biohydrogen production from crude glycerol by immobilized klebsiella sp. tr17 in a uasb reactor and...

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Biohydrogen production from crude glycerol byimmobilized Klebsiella sp. TR17 in a UASB reactorand bacterial quantification under non-sterileconditions

Teera Chookaewa, Sompong O-Thong b, Poonsuk Prasertsan a,c,*aDepartment of Industrial Biotechnology, Faculty of Agro-Industry, Prince of Songkla University, Songkhla 90112,

ThailandbDepartment of Biology, Faculty of Science, Thaksin University, Phatthalung 93110, ThailandcPalm Oil Products and Technology Research Center (POPTEC), Faculty of Agro-Industry,

Prince of Songkla University, Songkhla 90112, Thailand

a r t i c l e i n f o

Article history:

Received 9 September 2013

Received in revised form

8 April 2014

Accepted 12 April 2014

Available online xxx

Keywords:

Biohydrogen

Crude glycerol

UASB reactor

Fluorescence in situ hybridization

(FISH)

* Corresponding author. Department of IndusThailand. Fax: þ66 7455 8866.

E-mail address: [email protected]

Please cite this article in press as: ChookaTR17 in a UASB reactor and bacterial qu(2014), http://dx.doi.org/10.1016/j.ijhyden

http://dx.doi.org/10.1016/j.ijhydene.2014.04.00360-3199/Copyright ª 2014, Hydrogen Ener

a b s t r a c t

Biohydrogen production from crude glycerol by immobilized Klebsiella sp. TR17 was

investigated in an up-flow anaerobic sludge blanket (UASB) reactor. The reactor was

operated under non-sterile conditions at 40BC and initial pH 8.0 at different hydraulic

retention times (HRTs) (2e12 h) and glycerol concentrations (10e30 g/L). Decreasing the

HRT led to an increase in hydrogen production rate (HPR) and hydrogen yield (HY). The

highest HPR of 242.15 mmol H2/L/d and HY of 44.27 mmol H2/g glycerol consumed were

achieved at 4 h HRT and glycerol concentrations of 30 and 10 g/L, respectively. The main

soluble metabolite was 1,3-propanediol, which implies that Klebsiella sp. was dominant

among other microorganisms. Fluorescence in situ hybridization (FISH) revealed that the

microbial community was dominated by Klebsiella sp. with 56.96, 59.45, and 63.47% of total

DAPI binding cells, at glycerol concentrations of 10, 20, and 30 g/L, respectively.

Copyright ª 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

Introduction

Hydrogen has potential as a fuel for the future because it is

clean and has a high energy yield comparedwith hydrocarbon

fuels [1]. Among the biological methods of hydrogen produc-

tion, dark fermentation has various advantages such as its

ability to use a wide range of substrates and no requirement

trial Biotechnology, Facu

(P. Prasertsan).

ew T, et al., Biohydrogenantification under non-e.2014.04.083

83gy Publications, LLC. Publ

for a light source. Thus, thismethod is relatively energy saving

and environmentally friendly [2,3].

Crude glycerol is a by-product obtained from biodiesel

production. An increase in biodiesel production would inevi-

tably result in an increase in crude glycerol production [4].

Crude glycerol has high levels of impurities and its disposal is

costly and energy intensive [5]. In order to make biodiesel

productionmore sustainable, the conversion of crude glycerol

lty of Agro-Industry, Prince of Songkla University, Songkhla 90112,

production from crude glycerol by immobilized Klebsiella sp.sterile conditions, International Journal of Hydrogen Energy

ished by Elsevier Ltd. All rights reserved.

mailto:[email protected]

www.sciencedirect.com/science/journal/03603199

www.elsevier.com/locate/he

http://dx.doi.org/10.1016/j.ijhydene.2014.04.083



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 en e r g y x x x ( 2 0 1 4 ) 1e82

to a variety of value-added products, such as hydrogen [6], 1,3-

propanediol [7,8], 2,3-butanediol [9], and ethanol [10] has been

studied. Conversion of crude glycerol to hydrogen is an

attractive approach.

Investigations on hydrogen production from dark

fermentation have been focused on using pure cultures [11], in

which the genus Clostridium has beenmost studied for various

wastematerials such as foodwastes [12], palm oilmill effluent

[13], and molasses [14]. However, Clostridium is an obligate

anaerobe, requiring a strictly anaerobic condition which

makes it difficult to use for industrial production [15]. Thus,

using facultative bacteria for the conversion of crude glycerol

to hydrogen by dark fermentation is more appropriate.

Klebsiella sp. is able to convert crude glycerol to hydrogen at

a high rate and yield [16,17]. It is also easy to grow and will

produce various valuable by-products, such as 1,3-

propanediol, 2,3-butanediol [18], and ethanol [19]. To make it

more attractive for industrial applications, hydrogen should

be produced under non-sterile conditions to minimize pro-

duction costs. Under these conditions, the microorganisms

present in the reactor during operation should be quantified to

determine the dominant strains.

Up-flow anaerobic sludge blanket (UASB) reactor is an

effective process in wastewater treatment systems as it ex-

hibits high organic removal efficiency [20e22]. In addition, it

has also been employed for hydrogen production from various

substrates such as starch-wastewater [23], desugared

molasses [24], coffee drinkmanufacturing wastewater [1], and

cheese whey [25]. However, it has not been reported for

hydrogen production from crude glycerol.

The objective of this work is to investigate the hydrogen

production from crude glycerol in a UASB reactor using Kleb-

siella sp. TR17 immobilized on heat-pretreated methanogenic

granules under non-sterile conditions. Subsequently, the mi-

crobial communities in the UASB reactor were determined by

fluorescence in situ hybridization (FISH) in order to evaluate

the role of immobilized Klebsiella sp. TR17 in the fermentation

system.

Materials and methods

Microorganism and culture medium

Klebsiella sp. TR17 (accession number in Genbank AB647144)

was isolated from crude glycerol-contaminated soil. The op-

timum conditions for hydrogen production for this strain

were pH 8.0 and temperature at 40BC [19]. The culturemedium

contained: 11.14 g/L glycerol, 3.4 g/L K2HPO4, 2.47 g/L KH2PO4,

6.03 g/L NH4Cl, 0.2 g/L MgSO4$7H2O, 2.0 g/L yeast extract, 2.0 g/

L CaCO3, 5.0 mg/L FeSO4$7H2O, 2.0 mg/L CaCl2, and 2.0 mL/L

Table 1 e Oligonucleotide probes used for FISH technique.

Probe Specificity Sequence (50 to

EUB338 All bacteria GCTGCCTCCCGTAGGAG

Enterbact D Klebsiella sp. TGCTCTCGCGAGGTCGCT

a Formamide concentration in the hybridization buffer.b Sodium chloride concentration in the washing buffer.

Please cite this article in press as: Chookaew T, et al., BiohydrogenTR17 in a UASB reactor and bacterial quantification under non-(2014), http://dx.doi.org/10.1016/j.ijhydene.2014.04.083

trace element solution [26]. The crude glycerol with 50% purity

was obtained from the Biodiesel Pilot Plant at Prince of

Songkla University.

Experimental set-up and operation of UASB reactors

The 1.3 L UASB reactor (6 cm diameter � 47 cm height) was

made from glass with 1.0 L working volume and operated at

40BC with water internal jacket recirculation. Fresh medium

was fed from the bottom by a peristaltic pump while the

evolved gas and effluent were discharged from the top of the

reactor. The methanogenic granules were obtained from a

UASB reactor of a seafood wastewater treatment system

(Chotiwat Manufacturing Co., Ltd., Songkhla Province,

Thailand). The methanogenic granules were autoclaved at

121 �C for 30 min to kill methanogenic activity before being

used as carriers for immobilization of Klebsiella sp. TR17. For

the set-up, 440 mL of the heat-pretreated methanogenic

granules were transferred to each UASB reactors with 560 mL

of the inoculum (OD660 ¼ 0.5) [27]. After inoculation, the re-

actors were operated in batchmode for 24 h and fed with 10 g/

L pure glycerol, then the culturemediumwas re-circulated for

7 days at 12 h HRT (flow rate of 1.38 mL/min) in order to

enhance bacterial immobilization on the granules before

changing to crude glycerol. After reaching steady state, the

reactors were operated at the HRTs of 12, 10, 8, 6, 4, and 2 h,

respectively. The steady state of each HRT was established

when the value of the hydrogen production rate was less than

5% difference, and the final pH of the effluent was constant

[28]. The culture media containing glycerol concentrations of

10, 20, and 30 g/L were fed to each UASB reactor. The reactors

were monitored by examining the effluent every three days

for volatile suspended solids (VSS) concentration, and

measuring twice a day for soluble metabolic products and

glycerol residuals. Gas production and pH were measured

daily.

Fluorescence in situ hybridization (FISH)

The FISH technique was selected for detection and quantifi-

cation of Klebsiella sp. TR17 immobilized on heat-pretreated

methanogenic granules. The samples were taken from each

UASB reactor with different glycerol concentrations (10, 20,

30 g/L) at the end of the operation experiments. Table 1 shows

the list of the specific oligonucleotide probes and hybridiza-

tion conditions used in this study. Probes labeled with the

sulfoindocyanine dyes Cy3, EUB338 [29] and Enterbact D [30],

were used for the hybridization to target all bacteria and

Klebsiella sp., respectively. Fixation of samples started by

adding 375 mL of sludge samples to 1125 mL of 4% (v/v)

paraformaldehyde (pH 7.2). Then, the samples were mixed

30) FA (%)a NaCl (M)b Ref.

T 35 0.08 [29]

TCTCTT 0 0.90 [30]




i n t e rn a t i o n a l j o u rn a l o f h y d r o g e n en e r g y x x x ( 2 0 1 4 ) 1e8 3

and kept at 4BC for 4 h before being centrifuged at 13,000 g for

5 min. The supernatants were discarded and the cells were

washed twice in phosphate buffered saline (PBS). The cell

pellet was re-suspended in 150mL of filter-sterilized PBS, then

150mL of filter-sterilized 96% ethanolwas added. The samples

were mixed carefully and stored at �20BC [31]. The fixed

samples were further processed for FISH following the pro-

cedure as described by Amann et al. [32]. Quantitative deter-

mination was analyzed by counting 25 microscopic fields of

view per sample, and the dye 40,60-diamidino-2-phenilindol

(DAPI) stain was used to count the total number of cells

(total DAPI binding cells). The quantification of each bacterial

group was counted as the ratio of the area covered by samples

stained with probes and DAPI to the area covered by DAPI

Fig. 1 e Variation in (A) HPR, (B) HY, and (C) HC with respect

to different combinations of HRTs and glycerol

concentrations in the UASB reactors. In each panel,

symbols are C for 10 g/L, - for 20 g/L, and : for 30 g/L.


stained samples alone. Slides were viewed under a micro-

scope (Nikon Corporation, Japan) [24].

Analytical methods

The volume of gas production was measured every day by

using a gas meter with water replacement method.

Hydrogen content in the biogas was determined using an

Oldham MX2100 gas detector (Cambridge Sensotec Ltd., En-

gland) [33]. Glycerol and other metabolite products were

determined by HPLC [19]. VSS represented in the biomass

concentration were determined using the Standard Methods

[34]. The hydrogen production rate (mmol H2/L/d) was

calculated by measuring the total volume of hydrogen pro-

duced divided by the incubation time. The hydrogen yield

(mmol H2/g glycerol consumed) was calculated by measuring

Fig. 2 e Variation in (A) biomass concentration, (B) glycerol

conversion rate, and (C) final pH for different combinations

of HRTs and glycerol concentrations in the UASB reactors.

In each panel, symbols areC for 10 g/L,- for 20 g/L, and:

for 30 g/L.





the total volume of hydrogen produced divided by the glyc-

erol consumed (g/L). The glycerol conversion rate was

calculated by using the equation: [(IeF)/I] � 100%, in which I

and F are the initial and final glycerol concentrations (g/L),

respectively [6].

Results and discussion

Effect of HRTs and glycerol concentrations on hydrogenproduction in UASB reactors

The variation of HRTs and glycerol concentrations led to the

variation in hydrogen production rate (HPR), hydrogen yield

(HY), and hydrogen content (HC) (Fig. 1). The optimum HRT

Fig. 3 e Time course profile of soluble metabolic products durin

concentration, (B) 20 g/L glycerol concentration, and (C) 30 g/L gl

acid, D acetic acid, , 2,3-butanediol,+ ethanol, and C 1,3-pr


was at 4 h, giving the highest value for HPR (242.15 mmol H2/

L/d) and HY (44.27 mmol H2/g glycerol consumed). The value,

based on COD, was 11.95 mmol H2/g COD consumed

accounted for 58% of the theoretical yield. At 4 h HRT,

increasing glycerol concentrations (10, 20, and 30 g/L) resul-

ted in the increase of HPR (165.21, 210.44, and 242.15 mmol

H2/L/d, respectively) with the decrease of HY (44.27, 29.85,

and 29.00 mmol H2/g glycerol consumed, respectively) but

had no effect on HC (42, 46, and 43%, respectively). Limitation

of glycerol could lead to higher hydrogen yield as it favored

the conversion of pyruvate to acetyl CoA [19]. The result of

HC in this study was similar to that of Zhang et al. [35] and

Lin et al. [36]. It should be noted that the decline of HPR and

HY at 2 h HRT may possibly be attributed to too low mixing

and poor contact of glycerol with the microorganisms. This

g the operation of UASB reactors: (A) 10 g/L glycerol

ycerol concentration. In each panel, symbols are A succinic

opanediol.




Fig. 4 eMicrobial compositions of sludge samples obtained

from granules in hydrogen producing UASB reactors. The

error bars indicate the standard deviations from a triplicate

sampling analysis.


agrees with Liu et al. [37] who studied the effect of HRT (from

24 to 4 h) on fresh leachate biodegradation using the

expanded granular sludge bed (EGSB) reactor and found that

the lowest biodegradation was obtained at the lowest HRT

tested.

Biological hydrogen production varies depending on source

of substrate, bacterial strains, reactor types, and operating

conditions [13,16,19,27]. Compared with other UASB systems,

the maximum HPR (242.15 mmol H2/L/d or 10.1 mmol H2/L h)

from this study was higher than those from previous reports

such as from waste glycerol (6.2 mmol H2/L h) [38], glucose

(8.9 mmol H2/L h) [39], and pure glycerol (9 mmol H2/L h) [38].

However, it was lower than thatfrom a study using sucrose

(144.6 mmol H2/L h) [27].

Effect of HRTs and glycerol concentrations on biomassconcentration (VSS) and glycerol conversion rate in UASBreactors

The optimum HRT for growth at glycerol concentrations of

10 and 30 g/L was at 6 h while it was at 4 h HRT for 20 g/L

glycerol (Fig. 2A). The maximum growth (7.89, 9.15, and

17.47 g VSS/L) increased with increased glycerol concentra-

tions (at 10, 20, and 30 g/L, respectively). It should be noted

that the increase of biomass (1.2 and 2.2 folds) were lower

than the increase of glycerol concentrations (2 and 3 folds,

respectively).

Glycerol conversion rate tended to decrease with the

decrease of HRT. Therefore, its maximum value was obtained

at the maximum HRT tested (12 h HRT) with the values of

97.34, 79.88, and 64.65% at 10, 20, and 30 g/L glycerol, respec-

tively. On the contrary, increasing the glycerol concentration

from 10 to 30 g/L caused a decrease in the glycerol conversion

rate from 46.94 to 32.09%, at 4 h HRT (Fig. 2B). A decrease in

HRTs led to a decrease in glycerol conversion rate but an in-

crease in HPR and HY. The reason might be that higher HRTs

caused a lower substrate feeding rate and a longer time for

substrate remaining in the system, resulting in the higher

glycerol conversion rate [40].

During fermentation of glycerol to hydrogen, the increase

of final pH with the decrease of HRTs was observed at all

three glycerol concentrations tested (Fig. 2C). At 4 h HRT, the

final pH values were 6.3, 6.6, and 6.3 from 10, 20, and 30 g/L

glycerol concentrations, respectively. Klebsiella sp. TR17 uti-

lize glycerol and produce alcohol (2,3-butanediol, 1,3-

propanediol, and ethanol) and organic acids (such as suc-

cinic acid, acetic acid) (Fig. 3), the same as Klebsiella pneumo-

niae SU6 [7]. The oxidative pathway of glycerol provides

energy and reducing equivalents (NADH) for the biosynthesis.

The most energy-advantageous metabolite product of this

pathway is acetic acid, as its formation is connected with

NAD þ regeneration and coenzyme A recycling. However,

high acetic acid secretion leads to the pH drop and cell

growth inhibition by the accumulation of its undissociated

form [9]. It was reported that the accumulation of 2,3-

butanediol, acetic acid, and 1,3-propanediol was irregular in

the fermentation system without a pH control [9]. The pH-

controlled (pH 6.5e7.0) strategy was found to enhance 1,3-

propanediol from K. pneumoniae SU6 in fed-batch fermenta-

tion [7].


Effect of HRTs and glycerol concentrations on solublemetabolites production in UASB reactors

During UASB operation, Klebsiella sp. not only produced

hydrogen but alsosuccinic acid, acetic acid, 1,3-propanediol,

2,3-butanediol, and ethanol (Fig. 3). The maximum 1,3-

propanediol, as the main soluble metabolic product, was

achieved at 12 h HRT for all glycerol concentrations tested

and the maximum value was 9.0 g/L at 20 g/L glycerol. 1,3-

Propanediol is considered to be a favorable metabolite for

Klebsiella sp [19]. Thus, the presence of high concentrations of

1,3-propanediol in this study could imply the dominance of

Klebsiella sp. that successfully immobilized on heat-

pretreated anaerobic sludge granules in the UASB reactor

and played an important role for hydrogen production from

glycerol. It has been reported that when glycerol was in

excess (>20 g/L), more NADH2 was used for the formation of

1,3-propanediol than hydrogen production [19]. Thus, the

experimental results indicated that excessive glycerol at

higher HRTs should be implemented for production of 1,3-

propanediol. Decreasing HRTs also led to lower ethanol

concentrations as the HPR increased in all glycerol concen-

trations tested. This result coincides with Zhang et al. [35]

who reported that the concentration of ethanol decreased

(from 13 to 6 mM) when the HRTs decreased (from 4 to 0.5 h)

whereas the hydrogen production rate increased (from 0.4 to

2.2 L/L h).

Analysis of the microbial community by FISH

The FISH technique was used to monitor the contribution of

various microorganisms and for quantification of the selected

species under study in the three UASB reactors with different

glycerol concentrations. Microbial composition of the sludge

samples from the granules in UASB reactors after the end of

experiment was illustrated in Fig. 4. The microbial commu-

nity of UASB reactors fed with glycerol concentrations of 10,

20, and 30 g/L was found to contain Eubacteria with 75.13%,

77.05%, and 80.8% of total DAPI binding cells, respectively.

Among Eubacteria, Klebsiella sp. accounted for 56.96%,




Fig. 5 e Images of the hydrogen-producing sludge. (A), (C), and (E) are samples from UASB reactors with 10, 20, and 30 g/L of

glycerol, respectively, stained with DAPI for total cells. (B), (D), and (F) are samples from UASB reactors with 10, 20, and 30 g/L

of glycerol, respectively, probe Enterbact D hybridization and labeled with Cy3 for detected Klebsiella sp.


59.45%, and 63.47% of total DAPI binding cells, respectively.

The FISH images (Fig. 5) showed that Klebsiella sp. accounted

for more than 56% of total DAPI binding cells within the

glycerol concentrations tested (10e30 g/L). The main soluble

metabolic product in this study was 1,3-propanediol which

confirmed that Klebsiella sp. TR17 was dominant in the UASB

reactors.

Conclusion

The HPR and HY of the immobilized Klebsiella sp. TR17

increased with the decrease of HRTs under non-sterile con-

ditions in UASB reactors using crude glycerol as the substrate.

However, the glycerol conversion rate tended to decrease as

the HRTs decreased from 12 to 2 h. At 4 h HRT, HPR and HY

reached their maximum values of 242.15 mmol H2/L/d and

44.27 mmol H2/g glycerol consumed at 30 g/L and 10 g/L

respectively. Decreasing HRT and glycerol concentration

resulted in the decrease of soluble metabolites, in which 1,3-

propanediol was the main product. From the FISH


technique, the highest ratio of Klebsiella sp. and Eubacteria

(63.5% and 80.8% of total DAPI binding cells, respectively) were

achieved at 30 g/L glycerol.

Acknowledgment

The authors gratefully acknowledge the Royal Golden Jubilee

Ph.D Program of the Thailand Research Fund for the financial

support to Mr. Teera Chookaew under the Grant No. PHD/

0095/2551, the Graduate School and the Faculty of Agro-

Industry, Prince of Songkla University, Thailand.

r e f e r e n c e s

[1] Jung KW, Kim DH, Shin HS. Continuous fermentativehydrogen production from coffee drink manufacturingwastewater by applying UASB reactor. Int J Hydrogen Energy2010;35:13370e8.


http://refhub.elsevier.com/S0360-3199(14)01139-2/sref1








[2] Yossan S, O-Thong S, Prasertsan P. Effect of initial pH,nutrients and temperature on hydrogen production frompalm oil mill effluent using thermotolerant consortia andcorresponding microbial communities. Int J Hydrogen energy2012;37:13806e14.

[3] Chairattanamanokorn P, Penthamkeerati P, Reungsang A,Lo YC, Lu WB, Chang JS. Production of biohydrogen fromhydrolyzed bagasse with thermally preheated sludge. Int JHydrogen energy 2009;34:7612e7.

[4] Amaral PFF, Ferreira TF, Fontes GC, Coelho MAZ. Glycerolvalorization: new biotechnological routes. Food BioprodProcess 2009;87:179e86.

[5] Saenge C, Cheirsilp B, Suksaroge TT, Bourtoom T. Potentialuse of oleaginous red yeast Rhodotorula glutinis for thebioconversion of crude glycerol from biodiesel plant to lipidsand carotenoids. Process Biochem 2011;46:210e8.

[6] Markov SA, Averitt J, Waldron B. Bioreactor for glycerolconversion into H2 by bacterium Enterobacter aerogenes. Int JHydrogen energy 2011;36:262e6.

[7] Sattayasamitsathit S, Methacanon P, Prasertsan P. Enhance1,3-propanediol production from crude glycerol in batch andfed-batch fermentation with two-phase pH-controlledstrategy. Electron J Biotechn 2011;14:1e11.

[8] Mu Y, Teng H, Zhang DJ, Wang W, Xiu ZL. Microbialproduction of 1,3-propanediol by Klebsiella pneumoniae usingcrude glycerol from biodiesel preparations. Biotechnol Lett2006;28:1755e9.

[9] Petrov K, Petrova P. High production of 2,3-butanediol fromglycerol by Klebsiella pneumoniae G31. Appl MicrobiolBiotechnol 2009;84:659e65.

[10] Choi WJ, Hartono MR, Chan WH, Yeo SS. Ethanol productionfrom biodiesel-derived crude glycerol by newly isolatedKluyvera cryocrescens. Appl Microbiol Biotechnol2011;89:1255e64.

[11] Lee DJ, Show KY, Su A. Dark fermentation on biohydrogenproduction: pure culture. Bioresour Technol2011;102:8389e402.

[12] Kim JK, Nhat L, Chun YN, Kim SW. Hydrogen productionconditions from food waste by dark fermentation withClostridium beijerinckii KCTC 1785. Biotechnol Bioprocess Eng2008;13:499e504.

[13] Chong ML, Rahim RA, Shirai Y, Hassan MA. Biohydrogenproduction by Clostridium butyricum EB6 from palm oil milleffluent. Int J Hydrogen Energy 2009;34:764e71.

[14] Wang X, Jin B. Process optimization of biological hydrogenproduction from molasses by a newly isolated Clostridiumbutyricum W5. J Biosci Bioeng 2009;107:138e44.

[15] Marone A, Massini G, Patriarca C, Signorini A, Varrone C,Izzo G. Hydrogen production from vegetable waste bybioaugmentation of indigenous fermentative communities.Int J Hydrogen Energy 2012;37:5612e22.

[16] Liu F, Fang BS. Optimization of bio-hydrogen productionfrom biodiesel wastes by Klebsiella pneumoniae. J Biotechnol2007;2:274e80.

[17] Chen X, Sun Y, Xiu Z, Li X, Zhang D. Stoichiometric analysisof biological hydrogen production by fermentative bacteria.Int J Hydrogen Energy 2005;1:1e11.

[18] Sattayasamitsathit S, Prasertsan P, Methacanon P. Statisticaloptimization for simultaneous production of 1,3-propanedioland 2,3-butanediol using crude glycerol by newly bacterialisolate. Process Biochem 2011;46:608e14.

[19] Chookaew T, O-Thong S, Prasertsan P. Fermentativeproduction of hydrogen and soluble metabolites from crudeglycerol of biodiesel plant by the newly isolatedthermotolerant Klebsiella pneumoniae TR17. Int J HydrogenEnergy 2012;37:13314e22.

[20] Buzzini AP, Sakamoto IK, Varesche MB, Pires EC. Evaluationof the microbial diversity in an UASB reactor treating


wastewater from an unbleached pulp plant. Process Biochem2006;41:168e76.

[21] Khemkhao M, Nuntakumjorn B, Techkarnjanaruk S,Phalakornkule C. UASB performance and microbialadaptation during a transition from mesophilic tothermophilic treatment of palm oil mill effluent. J EnvironManage 2012;103:74e82.

[22] Lopes SIC, Dreissen C, Capela MI, Lens PNL. Comparison ofCSTR and UASB reactor configuration for the treatment ofsulfate rich wastewaters under acidifying conditions.Enzyme Microb Tech 2008;43:471e9.

[23] Akutsu Y, Lee DY, Chi YZ, Li YY, Harada H, Yu HQ.Thermophilic fermentative hydrogen production fromstarch-wastewater with bio-granules. Int J Hydrogen Energy2009;34:5061e71.

[24] Kongjan P, O-Thong S, Angelidaki I. Biohydrogen productionfrom desugared molassess (DM) using thermophilic mixedcultures immobilized on heat treated anaerobic sludgegranules. Int J Hydrogen Energy 2011;36:14261e9.

[25] Castello E, Santos CG, Iglesias T, Paolino G, Wenzel J,Borzacconi L, et al. Feasibility of biohydrogen productionfrom cheese whey using a UASB reactor: Links betweenmicrobial community and reactor performance. Int JHydrogen Energy 2009;34:5674e82.

[26] Chookaew T, O-Thong S, Prasertsan P. Statisticaloptimization of medium components affecting simultaneousfermentative hydrogen and ethanol production from crudeglycerol by thermotolerant Klebsiella sp. TR17. Int J HydrogenEnergy 2014;39:751e60.

[27] O-Thong S, Prasertsan P, Karakashev D, Angelidaki I. High-rate continuous hydrogen production byThermoanaerobacterium thermosaccharolyticum PSU-2immobilized on heat-pretreated methanogenic granules. IntJ Hydrogen Energy 2008;33:6498e508.

[28] Chang FY, Lin CY. Biohydrogen production using an up-flowanaerobic sludge blanket reactor. Int J Hydrogen Energy2004;29:33e9.

[29] Tsuneda S, Nagano T, Hoshino T, Ejiri Y, Noda N, Hirata A.Characterization of nitrifying granules produced in anaerobic upflow fluidized bed reactor. Water Res2003;37:4965e73.

[30] Ootsubo M, Shimizu T, Tanaka R, Sawabe T, Tajima K,Yoshimizu M, et al. Oligonucleotide probe for detectingEnterobacteriaceae by in situ hybridization. J Appl Microbiol2002;93:60e8.

[31] Ogue-Bon E, Khoo C, McCartney AL, Gibson GR, Rastall RA.In vitro effects of symbiotic fermentation on the caninefaecal microbiota. FEMS Microbiol Ecol 2010;73:587e600.

[32] Amann RI, Ludwig W, Schleifer KH. Phylogeneticidentification and in situ detection of individual microbialcells without cultivation. Microbiol Rev 1995;59:143e69.

[33] O-Thong S, Prasertsan P, Birkeland NK. Evaluation ofmethods for preparing hydrogen-producing seed inoculaunder thermophilic condition by process performance andmicrobial community analysis. Bioresour Technol2009;100:909e18.

[34] APHA. Standard methods for the examination of water andwaste water. 19th ed. Washington, DC: American PublicHealth Association; 1995.

[35] Zhang ZP, Tay JH, Show KY, Yan R, Liang DT, Lee DJ, et al.Biohydrogen production in a granular activated carbonanaerobic fluidized bed reactor. Int J Hydrogen Energy2007;32:185e91.

[36] Lin PJ, Chang JS, Yang LH, Lin CY, Wu SY, Lee KS.Enhancing the performance of pilot-scale fermentativehydrogen production by proper combinations of HRT andsubstrate concentration. Int J Hydrogen Energy 2011;36:14289e94.
















































































































































































[37] Liu Q, Zhang X, Yu L, Zhao A, Tai J, Liu J, et al. Fermentativehydrogen production from fresh leachate in batch andcontinuous bioreactors. Bioresour Technol 2011;102:5411e7.

[38] Reungsang A, Sittijunda S, O-thong S. Bio-hydrogenproduction from glycerol by immobilized Enterobacteraerogenes ATCC 13048 on heat-treated UASB granules asaffected by organic loading rate. Int J Hydrogen Energy2013;38:6970e9.


[39] Zhang H, Bruns MA, Logan BE. Biological hydrogenproduction by Clostridium acetobutylicum in an unsaturatedflow reactor. Water Res 2006;40:728e34.

[40] Hung CH, Lee KS, Cheng LH, Huang YH, Lin PJ, Chang JS.Quantitative analysis of a high-rate hydrogen-producingmicrobial community in anaerobic agitated granular sludgebed bioreactors using glucose as substrate. Appl MicrobiolBiotechnol 2007;75:693e701.
























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