amino acids, in the broth at different

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fermentation phases were also measured for metabolic flux analysis and 10 Page 11 of 44

performance comparisons. The detectable by-products are shown in Figs. 2A and

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2B. Pyruvate, lactate and acetate are the major organic acids excreted to the broth.

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Lactate excretion decreases with increased oxygen supply, and no lactate was

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detected under HOS. Highest concentrations of pyruvate (4.1 mmol/l) and acetate

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(41 mmol/l) were achieved under MOS. As part of the constraints of the model,

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the measured 8 kinds of amino acids (Fig.2 B) oscillate with respect to cultivation

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time due to their uptake and excretion rates. Glu reached the highest concentration

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under HOS and MOS condition, while concentration of Ala was maximum under

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LOS condition. As already shown in Fig. 1, LOS was not beneficial for cell

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growth and lipase production; therefore, metabolic flux analysis under LOS

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condition was not longer considered.

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3.2. Effect of aeration rate on metabolic flux

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The effect of oxygen supply levels on intracellular carbon flux distributions was

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investigated by metabolic flux analysis under MOS and HOS conditions. Lipase

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production was observed mainly in Phase III. In early Phase III (t=24 h), the cells

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began to produce lipase, while in late Phase III (t=27 h) lipase production started

19

to drop. Thus, the metabolic fluxes at the two time points were analyzed. The

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metabolic fluxes were estimated as described in Materials and Methods and

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Appendix A, and the results are shown in Fig. 3.

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During early Phase III (Fig. 3A), the metabolic flux to biomass synthesis (r 51) was

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enhanced 2.6-fold under HOS compared to MOS. The important intermediate R5P

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(r10) and the PP pathway are mainly used for the synthesis of nucleotides, cell 11 Page 12 of 44

components, and more than 33.33% of NADPH is required for bacterial growth.

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Additionally, as glucose is the main carbon source, the EMP pathway also needs

3

to be activated to produce precursors, such as G6P, F6P, T3P, PG3, and PEP for

4

amino acid production and cell synthesis. The metabolic flux of EMP under HOS

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was more active than under MOS. As shown in Fig. 3A, the metabolic flux toward

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Pyr (r9), the intermediate at the end of the EMP route and the precursor of the

7

subsequent TCA cycle, also was increased to 153% under HOS compared to MOS

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condition. The metabolic flux of the TCA cycle under HOS was less than that of

9

MOS during the early lipase production phase because more metabolic fluxes

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were required to be directed into the PP pathway for biomass synthesis

11

enhancement. In addition, the flux to biomass (r51) is controlled by the oxygen

12

supply level. In other words, the higher the oxygen supply level, the larger the r50

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value. The value of r51 was also higher under HOS than at MOS during the late

14

lipase production phase. Under HOS, r51 was increased by 44% in late Phase III

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compared to the early phase, whereas under MOS, r51 was decreased by 35% in

16

late Phase III compared to the early phase. The data above show that HOS is

17

favorable for cell growth.

18 19

Lipases are inducible enzymes [36] and thus are generally produced in the

20

presence of a lipid source, such as oil or any other inducer. Tributyrin was used

21

both as carbon source and as the inducer of lipase production. Therefore, the

22

higher the flux from tributyrin (v2), the higher was the flux to lipase (v52).

23

Obviously, in the early lipase production phase, under HOS, the tributyrin

24

consumption flux (r2) was increased by 37% compared to MOS. As a

25

consequence, flux to lipase (r51) was increased by 133% under HOS. When the 12 Page 13 of 44

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early lipase production phase elapses, r2 decreased under HOS but increased under

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MOS. In the late lipase production phase, the tributyrin consumption flux (v2) was

3

decreased by 18% under HOS and consequently, flux to lipase (v51) was

4

decreased by 15%.

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In the late lipase production phase, r10 decreased because of reduced energy and

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carbon source requirements for cell growth. The enhanced fluxes in the TCA

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cycle (v14, v15, v16, v17, and v18) coincided with the increased flux to lipase (v51).

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The TCA cycle had to be activated to satisfy the increased ATP requirements for

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both lipase synthesis and metabolic maintenance of the cells.

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The flux from αKG toward glutamate is important because aspartic family

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compounds occupy 26% of the amino acid composition of lipase A. αKG is

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therefore a key branch point in the TCA cycle [2]. Provided that TCA flux is still

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able to maintain the consequent energy generation and run the entire metabolic

16

network, more flux might be directed toward glutamate synthesis to accumulate

17

more lipase. Metabolic flux analysis indicated that during the various phases, the

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TCA fluxes prior to αKG node under both aeration strategies were almost the

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same. However, much more flux was directed out of TCA cycle at the αKG node

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for glutamate synthesis under the MOS strategy. Metabolic flux analysis results

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further supported the fact that MOS is beneficial for achieving a stable and

22

enhanced lipase accumulation during those phases.

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3.3. A novel oxygen supply strategy based on metabolic flux analysis

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13 Page 14 of 44

Considering the results of metabolic flux analysis, following conditions support

2

an enhanced lipase production: (1) high tributyrin consumption flux; (2) high

3

TCA fluxes throughout the fermentation; (3) sufficient ATP generation to

4

maintain the running of the entire network; and (4) less biomass fluxes throughout

5

the fermentation. Therefore, combination of HOS in the early fermentation phase

6

to achieve a high rate of lipase synthesis, and MOS in the late fermentation phase

7

to stabilize the lipase synthesis rate, seems to be a promising strategy to enhance

8

the overall lipase production. Consequently, the following two-stage oxygen

9

supply strategy was proposed and used in subsequent fermentations: controlling

10

aeration at 1.5 vvm during the first 24 h of the fermentation to achieve HOS

11

condition, then a stepwise reduction of 0.1 vvm per 30 min until aeration rate

12

reaches 1.0 vvm. Aeration, as well as agitation, is then maintained at this level

13

without further changes for the rest of the fermentation period. The careful

14

reduction of the oxygen supply was chosen to avoid big disturbances in

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fermentation performance and metabolism, as sudden DO changes caused by

16

rapid oxygen supply shifts are generally considered to be harmful to the

17

physiological state of a fermentation process [37]. The classification of the four

18

phases is based on the physiological and production states during fermentation.

19

Altering the oxygen supply modes will only change the physiological states

20

during each phase to some extent (particularly those in production phases) to

21

direct the entire fermentation to a desirable end result.

22 23

The fermentation curves, including concentration of biomass and glucose, as well

24

as lipase activity and DO levels, depending on the proposed oxygen supply

25

method are shown in Fig. 4. The curves obtained under HOS and MOS conditions 14 Page 15 of 44

are also depicted in the figure for comparison. As shown in Fig. 4D, applying the

2

proposed new oxygen supply strategy (TOS) led to an increase in final lipase

3

activity by 17% and 51%, when compared to separate HOS and MOS

4

fermentations, respectively. Lipase activity could be controlled at relatively stable

5

and high levels during the late fermentation stages.

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The changing patterns of DO under different oxygen supply strategies are given

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Fig. 4E. The sharp decrease and rebound in DO under MOS during the first 30

9

hours, particularly when DO level dropped to zero at about 30 h, are extremely

10

harmful to the fermentation performance. Applying TOS resulted in a more stable

11

and smoother DO course compared to MOS strategy and sudden changes and

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even the drop to zero were avoided. DO level was constant at about 20%

13

saturation from 36 h until the end of the fermentation.

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The constant DO level achieved by automatically changing either agitation or

16

aeration is generally considered to be the best and simplest way to control or

17

optimize aerobic fermentations [38, 39]. However, proper selection and changes

18

to the DO control levels can be a major challenge, as the relationship between DO

19

set point and the desirable metabolic flux profile are seldom clear or known. The

20

two-stage oxygen supply strategy incorporates and considers the desirable

21

metabolic flux profile, so enhanced lipase production was expected to be achieved

22

by this method.

23 24

The metabolic flux distributions under TOS condition are depicted in Fig. 5.

25

According to the prediction, the tributyrin consumption flux, TCA fluxes, and ATP 15 Page 16 of 44

generation flux were increased by 9%, 57% and 100%, respectively, while the

2

biomass flux decreased to 66.1%, compared to HOS condition. Therefore,

3

application of TOS to improve lipase production was further validated. In lipase

4

production phase, the flux directed toward glutamate synthesis at the αKG node in

5

the TCA cycle is significantly enhanced under the TOS strategy. More

6

importantly, ATP generation was not worsened by the enhanced metabolic

7

distribution at αKG node in TCA cycle. Instead, with the TOS strategy, the ATP

8

generation distribution increased throughout the fermentation period. In

9

conclusion, the TOS condition improves lipase production by enhancing the flux

10

distribution ratio directed toward glutamate synthesis during the late fermentation

11

phases and by maintaining the ATP generation rate throughout the fermentation at

12

a compatible level, similar to the early phases under HOS.

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The effect of the TOS condition on major by-metabolite formation was also

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15

investigated, and the results are summarized in Table 2. Under the TOS strategy,

16

the final concentrations of the two major detectable by-products, namely, acetate

17

and lactate, are basically compatible with those under MOS and HOS conditions.

18

The accumulation of some of the by-products was even reduced under the TOS

19

strategy.

20 21

TOS has been applied to the fermentations of cordycepin [40] and pyruvate [41]

22

to obtain maximal production levels. However, those reported TOS strategies are

23

mainly based on the experimental data of traditional dynamic models. As with

24

typical aerobic amino acid fermentation, many reports also demonstrated that

25

improvement of cell growth and lipase production could be achieved using 16 Page 17 of 44

different oxygen supply methods. However, reports on the development of

2

effective oxygen supply methods with the aid of the metabolic flux analysis or

3

models are seldom available. The novel TOS supply strategy was proposed to

4

improve lipase fermentation performance based on the metabolic flux model. This

5

strategy has been successfully applied and proven in the enhanced lipase

6

production.

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3.4. Lipase production in composite culture medium by oxygen supply

9

strategy

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Most of Bacillus species achieve maximum lipase activity in composite culture

12

medium. Therefore, fermentation was carried out in a composite medium to

13

investigate the application of TOS to industrial production. In the composite

14

culture medium, lipase activity increased significantly (Fig. 6); it reached 3.3

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15

U/ml under MOS, which is a 9-fold increase compared to synthetic medium. With

16

the proposed oxygen supply strategy (TOS), the final lipase activity reached 5.0

17

U/ml. The lipase activity could be controlled at relatively stable and high levels

18

during the late fermentation stages. The lipase activity remained nearly constant

19

for 8 h (from 66 h to 74 h) under TOS condition, while under HOS condition, the

20

lipase activity decreased sharply from 3.3 U/ml to 0.74 U/ml in 6 h.

21 22

4. Conclusion

23 24

Lipase production was coupled to the tributyrin uptake rate, TCA cycle and

25

formation of ATP and biomass. Thus, a TOS strategy that maintains a higher 17 Page 18 of 44

aeration rate in the early stage but step-wisely reduces it to a moderate level in the

2

late stages was proposed to provide the entail conditions for enhanced lipase

3

production. With this strategy, lipase activity reached 0.56 U/ml, which was the

4

highest Bacillus lipase activity in synthetic medium. Additionally, the stable

5

lipase production phase was prolonged. With the proposed TOS strategy, the

6

lipase activity reached 5.0 U/ml in composite medium, which indicates a potential

7

application in industrial lipase production.

Acknowledgements

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This work was supported by the National Science Foundation for Distinguished

12

Young Scholars of China (No. 21225626), the National Basic Research Program

13

of China (No.2011CB710800), the Key Program of National Natural Science

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Foundation of China (No. 20936002), and the National High Technology

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

Research and Development Program of China (No. 2012AA021704).

16 17 18 19

18 Page 19 of 44

Appendix A. Reactions used in metabolic flux model of B. sublilis.

2

Uptake reactions

3

1. Glc + ATP = G6P + ADP

4

2. Tributyrin = Glycerol + 3 Butyrate

5

3. Glycerol + ATP = PGA + NADH + ADP + Pi

6

4. Butyrate + ATP + 2 CoA = 2 AcCoA + FADH2 + NADH + AMP + 2 Pi

7

Embden-Meyerhof-Parns pathway

8

5. G6P = F6P

9

6. F6P + ATP = 2 PGA + ADP

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7. PGA + ADP + Pi = G3P + ATP + NADH

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8. G3P = PEP

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9. PEP + ADP = Pyr + ATP

13

Pentose phosphate pathway

14

10. G6P = R5P + 2 NADPH + CO2

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

11. 3 R5P = 2 F6P + PGA

16

12. R5P + G6P = E4P + F6P + CO2 + 2 NADPH

17

TCA cycle

18

13. Pyr + CoA = AcCoA + CO2 + NADH

19

14. AcCoA + OAA = α-KG + CO2 + NADPH + CoA

20

15. α-KG + Pi + ADP = Suc + CO2 + NADH + ATP

21

16. Suc = Fum + FADH2

22

17. Fum = Mal

23

18. Mal = OAA + NADH

24

Anapleoric reactions

25

19. Mal = Pyr + CO2 + NADPH 19 Page 20 of 44

3

Branches from glycolysis

4

22. Pyr + NADH = Lac

5

23. AcCoA + ADP + Pi = Ac + ATP + CoA

6

Biosynthesis of serine family amino acids

7

24. G3P + Glu = Ser + α-KG + NADH + Pi

8

25. Ser + THF= Gly + MetTHF

9

26. Ser + AcCoA + H2S = Cys + Ac + CoA

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11

27. OAA + Glu = Asp + α-KG

12

28. Asp + Gln + ATP = Asn + Glu + AMP + PPi

13

29. Asp + ATP + 2 NADPH + AcCoA + Cys + H2S + MTHF = Met + ADP + Pi +

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Pyr + Ac + NH3 + THF + CoA

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15 16

30. Asp + ATP + 2 NADPH + Pyr + AcCoA + Glu = Lys + ADP + Pi + Ac + α-KG + CO2 + CoA

17

31. Asp + 2 ATP + 2 NADPH = Thr + 2 ADP + 2 Pi

18

32. Thr + Pyr + NADPH + Glu = Ile + α-KG + NH3 + CO2

19

Biosynthesis of histidine

20

33. R5P + Gln + CO2 + 5 ATP + NADPH = His + 5 ADP + α-KG + 2 NADH + 6

21

Pi

22

Biosynthesis of aromatic family amino acids

23

34. 2 PEP + E4P + ATP + NADPH = Chor + ADP + 4Pi

24

35. Chor + NH3 + 2ATP+R5P + Ser = Trp + Pyr + PGA + CO2 + PPi+2ADP

25

36. Chor + Glu = Phe + α-KG + CO2 20 Page 21 of 44

37. Chor + Glu = Tyr + α-KG + NADH + CO2

2

Biosynthesis of alanine family amino acids

3

38. 2 Pyr + NADPH + AcCoA + Glu = Leu + α-KG + NADH + CO2 + CoA

4

39. 2 Pyr + NADPH + Glu = Val+α-KG + CO2

5

40. Pyr + Glu = Ala + α-KG

6

Biosynthesis of glutamic acid family amino acids

7

41. α-KG + NH3 + NADPH = Glu

8

42. 2 Glu + AcCoA + 2 ATP + NADPH + CO2 + Asp = Arg + α-KG + Ac + ADP +

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2 Pi + FUM + AMP + PPi + CoA

10

43. Glu + ATP + NH3 = Gln + ADP + Pi

11

44. Glu + ATP + 2 NADPH = Pro + ADP + Pi

12

C1

13

45. G3P + NADPH = NADH + CO2 + 2 C1

14

Electron transport system (P/O = 2)

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15

46. NADH + 1.3 ADP + 0.5 O2 + 1.3 Pi = 1.3 ATP

16

47. NADPH+ 1.3 ADP + 0.5 O2 + 1.3 Pi = 1.3 ATP

17

48. FADH2 + ADP + 0.5 O2 +0.8667 Pi = 0.8667ATP

18

Maintenance

19

49. ATP = ADP + Pi

20

50. ATP + AMP = 2 ADP

21

Biomass synthesis

22

51. 0.000154 G6P + 0.00019 F6P + 0.000194 PGA + 0.001395 G3P + 0.000711

23

PEP + 0.002492 Pyr + 0.002132 AcCoA + 0.038608 ATP + 0.000816 R5P +

24

0.016333 NADPH + 0.000308 E4P + 0.001071 a-KG + 0.001923 OAA +

25

0.000156 C1 = Biomass + 0.003595 NADH + 0.002205 CO2 + 0.002132 CoA 21 Page 22 of 44

1

Lipase synthesis

2

52. 16 Ala + 7 Arg + 17 Asn + 9 Asp + 7 Gln + 3 Glu + 24 Gly + 5 His + 13 Ile +

3

20 Leu + 14 Lys + 6 Met + 6 Phe + 5 Pro + 16 Ser + 12 Thr + 2 Trp + 9 Tyr +

4

21 Val + 913 ATP = Lip + 913 ADP + 913 Pi

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

22 Page 23 of 44

Appendix B. Metabolic abbreviations. Acetate

AcCoA

Acetyl coenzyme A

ADP

Adenosine 5’-diphosphate

Ala

Alanine

AMP

Adenosine monophosphate

Arg

Arginine

Asn

Asparagine

Asp

Aspartic acid

ATP

Adenosine 5’-triphosphate

C1

One-unit carbon

Chor

Chorismate

CoA

Coenzyme A

Cys

Cysteine

E4P

Erythrose 4-phosphate

F6P

Fructose 6-phosphate

FADH2

Flavine adeninedinucleotide (reduced)

Fum

Fumarate

Gln

Glutamine

Glu

Glutamic acid

Gly

Glycine

His

Histidine

Ile

Isoleucine

α-KG

α-ketoglutarate

G3P

3- phosphoglycerate

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

23 Page 24 of 44

Gluconate 6-phosphate

Glc

Glucose

Lac

Lactate

Leu

Leucine

Lys

Lysine

Mal

Malate

Met

Methionine

MetTHF

N5-N10-methylene-THF

MTHF

N5-methyl-THF

NADH

Nicotinamide-adeninedinucleotide (reduced)

NADPH

Nicotinamide-adeninedinucleotide phosphate (reduced)

OAA

Oxaloacetic acid

PGA

Phosphoglycerate

PEP

Phosphoenolpyruvate

Phe

Phenylalanine

Pi

Inorganic orthophosphate

Pro

Proline

Pyr

Pyruvate

R5P

Ribulose 5-phosphate

Ser

Serine

Suc

Succinate

THF

Tetrahydrofuran

Thr

Threonine

Trp

Tryptophan

Tyr

Tyrosine

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G6P

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24 Page 25 of 44

Val

Valine

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

25 Page 26 of 44

References

2

[1] I. Martínez, G.N. Bennett, K.-Y. San, Metabolic impact of the level of aeration

3

during cell growth on anaerobic succinate production by an engineered

4

Escherichia coli strain, Metabolic Engineering, 12 (2010) 499-509.

5

[2] H. Xu, W. Dou, H. Xu, X. Zhang, Z. Rao, Z. Shi, Z. Xu, A two-stage oxygen

6

supply strategy for enhanced l-arginine production by Corynebacterium

7

crenatum based on metabolic fluxes analysis, Biochemical Engineering

8

Journal, 43 (2009) 41-51.

us

cr

ip t

1

[3] Z. Chen, H.-J. Liu, J.-A. Zhang, D.-H. Liu, Cell physiology and metabolic flux

10

response of Klebsiella pneumoniae to aerobic conditions, Process

11

Biochemistry, 44 (2009) 862-868.

M

an

9

[4] K. Nakano, M. Rischke, S. Sato, H. Märkl, Influence of acetic acid on the

13

growth of K12 during high-cell-density cultivation in a dialysis reactor,

14

Applied Microbiology and Biotechnology, 48 (1997) 597-601.

d

12

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

[5] A.P. Zeng, T.G. Byun, C. Posten, W.D. Deckwer, Use of respiratory quotient

16

as a control parameter for optimum oxygen supply and scale-up of 2,3-

17

butanediol production under microaerobic conditions, Biotechnology and

18

Bioengineering, 44 (1994) 1107-1114.

19

[6] Y.S. Rahardjo, S. Sie, F.J. Weber, J. Tramper, A. Rinzema, Effect of low

20

oxygen concentrations on growth and alpha-amylase production of

21

Aspergillus oryzae in model solid-state fermentation systems, Biomol Eng,

22

21 (2005) 163-172.

23

[7] P. Calik, G. Calik, T.H. Ozdamar, Oxygen-transfer strategy and its regulation

24

effects in serine alkaline protease production by Bacillus licheniformis,

25

Biotechnol Bioeng, 69 (2000) 301-311. 26 Page 27 of 44

1

[8] F. Tabandeh, H.R.H. Moghaddam, B. Yakhchali, P. Shariati, M.T.H.

2

Mousavian, F. Ghasemi, Fed-Batch Fermentation of Bacillus Clausii for

3

Efficient Production of Alkaline Protease Using Different Feeding

4

Strategies, Chem Eng Commun, 198 (2011) 1063-1074. [9] E. Ducros, M. Ferrari, M. Pellegrino, C. Raspanti, C. Bogni, Effect of aeration

6

and agitation on the protease production by Staphylococcus aureus mutant

7

RC128 in a stirred tank bioreactor, Bioprocess Biosyst Eng, 32 (2009)

8

143-148.

us

cr

ip t

5

[10] H.-J. Kim, Y.-J. Lee, W. Gao, C.-H. Chung, C.-W. Son, J.-W. Lee, Statistical

10

optimization of fermentation conditions and comparison of their influences

11

on production of cellulases by a psychrophilic marine bacterium LBH-10

12

using

13

Engineering, 16 (2011) 542-548.

M

orthogonal

array

method,

Biotechnology

and

Bioprocess

d

14

an

9

[11] C. Asha Poorna, P. Prema, Production of cellulase-free endoxylanase from

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

novel alkalophilic thermotolerent Bacillus pumilus by solid-state

16

fermentation and its application in wastepaper recycling, Bioresource

17

Technology, 98 (2007) 485-490.

18

[12] J.Y. Chen, C.M. Wen, T.L. Chen, Effect of oxygen transfer on lipase

19

production by Acinetobacter radioresistens, Biotechnol Bioeng, 62 (1999)

20

311-316.

21 22 23 24 25

[13] D.V. Vadehra, L.G. Harmon, Factors affecting production of staphylococcal lipase, J Appl Bacteriol, 32 (1969) 147-150. [14] J.A. Alford, J.L. Smith, Production of microbial lipases for the study of triglyceride structure, J Am Oil Chem Soc, 42 (1965) 1038-1040. [15] R. Gulati, R.K. Saxena, R. Gupta, Fermentation and downstream processing 27 Page 28 of 44

1

of lipase from Aspergillus terreus, Process Biochemistry, 36 (2000) 149-

2

155. [16] T. Tan, M. Zhang, B. Wang, C. Ying, L. Deng, Screening of high lipase

4

producing Candida sp. and production of lipase by fermentation, Process

5

Biochemistry, 39 (2003) 459-465.

ip t

3

[17] P. Becker, I. Abu-Reesh, S. Markossian, G. Antranikian, H. Märkl,

7

Determination of the kinetic parameters during continuous cultivation of

8

the lipase-producing thermophile Bacillus sp. IHI-91 on olive oil, Applied

9

Microbiology and Biotechnology, 48 (1997) 184-190.

an

us

cr

6

[18] Y. Wang, K.C. Srivastava, G.-J. Shen, H.Y. Wang, Thermostable alkaline

11

lipase from a newly isolated thermophilic Bacillus, strain A30-1 (ATCC

12

53841), Journal of Fermentation and Bioengineering, 79 (1995) 433-438.

M

10

[19] V.R. Moller B, Wilke D, Foullois B. , Alkaline Bacillus lipases, coding DNA

14

sequences therefore and Bacillus which produce the lipase, (1991).

d

13

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

[20] A. Hiol, M.D. Jonzo, N. Rugani, D. Druet, L. Sarda, L.C. Comeau,

16

Purification and characterization of an extracellular lipase from a

17

thermophilic Rhizopus oryzae strain isolated from palm fruit, Enzyme and

18

Microbial Technology, 26 (2000) 421-430.

19

[21] M.Riaz, A. Shah, A.Hameed, F.Hasan, Characterization of lipase produced

20

by Bacillus sp. FH5 in immobilized and free state, Annals of Microbiology,

21

60 (2010) 169-175.

22

[22] S.Takaç, B.Marul, Effects of lipidic carbon sources on the extracellular

23

lipolytic activity of a newly isolated strain of Bacillus subtilis, Journal of

24

Industrial Microbiology and Biotechnology, 35 (2008) 1019-1025.

25

[23] S.Kumar, K. Kikon, A. Upadhyay,S. Kanwar, R. Gupta, Production, 28 Page 29 of 44

1

purification, and characterization of lipase from thermophilic and

2

alkaliphilic Bacillus coagulans BTS-3, Protein Expression and Purification,

3

41 (2005) 38-44.

4

[24] H. Huang, M.J. Jin, A.H. Xiao, Z. Gao, X. Liu, C. Peng, Enhancing

6

arachidonic acid production by Mortierella alpina ME-1 using improved

7

mycelium aging technology, Bioprocess and Biosystems Engineering, 32

8

(2009) 117-122.

cr

us

[25] L.H. Tang, L.M. Xia, Purification and partial characterization of a lipase

10

from

coagulans

ZJU318,

11

Biotechnology, 125 (2005) 139-146.

Applied

Biochemistry

and

M

Bacillus

an

9

ip t

5

[26] M. Dauner, J.E. Bailey, U. Sauer, Metabolic flux analysis with a

13

comprehensive isotopomer model in Bacillus subtilis, Biotechnol Bioeng,

14

76 (2001) 144-156.

d

12

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

[27] V. Dartois, A. Baulard, K. Schanck, C. Colson, Cloning, nucleotide sequence

16

and expression in Escherichia coli of a lipase gene from Bacillus subtilis

17

168, Biochimica et Biophysica Acta (BBA) - Gene Structure and

18

Expression, 1131 (1992) 253-260.

19

[28] P. Ozkan, B. Sariyar, F.O. Utkur, U. Akman, A. Hortacsu, Metabolic flux

20

analysis of recombinant protein overproduction in Escherichia coli,

21

Biochemical Engineering Journal, 22 (2005) 167-195.

22

[29] P. Çalik, G. Çalik, S. Takaç, T.H. Özdamar, Metabolic flux analysis for

23

serine alkaline protease fermentation by Bacillus licheniformis in a defined

24

medium: Effects of the oxygen transfer rate, Biotechnology and

25

Bioengineering, 64 (1999) 151-167. 29 Page 30 of 44

1

[30] Q. Wu, H. Xu, N. Shi, J. Yao, S. Li, P. Ouyang, Improvement of poly(γ-

2

glutamic acid) biosynthesis and redistribution of metabolic flux with the

3

presence of different additives in Bacillus subtilis CGMCC 0833, Applied

4

Microbiology and Biotechnology,79(2008) 527-535.

ip t

6

[31] G. Stephanopoulos, Metabolic Fluxes and Metabolic Engineering, Metabolic Engineering, 1 (1999) 1-11.

cr

5

[32] A. Marx, B.J. Eikmanns, H. Sahm, A.A. de Graaf, L. Eggeling, Response of

8

the Central Metabolism in Corynebacterium glutamicumto the use of an

9

NADH-Dependent Glutamate Dehydrogenase, Metabolic Engineering, 1

an

10

us

7

(1999) 35-48.

[33] H.W. Wisselink, C. Cipollina, B. Oud, B. Crimi, J.J. Heijnen, J.T. Pronk,

12

A.J.A. van Maris, Metabolome, transcriptome and metabolic flux analysis

13

of arabinose fermentation by engineered Saccharomyces cerevisiae,

14

Metabolic Engineering, 12 (2010) 537-551.

d

M

11

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

[34] P. ÇalIk, T.H. Özdamar, Mass flux balance-based model and metabolic

16

pathway engineering analysis for serine alkaline protease synthesis by

17

Bacillus licheniformis, Enzyme and Microbial Technology, 24 (1999) 621-

18

635.

19

[35] G.E. Rowe, A. Margaritis, N. Wei, Specific Oxygen Uptake Rate Variations

20

during Batch Fermentation of Bacillus thuringiensis Subspecies kurstaki

21

HD-1, Biotechnology Progress, 19 (2003) 1439-1443.

22

[36] M. Lotti, S. Monticelli, J. Luis Montesinos, S. Brocca, F. Valero, J. Lafuente,

23

Physiological control on the expression and secretion of Candida rugosa

24

lipase, Chemistry and Physics of Lipids, 93 (1998) 143-148.

25

[37] C. Kubicek, Organic acids Basic biotechnology, (2001). 30 Page 31 of 44

1

[38] M. Xu, Z. Rao, H. Xu, C. Lan, W. Dou, X. Zhang, H. Xu, J. Jin, Z. Xu,

2

Enhanced Production of Arginine by Expression of Hemoglobin Using a

3

Novel Expression System in Corynebacterium crenatum, Applied

4

Biochemistry and Biotechnology, 163 (2011) 707-719. [39] N.B. Jansen, M.C. Flickinger, G.T. Tsao, Production of 2,3-butanediol from

6

D-xylose by Klebsiella oxytoca ATCC 8724, Biotechnology and

7

Bioengineering, 26 (1984) 362-369.

cr

ip t

5

[40] X.-B. Mao, J.-J. Zhong, Hyperproduction of Cordycepin by Two-Stage

9

Dissolved Oxygen Control in Submerged Cultivation of Medicinal

10

Mushroom Cordyceps militaris in Bioreactors, Biotechnology Progress, 20

11

(2004) 1408-1413.

M

an

us

8

[41] X.-J. Ji, H. Huang, J. Du, J.-G. Zhu, L.-J. Ren, N. Hu, S. Li, Enhanced 2,3-

13

butanediol production by Klebsiella oxytoca using a two-stage agitation

14

speed control strategy, Bioresource Technology, 100 (2009) 3410-3414.

d

12

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15 16 17

31 Page 32 of 44

Fig.1. Effect of oxygen supply method on lipase fermentation performance. A:

2

Biomass, tributyrin, glucose and lipase during fermentation under different

3

oxygen supply conditions. B: Profiles of dissolved oxygen (DO), oxygen uptake

4

rate (OUR) and volumetric oxygen transfer coefficient (KLa) under different

5

oxygen supply conditions. 1: HOS (▲); 2: MOS (●); 3: LOS (■).

6

Fig. 2. Effects of aeration rate on lipase fermentation performance under different

7

oxygen supply conditions. 1: HOS (A1, A2); 2: MOS (B1, B2); 3: LOS (C1, C2).

8

Fig. 3. Metabolic flux distribution in different fermentation phases under HOS

9

and MOS conditions. A: Metabolic flux distribution at early lipase synthesis phase

10

(t=24 h). B: Metabolic flux distribution at late lipase synthesis phase (t=27 h).The

11

normalized metabolic fluxes of the respective reactions are symbolized by N:

12

F1/F2; N: code of respective reaction, F1: flux in HOS condition, F2: flux in MOS

13

condition.

14

Fig. 4. Comparison of lipase fermentation performance using the two-stage

15

oxygen supply strategy (TOS) with that under HOS and MOS conditions. 1: HOS

16

(▲); 2: MOS (●); 3: TOS (■). A: Biomass concentration. B: Tributyrin

17

concentration. C: Glucose concentration. D: Lipase activity. E: Changing patterns

18

of dissolved oxygen under different aeration rates.

19

Fig. 5. Metabolic flux distribution under TOS condition. The normalized

20

metabolic fluxes of the respective reactions are symbolized by N: F1; N: code of

21

respective reaction, F1: flux under TOS condition at t=27 h.

22

Fig. 6. Comparison of lipase fermentation performance under TOS condition with

23

that under HOS condition in composite culture medium. 1: HOS (●); 2: TOS (■).

24

A: Biomass concentration. B: Lipase activity.

d

M

an

us

cr

ip t

1

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

25

32 Page 33 of 44

1

Table 1. Aeration with approximate KLa and osygen uptakes rates used in the

3

cultivation of B. subtils. KLa

Oxygen uptake rate (mmol l-1 h-1)

(vvm)

(h-1)

24 h

27 h

0.5

55

3

5.25

1.0

115

11.97

20.58

1.5

232

23.94

34.02

us

cr

Aeration

ip t

2

an

4

d

M

5

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

33 Page 34 of 44

1

Table 2. By-product formation under two-stage oxygen supply (TOS), HOS, and

2

MOS strategiesa (fermentation time was 27 h) (concentration, mmol/l).

MOS

TOS

Lactate

0

15.49 11.29

Acetate

19.01 23.42 7.37

cr

a

HOS

fermentation time was 27 h

us

3

Control Strategies

ip t

By-product

d

M

an

4

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

34 Page 35 of 44

cr us an

1

d

M

B

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

ip t

A

2 3

Fig.1. Effect of oxygen supply method on lipase fermentation performance. A:

4

Biomass, tributyrin, glucose and lipase during fermentation under different

5

oxygen supply conditions. B: Profiles of dissolved oxygen (DO), oxygen uptake

6

rate (OUR) and volumetric oxygen transfer coefficient (KLa) under different

7

oxygen supply conditions. 1: HOS (▲); 2: MOS (●); 3: LOS (■).

35 Page 36 of 44

50

Concentration (mmol/L)

30

20

10

0

30

20

10

0 10

20

30

40

50

10

20

30

40

50

0

0.4 0.3 0.2

0.6 0.5

0.2 0.1 0.0

10

20

30

40

50

Glu Gln Arg Ala Tyr Val Cys Lys

0.7

0.3

0.0 0

0.8

0.4

0.1

20

0.6 0.5 0.4 0.3 0.2

us

0.5

10

30

40

50

Time (h)

Glu Gln Arg Ala Tyr Val Cys Lys

0.7

Concentration (mmol/L)

Concentration (mmol/L)

0.6

10

0.9

0.8

Glu Gln Arg Ala Tyr Val Cys Lys

0.7

20

Time (h) 0.9

0.8

30

0 0

Time (h) 0.9

1

cr

0

Pyruvate Lactate Acetate

40

Concentration (mmol/L)

Concentration (mmol/L)

Pyruvate Lactate Acetate

40

ip t

Pyruvate Lactate Acetate

40

0.1 0.0

0

10

30

Time (h)

40

50

0

10

20

30

40

50

Time (h)

d

M

an

Time (h)

20

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

50

Concentration (mmol/L)

50

36 Page 37 of 44

1 2 3 4

ip t

5

cr

6 7

us

8

an

9 10

13 14

d

12

M

11

Fig. 2. Effects of aeration rate on lipase fermentation performance under different

Ac ce pt e

1 C1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 C2 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

15

oxygen supply conditions. 1: HOS (A1, A2); 2: MOS (B1, B2); 3: LOS (C1, C2).

16

37 Page 38 of 44

Glucose

His 33: 0.019/0.008

1: 88.36/91.53

10: 92.05/88.59

G6P 5: 9.61/7.22

Tributyrate

R5P 12: 51.22 /18.56

11:163.52/95.65

E4P

F6P

2: 4.66/3.39

PGA

26: 43.67/20.85 Cys

45: 1.3/0.8

C1

1/0 .

G3P

8: 301.09/177.01 21: 0/0

Phe

0 .2

24: 79.65/37.33

cr

Ser

Trp

34: 3.88/2.95 Chor

PEP

5 .3/0.7 36: 1

37

us

Gly 25: 28.06/12.84

19

7: 380.77/214.14

Butyrate

35 :

Glyerol

ip t

6: 326.19/190.08 3: 4.66/3.39

an

13: 146.25/49.44

d

/0 .7 9

Suc 16: 73.63/13.16

51:

NADH

Transhydrogenation Reaction

46: 782.44/305.71

8

2/ 0.

326

Ile

Biomass

Lipase 48: 62.98/10.12

04

.51 /15 2

.89

Fum

52 ： 0.

32: 5.32/4.41

Gln

Pro

17: 74.02/14.31

Thr

01

31: 5.73/ 4.41

76

30 : 4 . 06 /3.

07 1. 5/

41 : 28: 0.011/0.009 27: 61.89/33.14 Asn Asp OAA 14: 85.28/17.46 α-KG 20.67 4.06/ 18: 10.19/15.47 29: 4 Met 15: 74.41/13.47 Mal

:0 .9 9

AcCOA

22: 96.48/103.17 Lac 42: 0.071/0.021 4 Glu Arg 4/67.3 .6 4 6 1

44

M

23: 29.39/20.44

Ac ce pt e

Leu .02 .04/0 39: 10.11/ 4.54 Val Pyr 40: 1 .05/0 .98 Ala 38 : 0

19: 64.23/54.11 Ac

Lys

Tyr

1 .7 /1

4: 4.66/3.39

7 .6 :3

9: 200.34/79.17

.6 :3 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

FADH2

NADPH 47: 160.71/120.53

ATP 49-50: 1163.47/485.79 ADP

Maintenance

1

38 Page 39 of 44

1

B Glucose

His 33: 0.021/0.011

1: 90.83/86.38 10: 91.37/87.51

G6P

E4P

F6P

2: 3.52/4.24.

12: 45.71 /30.19

ip t

5: 10.01/8.56

Tributyrate

R5P

11: 113.34/120.23

6: 210.33/178.72 3: 3.52/4.24

PGA

cr

Glyerol

Trp

7: 296.54/208.46

Butyrate 24: 56.59/76.19

45: 2.12/0.93 G3P C1

/0 .3 2

34: 2.97/8.19 Chor

PEP

36: 1

.95 .23/0

37

an

21: 0/0

35

8: 214.34/189.53

26: 27.36/36.96 Cys

Phe

:0 .1 9

Ser

us

Gly 25: 18.99/21.73

5 .7 :1

Tyr

7 .8 /2

9: 119.34/85.82

Leu .05 .04/0 38: 0 39: 7.78/ 12.19 Val Pyr 40: 0 .96/1 13: 112.25/135.16 . 21 Ala

4: 3.52/4.24

M

19: 52.19/89.73

Fum

Biomass

/1 .8 1

NADH Lipase

9/ 0. 03

.0 2

51:

471

Ile

Suc 16: 85.12/88.78

4

Thr

.21 /98 .65

32: 4.52/5.31

Gln

Pro

17: 52.12/88.78

52 :0

31: 4.522/ 5.31

57

2/3 .

3 .1

30 :

Lys

1

28: 0.008/0.012 27: 41.45/56.31 Asn Asp OAA 14: 85.52/88.78 α-KG 4 5 . 2 3 4.61/ 18: 85.12/88.78 29 : 2 Met 15: 85.52/88.78 Mal

4 3. 9/ .0

Ac ce pt e

AcCOA

22: 102.23/67.45 Lac 42: 0.034/0.053 42 Glu Arg . 7 8 / 3.11 41: 4 :0 .7 2

d

23: 45.21/19.86

44

Ac

:2 43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

Transhydrogenation Reaction

46: 534.98/698.46 48: 43.12/34.67 FADH2

NADPH 47: 98.87/142.45

ATP 49-50: 809.76/598.97 ADP

Maintenance

2 3 4

Fig. 3. Metabolic flux distribution in different fermentation phases under HOS

5

and MOS conditions. A: Metabolic flux distribution at early lipase synthesis phase

6

(t=24 h). B: Metabolic flux distribution at late lipase synthesis phase (t=27 h).The

7

normalized metabolic fluxes of the respective reactions are symbolized by N: 39 Page 40 of 44

1

F1/F2; N: code of respective reaction, F1: flux in HOS condition, F2: flux in MOS

2

condition.

d

M

an

us

cr

ip t

3

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

40 Page 41 of 44

ip t cr us an M

1

Fig. 4. Comparison of lipase fermentation performance using the two-stage

3

oxygen supply strategy (TOS) with that under HOS and MOS conditions. 1: HOS

d

2

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

4

(▲); 2: MOS (●); 3: TOS (■). A: Biomass concentration. B: Tributyrin

5

concentration. C: Glucose concentration. D: Lipase activity. E: Changing patterns

6

of dissolved oxygen under different aeration rates.

41 Page 42 of 44

Glucose

His 33: 0.018

1: 85.02 10: 85.75

G6P

R5P

5: 118.32

11: 153.25

12: 48.19

Tributyrate E4P

F6P 2: 5.99 3: 5.99

Glyerol

ip t

6: 305.33 PGA

Trp

7: 359.49 25: 32.76

24: 93.07

Ser

G3P

45: 1.32

C1

8: 266.39

35

26: 51.58 Cys

21: 0

34: 4.46

36 : 1

Chor

5 .1 :4 37

us

PEP

an

19: 57.45 Ac

Pyr

13: 206.15

38: 0

.06

40 : 1

.35

Tyr

9: 171.29

4: 5.99

Phe

:0 .2 5

Gly

cr

Butyrate

Leu 39: 11.07

.31

Val

Ala

22: 55.41

M

23: 38.79

Lac

AcCOA

:0 .6 8

42: 0.061

Arg

Gln

Pro

15: 122.76

Mal

17: 122.24 Suc 16: 121.68 Biomass

NADH

Lipase 58

51 :

159

.77

32: 1.48

Fum

Transhydrogenation Reaction

46: 1114.98 48: 105.27

52 :0

30 :3 . 72

Ile

44

18: 65.35

Thr

Lys

α-KG

OAA

.0

.16 9 : 52

d

27: 63.99

31: 4.58

2

Asp

Ac ce pt e

Met

28: 0.022

Glu

5

3 .0 :4

Asn

14: 134.62

85.2 41: 1

43

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

FADH2

NADPH 47: 155.87 ATP 49-50: 1549.90 ADP

Maintenance

1 2 3

Fig. 5. Metabolic flux distribution under TOS condition. The normalized

4

metabolic fluxes of the respective reactions are symbolized by N: F1; N: code of

5

respective

reaction,

F1 :

flux

under

TOS

condition

at

t=27

h.

42 Page 43 of 44

9 A

7

ip t

6 5

cr

Biomass (g/l)

8

3 0

20

40

60

80

us

4

100

120

140

an

Time (h)

M

1

B

d

Lipase activity (U/ml)

5 4

Ac ce pt e

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

3 2 1 0

0

20

40

60

80

100

120

140

Time (h)

2 3

Fig. 6. Comparison of lipase fermentation performance under TOS condition with

4

that under HOS condition in composite culture medium. 1: HOS (●); 2: TOS (■).

5

A: Biomass concentration. B: Lipase activity.

43 Page 44 of 44