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Biochemical Engineering Journal 39 (2008) 581–585

Short communication

Quantification of a specific bacterial strain in an anaerobic mixedculture for biohydrogen production by the aerobic fluorescence

recovery (AFR) technique

Chong Zhang, Xin-Hui Xing ∗Department of Chemical Engineering, Institute of Biochemical Engineering, Tsinghua University, Tsinghua Yuan, Beijing 100084, PR China

Received 11 October 2007; received in revised form 1 November 2007; accepted 1 November 2007

bstract

To analyze a mixed culture consisting of gfp-harbored Enterobacter aerogenes and Clostridium paraputrificum for hydrogen production, aethod by an aerobic fluorescence recovery (AFR) of GFP expressed under anaerobic conditions has been studied. The concentrations of the

espective strain in the mixed culture were calculated based on the fluorescence after AFR. E. aerogenes grew in the early stages because of its

nsensitivity to the residual oxygen in the medium. C. paraputrificum became the dominant strain in the cultivation period after the consumptionf oxygen. According to the repartition of the two strains in the mixed culture and the hydrogen production profile of the respective pure cultures,he hydrogen production by the mixed culture could be calculated.

2007 Elsevier B.V. All rights reserved.

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eywords: Anaerobic processes; Bioprocess monitoring; Microbial growth; Fe

. Introduction

As a sustainable and clean production technology, bio-ydrogen has been paid much attention [1]. Microbial darkermentation is a promising way to produce hydrogen fromiomass or biowastes, because of its high rate of hydrogen pro-uction, the feasibility of process design and control, as well asts potential for integration with waste treatment [2].

Most hydrogen fermentation has been conducted using mixedulture systems [3]. Maintaining the high cell retention of theydrogen-producing strains and control of the cooperative inter-ction between the different strains are the key issues for theioprocess development. Previous researchers have focused onhe optimization of operating parameters in the mixed cultures4–6]. PCR-denatured gradient gel electrophoresis (DGGE) cane successfully applied to qualitatively identify the influences

f operating parameters on the bacterial communities [7]. How-ver, these techniques cannot reveal the dynamic information ofmixed culture.

∗ Corresponding author. Tel.: +86 10 6279 4771/66277 2294;ax: +86 10 6277 0304.

E-mail address: xhxing@tsinghua.edu.cn (X.-H. Xing).

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369-703X/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.bej.2007.11.001

ation; Biohydrogen; GFP

Molecular biology techniques based on fluorescence tracingnd quantitative PCR make it possible to open the “black box”f mixed culture systems. Fluorescence in situ hybridizationnd quantitative PCR methods have been applied to hydrogen-roducing bioreactor, revealing the structure of the microbialommunity [8–10]. However, these methods are expensive andeed complicated procedures, which make the detection timeong, thereby hampering the rapid quantification. Fluorescence

ethods based on GFP have been applied as both qualitativend quantitative detection methods for bioprocesses. The mosterious obstacle to the application of GFP in anaerobic culturess that the mature GFP requires oxygen for the formation of theuorophore to exhibit fluorescence [11]. To solve this problem,ne simple approach is to allow the anaerobically expressed GFPo react with oxygen by introducing air in a bacterial sample toevelop the fluorophore. This method, called aerobic fluores-ence recovery (AFR), has been proved to be effective for theapid and non-disruptive cell quantification of the gfp-harboredydrogen-producing strain, Enterobacter aerogenes PUCKG12]. Using AFR, the fluorescence of GFP expressed under the

naerobic conditions can be recovered, which is proportional tohe cell density.

Since E. aerogenes is a facultative anaerobe, it can removeesidual O2 in a mixed culture of Clostridium butyricum and

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Thus, the following equations can be written

RFUEA = KEAODEA (1)

RFUCP = KCPODCP (2)

82 C. Zhang, X.-H. Xing / Biochemical

. aerogenes for effective H2 production without the addition ofny reducing agents to the medium [13]. In this study, a modelixed culture system consisting of E. aerogenes PUCKG and. paraputrificum M-21 (a strictly anaerobic, mesophilic andhitinolytic strain [14]) was constructed and operated withouteoxygenation to produce hydrogen from lactose. The aim ofhis study was to develop a method based on the AFR techniqueo rapidly quantify the specific strain in the anaerobic mixedulture and its contribution to the hydrogen production.

. Materials and methods

.1. Bacterial strains and media

E. aerogenes pUCGK was constructed in our previous work12]. C. paraputrificum M-21 was a gift from Professor Ohmiyaunio of Mie University, Japan. Lactose medium (per liter:5 g lactose, 5 g tryptone, 14 g K2HPO4, 6 g KH2PO4, 2 gNH4)2SO4, and 0.2 g MgSO4·7H2O) was used for cultivation.

.2. Bacterial batch cultivation

All cultivations of E. aerogenes PUCKG and C. paraputrifi-um M-21 were carried out on a reciprocal shaker at 150 rpm and7 ◦C. A 60-mL anaerobic bottle containing 20 mL of lactoseedium (30 �g/mL kanamycin was added for the cultivation

f E. aerogenes pUCGK) was previously degassed to ensurenaerobic conditions, and was subsequently inoculated with a-d seeding culture of each strain at an inoculum size of 2.4%v/v).

.3. H2 production in a fermenter

For H2 production by a pure culture of C. paraputrificum-21 or E. aerogenes pUCGK, 200 mL of precultured bacterial

ells (OD600 = 2.0) were inoculated in a 5-L fermenter contain-ng 3 L of lactose medium. Anaerobic cultivation was carriedut at 37 ◦C and a constant pH of 6.86 without replacing theas phase in the headspace of the reactor with nitrogen. Theas evolved from the fermenter was collected and the volumeas measured by means of an inverted measuring cylinder over a0% NaOH solution (for the complete absorption of CO2). When2 production was conducted using a mixed culture, 100 mL ofrecultured cells of C. paraputrificum M-21 (OD600 = 2.0) and00 mL of precultured E. aerogenes PUCKG (OD600 = 2.0) werenoculated together into the 5-L fermenter containing 3 L of lac-ose medium and cultivated under the same conditions as for theure cultures. Since H2 can only be evolved when the cultiva-ion becomes the anaerobic condition (ORP less than −150 mV)15], dissolved oxygen was not detected in the present study.

.4. AFR treatment of the cultivated cells

Aliquots (10 mL) of the culture broth taken at different cul-ure times were first centrifuged at 10,200 × g for 10 min. Theentrifuged cells were washed twice with 100 mM PBS bufferpH 7.0). They were then resuspended in PBS buffer to reach

FitC

eering Journal 39 (2008) 581–585

D600 = 0.8 and subsequently transferred to an unsealed tube toe incubated on a reciprocal shaker at 150 rpm and 37 ◦C for 1 hntil the fluorescence intensity was recovered to the maximumalue.

.5. Analytical methods

The OD600 for evaluating the cell concentration was mea-ured with a spectrophotometer (Shimadzu UV-1206, Japan) andhe measurement error was less than 1.0%. The components ofhe gas evolved were analyzed using a gas chromatograph (Shi-

adzu GC8A, Japan) with a Parapak Q 80–100 mesh columnnd a TCD detector. For the measurement of fluorescence inten-ity, a fluorescence spectrophotometer (Hitachi F-2500, Japan)as used, with excitation at 488 nm and emission at 510 nm. The

elative error for the fluorescence detection was 3.370%.

. Results and discussion

.1. Establishment of a method for quantification ofonstituent strains in a model mixed system

The anaerobically cultivated E. aerogenes PUCKG and. paraputrificum M-21 cells were resuspended in phosphateuffer, respectively. The fluorescence of E. aerogenes PUCKGas then recovered by the AFR method. E. aerogenes PUCKGith the expressed GFP exhibited high fluorescence intensity byFR, while the nonfluorescent C. paraputrificum M-21 showed

ow fluorescence intensity, mainly because of the light-scatteringffect by the cells [16]. Nevertheless, the fluorescence intensi-ies of each strain exhibited good linear relationships with OD600Fig. 1).

ig. 1. Standard curve of the relationship between the maximum fluorescencentensity (RFU) and the optical density (OD) of Enterobacter aerogenes cul-ivated anaerobically without the deoxygenation at the beginning (�) andlostridium paraputrificum M-21 (�). The cells cultivated 10 h were used.

Engineering Journal 39 (2008) 581–585 583

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Table 1Comparison of calculated and real optical densitiesa

Added EA,OD600

Added CP,OD600

RFUmix CalculatedEA, OD600

Relativeerror (%)

0.80 0 41.86 0.83 3.20.70 0.10 37.68 0.71 1.60.60 0.20 35.49 0.65 6.70.50 0.30 31.38 0.54 5.30.40 0.40 28.02 0.45 6.50.30 0.50 23.91 0.34 5.10.20 0.60 19.87 0.23 4.00.10 0.70 15.39 0.11 1.40 0.8 11.36 0 –

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C. Zhang, X.-H. Xing / Biochemical

here RFUEA and RFUCP represent the fluorescence intensitiesf E. aerogenes and C. paraputrificum; ODEA and ODCP rep-esent OD600 of E. aerogenes and C. paraputrificum; and KEAnd KCP represent the coefficients between the RFU and the ODf E. aerogenes and C. paraputrificum. From the model experi-ent shown in Fig. 1, KEA and KCP were determined to be 51.1

nd 14.1, respectively.When the different types of cells were mixed together, the

uorescence intensity and the optical density of the mixed cul-ure, RFUmix and ODmix were used to denote the sums of theifferent strains, that is

FUmix = RFUEA + RFUCP (3)

Dmix = ODEA + ODCP (4)

rom the above Eqs. (1)–(4), the following equation can bebtained

KEA KCP

1 1

)(ODEA

ODCP

)=(

RFUmix

ODmix

)(5)

Thus, by detecting RFUmix and ODmix, OD600 of the specifictrains in the mixed culture can be calculated.

As shown in Table 1, the RFU and OD of mixed strains with

ifferent ratios of ODEA and ODCP were detected. The cell con-entration of E. aerogenes PUCKG was calculated using Eq. (5),nd the relative error compared with the actual cell concentrationas less than 6.7% for all the ratios of ODEA and ODCP studied.

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ig. 2. Profiles of cell optical density, fluorescence intensity, and total gas volume duhase in the headspace of the fermenter with nitrogen. Symbols: (�) hydrogen volulture of E. aerogenes; (B) pure culture of C. paraputrificum M-21; (C) mixed cultu

a The parameters used for the calculation were as follows: KEA = 51.1;

CP = 14.1.

hus, the concentration of the specific strain in the model mixedystem could be quantified by the above method.

.2. Analysis of the repartition of the constituent strains inhe anaerobic mixed culture of E. aerogenes and C.araputrificum for hydrogen production

E. aerogenes PUCKG and C. paraputrificum M-21 were inoc-

lated into a 5-L fermenter without replacing the gas phase inhe head space of the fermenter with nitrogen. Pure cultures ofhe respective strains were also performed as controls under theame conditions as those of the mixed culture.

ring the whole course of anaerobic cultivation without replacement of the gasume; (�) OD600; (©) maximum fluorescence intensity of the cells. (A) Purere of E. aerogenes and C. paraputrificum M-21.

5 Engineering Journal 39 (2008) 581–585

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Fig. 3. Contribution of E. aerogenes and C. paraputrificum M-21 to hydrogenproduction during the first 12 h of the mixed culture.

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84 C. Zhang, X.-H. Xing / Biochemical

As shown in Fig. 2A, as E. aerogenes PUCKG is a facul-ative anaerobe, the residual oxygen did not inhibit the growthf the strain. In the first 6 h after the inoculation, the growth ofhis strain was fast because of its aerobic metabolism; mean-hile, no hydrogen was produced in this period. After all the

esidual oxygen had been consumed, the metabolism switchedo the anaerobic fermentation, and consequently, the productionf hydrogen commenced. For ease of comparison with the otherwo cultivations, only the first 25 h of cultivation of E. aero-enes PUCKG was monitored. After 25 h of cultivation, 0.9 Lf hydrogen had been produced. The maximum fluorescencentensity after AFR was proportional to the cell concentrationnd the parameter KEA was calculated to be 50.9, which waslose to the value obtained from Fig. 1.

As shown in Fig. 2B, when the pure culture of C. parapu-rificum M-21 was performed, growth of the cells was inhibiteduring the first 3 h because of the residual oxygen in the fer-enter. However, C. paraputrificum M-21 was not so strictly

ensitive to oxygen compared with C. butyricum; it could adapto the culture conditions, and began to grow after 3 h. After 3 h,rowth of this strain was much higher than that of E. aerogenesUCKG; its cell concentration reached a maximum OD600 of.20 at 10 h and then decreased thereafter, probably as a resultf the formation of spores when the nutrients were depleted.he final volume of hydrogen was 4 L after 25 h of cultivation.he fluorescence intensity detected during the cultivation of C.araputrificum M-21 was mainly caused by the light-scatteringffect of these fast-growing cells, and was proved to be depen-ent on OD600 before 10 h. The parameter KCP was calculatedo be 21.3.

In the mixed culture, the two strains were inoculated in aatio of 1:1 in terms of OD600. As shown in Fig. 2C, the cellsrew slowly during the first 8 h; thereafter, they began to grow atigh speed and reached the maximum OD at 14 h. The hydrogenroduced after 25 h was 3.8 L, slightly less than that producedy the pure culture of C. paraputrificum M-21.

To analyze the repartition of the two strains in the mixed cul-ure, ODmix and RFUmix were used to calculate the ODs of theespective strains by using Eq. (5) and the values of KEA andCP obtained for the respective pure cultures. The results were

hown in Fig. 3. As dead cells will influence the detection of flu-rescence intensity, only the data during the first 12 h were used.n the initial stage, the estimated proportion of E. aerogenes was0%, which agreed well with the actual proportion at the inocu-ation. This result indicated the validity of the calculation basedn Eq. (5). The residual oxygen in the fermenter enhances therowth of E. aerogenes PUCKG and inhibits the growth of C.araputrificum M-21; thus, during the first 3 h, the proportionf E. aerogenes PUCKG increased up to 67% as a result. Whenhe residual oxygen had been consumed, which could allow therowth of C. paraputrificum M-21, the proportion of E. aero-enes was reduced from 67% to 15% as the growth rate of C.araputrificum M-21 was much higher than that of E. aerogenes

UCKG (Fig. 2).

With the repartition of the two strains before 12 h, the ODalues of respective strains could be calculated. If the celloncentration of the respective strains in the mixed culture

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ig. 4. Comparison of the produced hydrogen volume and the calculated datauring the first 12 h of mixed culture. Symbols: (�) the experimental value; (©)he calculated value.

as known, the corresponding hydrogen production could bebtained from Fig. 2A and B. Then the total hydrogen produc-ion could be calculated as the sum of the hydrogen produced byhe respective strains. As shown in Fig. 4, the calculated hydro-en volume was identical with the experimental value in theixed culture. This result indicated that the effect of population

hanges on hydrogen production in the mixed culture could beasily studied.

. Conclusions

In the present study, the anaerobic mixed culture consisting of. aerogenes PUCKG and C. paraputrificum M-21 was success-

ully analyzed by a method based on AFR of GFP. E. aerogenesUCKG grew in the early stages, while C. paraputrificum, whichad a higher growth rate than that of E. aerogenes, becamehe dominant strain in the subsequent cultivation period after

xhaustion of oxygen. Moreover, according to the repartition ofhe two strains in the mixed culture, the hydrogen productionrocess could be calculated.

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cknowledgements

This work was supported by the Natural Science Foundationf China (No. 20336010, 20176025) and the National Basicesearch Program of China (973 Plan) (No. 2003CB716003).

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