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Regulation of hydrogen production by Enterobacter aerogenesby external NADH and NADD

Chong Zhang, Kun Ma, Xin-Hui Xing*

Department of Chemical Engineering, Tsinghua University, Beijing 100084, PR China

a r t i c l e i n f o

Article history:

Received 4 September 2007

Received in revised form

20 August 2008

Accepted 24 November 2008

Available online 25 December 2008

Keywords:

Enterobacter aerogenes

NADH (NADþ)

Oxidoreduction reaction

Hydrogenase

Hydrogen production

* Corresponding author. Department of CheBeijing 100084, PR China. Tel.: þ86 10 6279 4

E-mail address: xhxing@tsinghua.edu.cn0360-3199/$ – see front matter ª 2008 Interndoi:10.1016/j.ijhydene.2008.11.070

a b s t r a c t

Experiments involving the addition of external nicotinamide adenine dinucleotide,

reduced form (NADH) or nicotinamide adenine dinucleotide (NADþ) have been designed to

examine how the hydrogen in Enterobacter aerogenes is liberated by NADH or NADþ. The

addition of external NADH or NADþ was found to regulate hydrogen production by

E. aerogenes in resting cells, batch cultures, and chemostat cultures. Particularly in

chemostat cultivation, with the external addition of NADH, hydrogen production via the

NADH pathway was decreased, while that via the formate pathway was increased; in the

end, the overall hydrogen p was decreased. The addition of NADþ, on the other hand, gave

the opposite results. The membrane-bound hydrogenase was found to play a central role in

regulating hydrogen production. The occurrence of NADH oxidation (NADþ reduction) on

the cell membrane resulted in an electron flow across the membrane; this changed the

oxidation state and metabolic pattern of the cells, which eventually affected the hydrogen

evolution.

ª 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights

reserved.

1. Introduction membrane-bound protein [3], but little has been discovered

Enterobacter aerogenes, a facultative anaerobic bacterium, has

high potential for the production of hydrogen, which is an

environmentally friendly energy carrier that may find wide-

spread use in the future [1]. The production of hydrogen by E.

aerogenes is highly dependent on NADH and NADþ, and thus

engineering the metabolic reactions mediated by these

cofactors would have to be a key feature in any development

of an enhanced hydrogen production process. Since the

unique NADH pathway in E. aerogenes is reportedly responsible

for hydrogen production, elucidating how NADH affects

hydrogen evolution in the complicated metabolic network

inside the cells is critical for improving the hydrogen yield [2].

The NADH-dependent hydrogenase responsible for hydrogen

production in this bacterium is reportedly a putative

mical Engineering, Tsin771; fax: þ86 10 6277 030(X.-H. Xing).ational Association for H

about the enzyme itself, the reaction mechanism, or its related

genes. Moreover, increasing evidence has indicated that the

levels of NADH and NADþ are not only involved in the regu-

lation of numerous intracellular NADH- and NADþ-dependent

enzymes, but can also change the metabolic flux [4].

Since cofactors play an essential role in a large number of

biochemical reactions in cells, their manipulation can result

in their potential use as tools to achieve the desired metabolic

engineering goals. Berrios-Rivera et al. have proposed the

concept of ‘‘cofactor engineering’’, which integrates dynamics

and the overall control structure of cofactors on metabolism

to achieve the desired goal of metabolic regulation [5–8]. For

example, Escherichia coli capable of overexpressing an NADþ-

dependent formate dehydrogenase (FDH) from Candida boidinii

has been constructed; this FDH can generate higher NADH

ghua University, Room 450, Gong-Wu Building, Tsinghua Yuan,4.

ydrogen Energy. Published by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 2 1227

yields and NADH/NADþ ratios inside the cells, and conse-

quently reduce the intracellular environment [7]. In addition,

Berrios-Rivera et al. have also shown that this NADþ cofactor

manipulation system can be used to improve the production

of ethanol and 1,2-propanediol [6].

‘‘Cofactor engineering’’ is an effective tool for changing the

availability of NADH or NADþ inside cells, but genetic engi-

neering tools are always employed for this purpose. It is

therefore difficult to reach the desired goals because of the

complexity of the distribution of the cofactors inside the cells,

especially with strains for which little genetic information is

available. A promising alternative approach is to regulate the

internal cofactor status and the metabolic flux by an extra-

cellular control strategy, which may involve altering the redox

state of carbon sources, controlling the oxidoreduction

potential (ORP), adding additional electron donors or accep-

tors, and the like.

Nakashimada et al. have studied the hydrogen production

process of E. aerogenes by changing the degree of reduction of

the substrates (Cave) [3]. H2 yields were found to increase

linearly with Cave, indicating that the extracellular redox state

of the substrates directly affects H2 production by E. aerogenes.

Studies on E. coli have also led to the same conclusion that

substrates with different oxidation states can yield specific

product profiles [9,10].

Fermentation of the wild strain of E. coli K-12 at different

ORP levels has shown that at a low ORP level (�100 mV),

formate, acetate, ethanol, and lactate were produced in molar

proportions of approximately 2.5:1:1:0.3, while under highly

reducing conditions (�320 mV), these four metabolites were

produced in molar proportions of 2:0.6:1:2 [4]. Moreover, ORP

has been used as a controlling factor to regulate 1,3-pro-

panediol production by the fermentation of Klebsiella pneu-

moniae under anaerobic conditions [11].

Electrically reduced neutral red added as an electron donor

during the growth of Actinobacillus succinogenes in an electro-

chemical bioreactor has been reported to enhance glucose

consumption, bacterial growth, and succinate production

by about 20%, while decreasing acetate production by about

50% [12]. The exogenous oxidation of NADH by 2-amino-3-

carboxy-1,4-naphthoquinone (ACNQ)/Fe(CN)63� has also been

reported to result in a remarkable increase in pyruvate

production and a decrease in lactate production by bifido-

bacteria cells [13].

The above examples highlight the possibilities of an

extracellular control approach in altering intracellular oxi-

doreduction reactions inside cells. Compared to altering the

NADH (NADþ) status by genetic modification, extracellular

control is more convenient and easily affects the metabolic

networks. Although many extracellular control methods have

been used, to the best of our knowledge there has not hitherto

been any study on the use of extracellular NADH or NADþ to

control the intracellular metabolism of bacterial cultivations.

Furthermore, from a biotechnological point of view, if NADH

or NADþ outside of bacterial cells can modify the intracellular

metabolic fluxes, their use should offer a promising approach

for the control of bioprocesses because a series of cofactor

regeneration systems has been proposed [14,15], and these

systems can be used as inexpensive whole-cell suppliers of

NADH or NADþ outside of the target cells.

In the present work, we have examined the effects of

external NADH and NADþ on the regulation of hydrogen

production by E. aerogenes in resting cells, batch cultures, and

chemostat cultures. The metabolic change in chemostat

cultivation upon the addition of external NADH (NADþ) has

been studied, and the reason for the change in the hydrogen

generation pathway has been explored. Finally, a possible

mechanism that rationalizes the influences of external NADH

(NADþ) on the metabolism of E. aerogenes is discussed.

2. Materials and methods

2.1. Bacterial strains and media

E. aerogenes IAM 1183 purchased from the Institute of Applied

Microbiology of the University of Tokyo, Japan, was used in

the present study. Glucose medium (per liter: 15 g glucose, 5 g

tryptone, 14 g K2HPO4, 6 g KH2PO4, 2 g (NH4)2SO4, and 0.2 g

MgSO4$7 H2O) was used for the bacterial cultivation.

2.2. Effects of external NADH on hydrogen production byE. aerogenes resting cells

E. aerogenes cells were collected according to the following

procedure: after 12 h of anaerobic batch cultivation, 50 mL of

culture broth was centrifuged (10,200� g for 5 min at 4 �C) and

the cells were collected and resuspended in buffer A (50 mM

triethanolamine (TEA) buffer, pH 6.80, 5 mM dithiothreitol).

This procedure was repeated three times to wash the cells,

and finally they were resuspended in buffer A and the optical

density at 600 nm (OD600) was adjusted to 0.8–1.0. Fifty milli-

liters of this resuspended cell solution was added to a 60 mL

anaerobic bottle. After 10 mg of NADH had been added (no

addition of NADH was used as a control), the reaction mixture

was degassed to achieve anaerobic conditions (purging with

nitrogen for 15 min) and then incubated for 8 h at 37 �C and

170 rpm. The H2 produced was measured every 2 h.

2.3. Batch cultivation

A 50 mL anaerobic bottle containing 20 mL of glucose

medium was first degassed to anaerobic conditions (purging

with nitrogen gas prior to tubing, head-gas replacement, and

then autoclaving), and was subsequently inoculated with

a 1-d seeding culture at an inoculum size of 2.4% (v/v). Batch

cultivation was carried out on a reciprocal shaker at 150 rpm

and 37 �C for 12 h.

2.4. Chemostat cultivation

Anaerobic chemostat cultivation was performed using the

equipment shown in Fig. 1. The volume of the bioreactor was

400 mL, with a working volume of 200 mL. The medium was

previously degassed to anaerobic conditions (purging with

nitrogen gas prior to tubing, head-gas replacement, and then

autoclaving). After inoculation with seeding E. aerogenes at an

inoculum size of 2%, the bioreactor was run in batch mode for

10 h to grow the cells, and was then switched to the contin-

uous culture mode until the chemostat state was reached. The

Pump

Gas CollectorMagnetic stirrer / heaterCulture brothWaste

Chemostat

reactor

N2

sparging

Sample point

Gas outlet

Broth inlet

Waste outlet

Fig. 1 – Schematic representation of the chemostat cultivation equipment.

0 2 4 6 80

1000

2000

3000

4000

5000 With NADHWithout NADH

Pro

du

ced

H

yd

ro

gen

( M

)

Time (h)

Fig. 2 – Effects of external NADH on hydrogen production

by E. aerogenes resting cells. Experiments were carried out

in 60 mL bottles (50 mM TEA buffer, pH 6.80, 10 mg glucose,

37 8C, 170 rpm, anaerobic) (n [ 3).

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 21228

gas evolved was collected in a gas collector (cylinder) as

shown in Fig. 1, which was filled with saturated NaCl solution.

Following preliminary cultivation experiments at different

dilution rates, the dilution rate was chosen as 0.32/h for all of

the experiments described in this paper. Under the anaerobic

conditions, the continuous culture reached a steady state

after five or six residence times. The temperature and agita-

tion speed were maintained at 37 �C and 150 rpm, respec-

tively. Once the continuous culture reached the steady state,

NADH (NADþ) was added to the inlet culture broth at

a concentration of 0.7 mM. The change in metabolic flux was

detected in the ensuing 4 h following this addition of NADH

(NADþ).

2.5. Analytical methods

The amount of gas produced was measured with a measuring

cylinder as shown in Fig. 1. OD600 was measured with a spec-

trophotometer (Shimadzu UV-1206, Japan) to measure the cell

concentration on a dry cell weight basis.

2.5.1. Glucose concentrationOne milliliter of dinitrosalicylic acid solution was added to

1 mL of the sample solution. The mixture was heated in

a boiling water bath for 5 min, and then 4 mL of water was

added. The absorbance of the sample was measured at

510 nm.

2.5.2. Gas componentsThe components of the gas evolved were analyzed on a gas

chromatograph (Shimadzu GC8A, Japan), equipped with

a Parapak Q column (80–100 meshes) and a thermal conduc-

tivity detector (TCD), with N2 as the carrier gas. The working

temperatures of the column and the TCD were 80 �C and

120 �C, respectively.

2.5.3. MetabolitesA 10 mL sample drawn from the chemostat cultures was

centrifuged (10,200� g for 5 min at 4 �C), and the supernatant

was frozen at �20 �C until the analysis. Pyruvate, acetate,

ethanol, succinate, formate, lactate, and 2,3-butanediol were

determined at 40 �C on a high-pressure liquid chromatograph

(HPLC-10A, Shimadzu) equipped with an SCR-102G organic

acid analysis column (Shimadzu) and an RID-10A detector

(Shimadzu). The mobile phase was 0.1% aqueous perchloric

acid solution.

2.6. Assay of NADH and NADþ [4]

2.6.1. Pre-treatmentAliquots of 5 mL were withdrawn from the chemostat cultures

after a steady state had been attained, and the tubes were

immediately immersed in an ice bath. For the assay of NADþ,

a variable volume of HCl (6 N) was added to yield a final pH of

1.2, and the HCl-treated sample was subsequently incubated

at 50 �C for 10 min prior to neutralization with KOH to a pH of

6.5–7 with vigorous agitation. For the assay of NADH, a vari-

able volume of KOH (10 N) was added to yield a final pH of 12.5,

and the KOH-treated culture (pH 12.5) was subsequently

incubated for 10 min at room temperature (25 �C). The NADH

(NADþ) concentrations of the treated samples were measured

within 24 h.

2.6.2. MeasurementsNADH (NADþ) was measured by coupling the appropriate

enzyme assays with fluorimetric determination of the coen-

zyme NADH [4]. Emission was measured at 460 nm after

0 2 4 60.8

0.9

1.0

1.1

1.2

NADH Concentration (mM)

OD

600 (-)

16

18

20

22

24

Hyd

ro

gen

V

olu

me (m

l/20m

l b

ro

th

)

OD600Hydrogen Volume

Fig. 3 – Influence of extracellular NADH on hydrogen

production in the batch cultivation of E. aerogenes (n [ 3).

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 2 1229

excitation at 350 nm with a fluorescence spectrofluorimeter

(Hitachi F-2500, Japan).

NADþ was assayed in a reaction mixture containing pyro-

phosphate buffer (50 mM, pH 8.8), semicarbazide (2.5 g/L),

absolute ethanol (80 mM), acid extract, and alcohol dehydro-

genase (0.5 U/mL). Furthermore, NADH was assayed in

a reaction mixture containing triethanolamine buffer

(200 mM, pH 7), pyruvate (5 mM), alkaline extract, and LDH

(20 U/mL).

The error in the assays of NADH and NADþ was less

than 6%.

3. Results and discussion

3.1. Effects of external NADH on hydrogen production byE. aerogenes resting cells

E. aerogenes cells were suspended in TEA buffer, and a trace

amount of glucose was used as the sole carbon source to

Glucose

2Pi2NAD+

2NADH2 Phosphoenolpyruvate

2ADP

2ATP2 Pyruvate

2 Lactate

2NAD+

2NADH

CO2

a-acetolactate

CO2Acetoin

2NADH

2NAD+

2,3-butnediol

2CoA-SH

2 Acetyl-CoA2NADH

2CoA-SH+2NAD+

2 Acetaldehyde

2 Ethanol

2NADH

2NAD+2 Acetylphosphate

2Pi

2CoA-SH

2 Acetate

2ATP

2ADP

2 For

2 CO2

2C

2

Fig. 4 – Flux patterns for the anaerobic metabolism of

produce hydrogen. As shown in Fig. 2, glucose could be used to

produce hydrogen in the resting cell system, and with the

addition of NADH, hydrogen production by E. aerogenes was

enhanced accordingly. After a period of 8 h, 0.6 mM more

hydrogen was produced with the addition of NADH compared

with the control. The increase in the amount of hydrogen

(0.6 mM) was much higher than that with added NADH

(0.014 mM). This result indicated that external NADH affected

the hydrogen production by the cells.

3.2. Batch cultivation

NADH in various concentrations in the range 0.5–5 mM was

initially added to the culture medium to study its effects on

the amount of hydrogen produced and the cell concentration

during the batch cultivation of E. aerogenes. As shown in Fig. 3,

after 12 h of cultivation, external NADH of the appropriate

concentration enhanced hydrogen production and cell

concentration, giving optimal results at a concentration of

0.7 mM. Much higher concentrations, such as 5 mM, were

found to inhibit hydrogen production and cell growth. With

the addition of 0.7 mM NADH, the yield of hydrogen from

glucose increased from 0.82 to 1.10 mol H2/mol glucose. Also,

hydrogen production increased from 10.3 to 13.5 mL/OD,

indicating that the external NADH increased the hydrogen

production capability of E. aerogenes.

Tanisho et al. were the first to report that the NADH

pathway plays an important role in hydrogen production by E.

aerogenes [1,2]. If all of the added NADH were used in the NADH

pathway, an additional 0.7 mM of hydrogen (0.17 mL in the

present system) would be produced. However, the actual

increase in the amount of hydrogen produced was 3 mL

(Fig. 3), which was much larger than the presumed value. This

result was identical to that obtained for the resting cells.

Thus, in both the resting cells system and the batch culti-

vation mode, external NADH is not only used by the NADH

pathway to produce hydrogen, but also affects the intracel-

lular metabolism of E. aerogenes. In fact, with the addition of

mate

2 H2

2 H+

H2

2NAD+

2NADH

2 Acetyl-CoA

oA-SH 2CO2

NAD+ 2NADH

4 CO2

TCA Cycle

Biomas

2ATP2H2O

2ADP 6NADH

6NAD+2FAD

2FADH22CoA-SH

2 Oxaloacetate

2 Malate

2 Fumarate

2 Succinate

2NADH

2NAD+

2H2O

2NADH

2NAD+

E. aerogenes (modified from previous reports [16]).

Table 1 – Changes in metabolic fluxes after the addition of NADH and NADD in a chemostat culture.

Time after NADH addition (h) Time after NADþ addition (h)

0 1 4 0 1 4

Dry weight (g/L) 0.22 0.20 0.19 0.23 0.24 0.22

Glucose consumed (g/L) 11.23 10.54 10.46 11.16 11.20 11.33

Succinate (mM) 0.89 0.74 0.29 0.91 0.85 0.72

Lactate (mM) 50.94 54.86 54.99 48.88 45.32 36.31

Formate (mM) 3.56 2.53 4.12 3.86 4.45 5.03

Acetate (mM) 12.35 12.78 13.34 12.20 12.08 11.25

2,3-Butanediol (mM) 2.22 2.37 2.24 2.39 2.34 2.14

Ethanol (mM) 22.16 25.49 25.84 22.66 22.34 20.96

CO2 (mM)a 34.50 39.73 39.24 34.87 33.79 30.76

H2 (mM) 88.79 78.66 76.16 87.20 97.41 95.59

a Calculated using theoretical flux (CO2¼ ethanolþ acetate� formate�CO2 consumed by succinate productionþ 2� 2,3-butanediol).

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 21230

NADH, the concentrations of the final metabolites were

changed (data not shown).

3.3. Chemostat cultivation

To obtain a better understanding of the effect of externally

added NADH (NADþ) on the intracellular metabolism with

emphasis on H2 production, a further experiment on anaer-

obic chemostat cultivation was designed. At a dilution rate of

0.32/h, continuous culture was initiated after a 12 h batch

cultivation, and a steady state was attained after 24 h of

cultivation, whereupon the OD600, pH, and glucose concen-

tration were 1.752, 5.80, and 11.2 g/L, respectively. At the

steady state, the hydrogen productivity reached 46 mL/h (data

not shown). Once the system reached the steady state, NADH

or NADþwas added to the inlet culture broth. Since NADH and

NADþ were unstable in the inlet culture broth, it was difficult

to study the performance of the system in the new stable state

that was reached after the addition of the target cofactor.

Thus, the metabolic shift during the ensuing 4 h following the

addition of the cofactors was studied continuously. Cultiva-

tion without the addition of the cofactors was used as

a control.

The metabolic flux of E. aerogenes was adapted from that

reported in the literature [16] and is shown in Fig. 4. Consid-

ering the hydrogen formation pathway, the anaerobic

fermentation products of E. aerogenes were pyruvate, succi-

nate, lactate, 2,3-butanediol, acetate, ethanol, CO2, and H2.

Table 2 – Influences of external NADH and NADD on hydrogen

Time after NADH addi

0 1 4

H2 yield (mol H2/mol glucose) 1.42 1.41 1.31

H2 (mM) 88.79 78.66 76.16

H2 produced by formate pathwaya 30.95 35.74 35.06

H2 produced by NADH pathwayb 56.47 43.15 41.77

a H2 produced by the formate pathway¼ (acetateþ ethanol� formate).

b H2 produced by the NADH pathway was calculated from the s

NADH [H2 produced by the NADH pathway¼ (2� glucoseþCO2

�(2� ethanolþ 2� succinateþ lactateþ 2,3-butanediolþH2�CO2)].

The addition of NADH or NADþ obviously modified the

fermentation spectrum (Table 1). These data obtained for the

continuous culture imply that the presence of extracellular

NADH (NADþ) affected the metabolic fluxes. In particular, the

hydrogen yield decreased from 1.42 to 1.10 mol H2/mol

glucose upon the addition of NADH. However, upon the

addition of NADþ, the hydrogen yield interestingly increased

from 1.41 to 1.52 mol H2/mol glucose.

In previous studies, it has been found that the hydrogen

production pathway in E. aerogenes comprises two routes [1,2]:

production from formate and conversion from excess NADH.

Based on the concentrations of the metabolic products shown

in Table 1, the amount of hydrogen produced via the formate

and NADH pathways was calculated. As shown in Table 2, the

sum total of hydrogen produced by both pathways corre-

sponded almost exactly to the detected values. With the

addition of NADH, hydrogen produced via the formate

pathway was increased, while that produced via the NADH

pathway was decreased. The net result of these two effects

was that hydrogen production by E. aerogenes was decreased

after the addition of external NADH. The effect of external

NADþ was opposite to that of NADH.

To further illustrate the intracellular metabolism change in

depth, Table 3 lists the parameters reflecting the intracellular

metabolic state of the cells. The oxidation state inside the cells

was less than zero, indicating the presence of excess reducing

power, which corresponded exactly to the hydrogen produc-

tion in this system. With the addition of NADH, the oxidation

production by E. aerogenes.

tion (h) Time after NADþ addition (h)

Trend 0 1 4 Trend

Y 1.41 1.57 1.52 [

Y 87.20 97.41 95.59 [

[ 31 29.97 27.18 Y

Y 56.63 68.48 71.31 [

um of the pathways producing NADH minus those consuming

þCO2 consumed by succinate production� 2� 2,3-butanediol)

Table 3 – Effects of external NADH and NADD on the intracellular metabolism of E. aerogenes.

Time after NADH addition (h) Time after NADþ addition (h)

0 1 4 0 1 4

NADH (mM/g DW) 5.41 6.13 6.70 3.61 3.78 3.18

NADþ (mM/g DW) 19.65 19.83 19.79 13.89 14.81 14.90

NADHþNADþ (mM/g DW) 25.06 25.96 26.49 17.50 18.59 18.08

NADH/NADþ 0.28 0.31 0.34 0.26 0.26 0.21

Ac/Et 1.79 1.99 1.93 1.86 1.85 1.86

ATP yielda 1.67 1.93 1.97 1.67 1.59 1.37

Oxidation stateb �1.06 �1.05 �0.89 �1.05 �1.22 �1.22

a ATP yield values are in moles of ATP produced (lactateþ acetateþ formateþ 2,3-butanediolþCO2þCO2 consumed by succinate production)

per mole of glucose consumed.

b Oxidation state balance was calculated as the sum of products with positive oxidation states and those with negative oxidation states per

mole of glucose consumed. A molecule with a formula of CxHyOz has its own oxidation state of z� 0.5y, and the oxidation state of every

fermentative product in the cultivation of E. aerogenes can be calculated by this equation. Specifically, using the average composition formula of

E. coli for the estimation of E. aerogenes, C4.2H8O1.25N0.68P0.1, and 4% for trace elements (100 g/mol), the oxidation state of the biomass is equal to

�1.5 [4].

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 2 1231

state, ATP yield, and Ac/Et ratio gradually increased. However,

overall, NADþ addition gave the opposite results as compared

to NADH addition. These three parameters indicated the

increase in the oxidation equivalent in the cells, which could

explain why the excess NADH (NADH pathway) was reduced

after NADH was added.

For the change in intracellular NADH (NADþ) (Table 3),

external NADH addition had a slight effect on the internal

NADþ, but this increased the intracellular NADH and the

overall NADH/NADþ ratio. In contrast, the addition of NADþ

led to the opposite effects. A change in the intracellular

NADH/NADþ ratio would influence various metabolic reac-

tions. From the viewpoint of reaction engineering, a higher

NADH/NADþ ratio benefits the reactions that use NADH as the

cofactor, while a lower NADH/NADþ ratio benefits the reac-

tions that use NADþ as the cofactor. Thus, upon the addition

of NADH, the route for ethanol production was enhanced.

Considering the increase in the Ac/Et ratio, the sum of ethanol

and acetate was increased. As the sum of ethanol and acetate

was equal to that of formate and hydrogen, ignoring the

influence of unused formate (which was very small), hydrogen

production via the formate pathway was enhanced.

0 1 2 3 4-50

0

50

100

150

200

250

300

350

time (h)

C( M

)

CNADH-out (NADH addition)CNAD-out (NADH addition)CNADH-out (NAD addition)CNAD-out (NAD addition)

Fig. 5 – External NADH (NADD) profile with time after the

addition of NADH (NADD) (n [ 3).

To understand how the external cofactor can affect the

intracellular metabolism of E. aerogenes, the change in the

extracellular NADH (NADþ) was measured. As shown in Fig. 5,

upon the addition of NADH, the concentration of extracellular

NADH and NADþ in the culture broth gradually increased, and

24% of the added NADH was transformed to NADþ after 4 h. As

the cofactor was introduced into the inlet of the bioreactor,

with the continuous addition of fresh medium, the NADH

concentration increased. Of interest here is the increase in

NADþ, which indicated the oxidation of NADH outside of the

cell. Meanwhile, with the addition of NADþ, 4% of the input

NADþ was found to be transformed to NADH. Nakashimada

et al. have observed NAD(P)H-dependent hydrogen formation

in the cell-free extract of E. aerogenes AY-2, and hydrogenase

activity was found in the cell membrane and not in the cyto-

plasmic fraction [3], implying the direct conversion of NADH

to NADþ and H2 by a membrane-bound hydrogenase. Thus,

the membrane-bound hydrogenase would seem to play

a central role in transforming external NADH (NADþ). The

occurrence of such an oxidoreduction reaction on the cell

membrane resulted in an electron flow across the membrane,

and ultimately changed the oxidation state and metabolic

pattern of the cell.

4. Conclusions

The present study has shown that the addition of external

NADH or NADþ can regulate hydrogen production by E. aero-

genes in resting cells, batch cultures, and chemostat cultures.

In particular, in chemostat cultivation, the addition of NADH

led to a decrease in hydrogen production via the NADH

pathway and an increase in that via the formate pathway,

with the net overall effect being a decrease in production. On

the other hand, NADþ addition gave the opposite results. The

membrane-bound hydrogenase probably played a central role

in this regulation. The occurrence of NADH oxidation (NADþ

reduction) on the cell membrane resulted in an electron flow

across the membrane, ultimately changing the oxidation state

and metabolic pattern of the cell.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 4 ( 2 0 0 9 ) 1 2 2 6 – 1 2 3 21232

Acknowledgements

This work was supported by a project of the Natural Science

Foundation of China (grant no. 20336010, 20676071 and

20806046) and the National Basic Research Program of China

(973 Plan) (grant no. 2003CB716003 and 2009CB724702).

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