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Comparative Evaluation of Bio-Hydrogen Production From Cheese WheyWastewater Under Thermophilic andMesophilic Anaerobic ConditionsN. Azbar a , F. T. Dokgöz a , T. Keskin a , R. Eltem a , K. S. Korkmaz a

, Y. Gezgin a , Z. Akbal a , S. Öncel a , M. C. Dalay a , Ç. Gönen a & F.Tutuk aa Ege University, Faculty of Engineering, Bioengineering Department,Izmir, Turkey

Version of record first published: 07 Apr 2009.

To cite this article: N. Azbar, F. T. Dokgöz, T. Keskin, R. Eltem, K. S. Korkmaz, Y. Gezgin, Z.Akbal, S. Öncel, M. C. Dalay, Ç. Gönen & F. Tutuk (2009): Comparative Evaluation of Bio-HydrogenProduction From Cheese Whey Wastewater Under Thermophilic and Mesophilic Anaerobic Conditions,International Journal of Green Energy, 6:2, 192-200

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COMPARATIVE EVALUATION OF BIO-HYDROGENPRODUCTION FROM CHEESE WHEY WASTEWATERUNDER THERMOPHILIC AND MESOPHILIC ANAEROBICCONDITIONS

N. Azbar, F. T. Dokgoz, T. Keskin, R. Eltem, K. S. Korkmaz,Y. Gezgin, Z. Akbal, S. Oncel, M. C. Dalay, C. Gonen,and F. TutukEge University, Faculty of Engineering, Bioengineering Department, Izmir, Turkey

Hydrogen production from cheese whey wastewater via dark fermentation was conducted

using mixed culture under mesophilic (36�C � 1) and thermophilic (55�C � 1) conditions,

respectively. The hydrogen yields and specific hydrogen production rates were found as

follows: mesophilic: 9.2 mmol H2 /g COD (chemical oxygen demand) and 5.1 mL H2 /g VSS

h; thermophilic: 8.1 mmol H2 / g COD and 1.1 mL H2 /g VSS h. The reaction mixture for the

mesophilic condition was composed of acetate (0.3–14.7%) and iso-butyrate (85–98%), plus

other volatile fatty acids. On the other hand, the reactor mixture for the thermophilic

condition was composed of acetate (1–43%) and iso-butyrate (29–46%).

Keywords: Anaerobic dark fermentation; Cheese whey; Granular sludge; Hydrogen

production; Mesophilic; Thermophilic; pH effect

INTRODUCTION

Hydrogen with high energy content as compared to conventional fossil fuels is

considered as the ultimate energy carrier for the future by virtue of the fact that it is

renewable, does not evolve the ‘‘greenhouse gas’’ CO2 during combustion, and is easily

converted to electricity by fuel cells. Biological methods for hydrogen production offer

distinct advantages as compared to chemical methods (steam reforming of hydrocarbons,

partial oxidation of fossil fuels at high temperatures etc.) which are energy-intensive

and expensive. On the other hand, hydrogen is not readily available in nature and further

studies are required to develop cost-effective methods for sustainable hydrogen production

methods. Carbohydrate-rich starch- or cellulose-containing organic wastes are attractive

candidate raw materials for this purpose, including cheese whey wastewater.

Among the various bio-hydrogen production methods (direct biophotolysis, indirect

biophotolysis, photo-fermentation), anaerobic dark fermentation of carbohydrate-rich

wastes seems to be a promising alternative. Pure cultures are known to produce hydrogen

from carbohydrates including species of Enterobacter (Fabiano 2002), Bacillus (Kalia 1994),

International Journal of Green Energy, 6: 192–200, 2009

Copyright � Taylor & Francis Group, LLC

ISSN: 1543-5075 print / 1543-5083 online

DOI: 10.1080/15435070902785027

Address correspondence to N. Azbar, Bioengineering Department, Ege University, Bornova-lzmir 35100,

Turkey. E-mail: nuri.azbar@ege.edu.tr

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and Clostridium (Hawkes 2002). Mixed cultures enriched from natural environments are

reported to contain mostly Clostridia (Lay 1999; Mizuno 2002). Heat-treated biological

materials (sewage sludge, compost material, soil etc.) are frequently used for inoculation

purpose in bio-hydrogen production studies as a ready source of hydrogen-producing

mixed microflora (Khanal 2004; Lay 1999).

A variety of alternative organic substrates including bean curd manufacturing

waste; rice and wheat wastewater; molasses and sugary wastewater; waste-activated

sludge; municipal solid waste; starch wastewater; cafeteria food waste; peptone degrada-

tion; lignocellulose materials, such as rice straw, coir, and sugarcane bagasse; paper

sludge; and cheese whey have been used for hydrogen production (Ferchichi et al.

2005a,b).

It is estimated that 137.9 million tons of cheese whey, which is the lactose-rich

(about 5%) byproduct of the cheese manufacturing industry, was produced worldwide

in 1998 (Gahly 2000). Even though there are a number of technological developments

in the transformation of whey to other useful products, utilization of cheese whey

wastewater in order to produce bio-hydrogen offers new use for this waste, which is a

significant problem in the dairy industry. Currently, very limited number of papers on

the use of cheese whey for bio-hydrogen production may be found in the literature.

Ferchichi (2005) carried out a series of batch tests using pure culture of Clostridium

saccharoperbutylacetonicum ATCC 27021 at varying initial pH conditions (pH between

5 and 10). There is no data regarding the comparative evaluation of bio-hydrogen

production from crude cheese whey using heat-treated anaerobic granular biomass;

therefore this study was conducted to investigate the feasibility of using an acidophilic

anaerobic dark fermentation process for hydrogen production from cheese whey

wastewater at both mesophilic (35�C) and thermophilic (55�C) temperature at laboratory

conditions.

MATERIALS AND METHODS

Characterization of Cheese Whey Used in this Study

Fresh raw cheese whey was obtained on a weekly basis from a large dairy facility in

Izmir, Turkey. It had a pH of 4.7, Chemical Oxygen Demand (COD) of 86.3 g/l, total sugars

(as lactose) of 42.6 g/l, suspended solids of 6.9 g/l, and a total nitrogen of 0.2 g/l. Cheese

whey was kept at 4�C until used.

Experimental Procedure and Set-Up

The BHP test (biochemical hydrogen potential), which is the modified version of

biochemical methane potential (BMP) developed by Owen (1979), was employed. Cheese

whey used in this study was diluted to 50% (v/v) with deionized water and supplemented

per liter of the medium with 1.25 g NaHCO3; 2.5 g NH4Cl; 0.25 g KH2PO4; 0.25 g CaCl2,

0.032 g NiSO4; 0.32 g MgSO4.7H2O; 0.02 g FeCl3; 0.072 g Na2BO4.H2O; 0.0144 g

Na2MoO4.2H2O; 0.023 g ZnCl2; 0.021 g CoCl2.6H2O; 0.01 g CuCl2.6H20; 0.03 g

MnCl2.4H2O; and 0.05 g yeast extract. Batch experiments were conducted in triplicate in

100 ml culture bottles. Each bottle prior to experimentation was inoculated with pretreated

anaerobic sludge (working volume of 60 ml; VSS: 10 g/L in the bottle). Anaerobic mixed

microflora acquired from an operating field scale up-flow anaerobic sludge blanket

BIO-HYDROGEN PRODUCTION FROM CHEESE WHEY WASTEWATER 193

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(UASB) reactor treating brewery effluent for over 5 years was used as parent inoculum

for producing H2 after pretreatment as follows: dewatered anaerobic sludge (pH: 7.5;

SS: 100 g/L) was subjected to heat-shock pretreatment at 85�C for 45 min to enrich

H2 producers but to deactivate the hydrogentrophic methanogens. Cheese whey (CW)

was also subjected to heat treatment at 85�C for 30 min to eliminate lactic acid bacteria,

which may inhibit the biohydrogen production (Noike 2002). The feed material (CW) for

batch reactors was prepared based on the COD concentrations. Aqueous phase pH before

feeding was adjusted using either 2N HCl or 2 N KOH solutions to desired initial levels

(between 4.5 and 7.5). The culture pH was not controlled during fermentation. After

loading the inoculum, the bottles were flushed with nitrogen for 3 minutes tightly capped

with butyl rubber septum and aluminum caps under aseptic conditions. The mesophilic

experiments were performed at a constant mesophilic temperature at 36�1�C in a

temperature-controlled room, and the thermophilic experiments were conducted in an

incubator at 55�1�C. These culture bottles were then placed in a reciprocating shaker at

150 rpm. Total gas production was measured by glass syringe. When needed, hydrogen

content of biogas in the headspace was determined as follows. A known volume of the

headspace biogas was withdrawn by a syringe and injected into another serum bottle

that contained 20 g/l KOH. This serum bottle was then shaken for about 3 minutes to be

able to have all the CO2 absorbed in the KOH solution. H2 content measurements for

headspace gas mentioned above were also confirmed by GC analysis. The ratio of total

biogas volume before and after absorption by KOH provided the percentage of H2 in the

headspace gas. The control serum bottles, which contained only biomass but no organic

wastes (control bottles), were also run in all experiments to determine the background gas

production.

Analytical Methods

Samples were centrifuged at 5000 rpm for 15 min before they were analyzed. Chemical

oxygen demand (COD) and suspended solids were measured according to the standard methods

(APHA, AWWA 2001). Total nitrogen was measured with MERK 14 555 (N, 10–150 mg/L)

test kits. Total sugar concentration was estimated as lactose by the phenol sulfuric acid method

described by Dubois (1956). Volatile fatty acids (acetate, propiyonat, butyrate, isobutyrate,

isovalate, valate, isocaprionate, caprionate, and heptanoic acid) and alcohols (ethanol, acetone,

and butanol) in the mixed liquor were analyzed using a GC (6890N Agilent) equipped with a

flame ionization detector and DB-FFAP 30 m · 0,32 mm · 0,25 mm capillary column (J&W

Scientific). Mixed liquor sample of 1.5 ml was first acidified with phosphoric acid and then

filtered through a 0.2 �m membrane before analyzed. The initial temperature of the column

was 40�C for 3 min followed with a ramp of 20�C/min to 60�C for 3 min and then increased

at 30�C/min to 120�C for 4 min and reach a final temperature with ramp of 30�C/min to 240�Cfor 6 min. The temperatures of the injector and detector were both 240�C. Helium was used as

the carrier gas at constant pressure of 103 kPa. The volume of biogas produced in each serum

bottle was measured using a gas-tight syringe. H2 content of the headspace gas was confirmed

by injecting 5 ml bioreactor gas sample into the gas chromatograph (GC) (6890N Agilent)

equipped with a thermal conductivity detector and Hayesep D 80/100 packed column. Injector,

detector, and column temperatures were kept at 120�C, 140�C, and 35�C, respectively. Argon

was used as the carrier gas at a flow rate of 20 ml/min.

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RESULTS AND DISCUSSION

The aim of this study was to comparatively evaluate the feasibility of various

temperatures and initial pH values on the H2 evolution by utilizing cheese whey wastewater,

which was not studied in this respect for this substrate. For this purpose, BHP test was

employed. All experiments were carried out batchwise, and control experiments were

operated in parallel to take background gas production into account.

BHP test results for pretreated anaerobic mixed culture are presented in Figures 1a and 1b

for both mesophilic and thermophilic conditions as a function of the initial pH values,

respectively. Although hydrogen production occurred within the pH range investigated, it is

apparent that cumulative hydrogen production was pH-dependent. For mesophilic condition,

hydrogen production peaked at 100 ml at pH 6.5. On the other hand, for thermophilic condition,

the volume of hydrogen production was highest at 64 ml at pH 5.5. The total gas production

decreased with increasing temperature (thermophilic condition). Authors assume two reasons

for this: the first is thought to be the inoculum characteristics, since mesophilic anaerobic

granular sludge was used as the inoculum to grow thermophilic bacteria needed in the

experiments at 55�C, and secondly, propionic acid production (which is H2 consuming

VFA), is significantly higher in thermophilic experiments compared to mesophilic experiments

(Table 2). For both conditions very short lag phases were observed (Figure 1b), except at

pH 4.5. This finding was in close agreement with the results obtained by Khanal (2004).

0

20

40

60

80

100

120

4.5 5.5 6.5 7.5initial pH

H2

(ml)

36±1 ºC 55±1 ºC

Figure 1–a Relationship between the initial pH and volume of H2 produced. (mesophilic: CODinitial = 13,8 g/l;

Xinitial = 4,2 g/l; Thermophilic: CODinitial = 11.26 g/lt; Xinitial=4,2 g/l).

0

20

40

60

80

100

0 20 40 60 80 100Time (hr)

Cum

ulat

ive

Hyd

roge

n (m

l)

pH 4.5 pH 5.5 pH 6.5 pH 7.5

0

20

40

60

80

100

0 20 40 60 80 100 120 140 160 180 200 220Time (hr)

Cum

ulat

ive

Hyd

roge

n (m

l)

pH 4.5 pH 5.5 pH 6.5 pH 7.5

ii)i)

Figure 1–b Cumulative hydrogen production in i) mesophilic ii) thermophilic.

BIO-HYDROGEN PRODUCTION FROM CHEESE WHEY WASTEWATER 195

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Table 1 summarizes hydrogen yield and hydrogen production rate of both conditions

at different initial pH values. Results show that the highest hydrogen yield for mesophilic

condition was 233 ml/min (9.2 mmol H2/g COD) at pH 5.5. On the other hand, hydrogen

yield of thermophilic condition was 220 ml H2/g COD (8.1 mmol H2/g COD) at pH 4.5.

Although hydrogen yields of thermophilic condition were higher than the mesophilic

condition at all tested pHs, specific hydrogen production and hydrogen production rate of

this condition was lower than in the mesophilic condition. Among the studied factors

(temperature and initial pH), it seems increasing initial pH values (except for thermo-

philic conditions at an initial pH of 7.5) resulted in lower H2 production per mass of COD

utilized.

With the anaerobic degradation of cheese whey in mesophilic and thermophilic

conditions, H2 and CO2 were produced as gaseous products. Although the biogas

produced in mesophilic condition was free of methane gas, small percentage of

methane production (1–13 %) at all pH values was determined for thermophilic

condition (Figure 2). The hydrogen content of biogas produced in mesophilic and

thermophilic experiments were 38–50% and 14–40%, respectively. The biogas pro-

duction occurred within the pH range investigation, but percentage of hydrogen was

pH-dependent.

Figure 2 shows the volumetric percentages of headspace in the bottles. As tem-

perature increased from 35�C up to 55�C, H2 percentages in the gas phase decreased. H2

percentages in the gas phase for mesophilic experiments ranges between 35–40%

whereas it was between 10–40% for thermophilic experiments. These results were

more pronounced in the experiments where initial pHs were more alkaline (thermophilic

experiments).

Figure 3 shows that all final pHs were lower than initial pH except pH 4.5

for mesophilic condition. The final pH values were found to be in the range of

4.1–6.8.

Table 2 summarizes the distribution of VFAs produced in batches at various pH

values. It shows that isobutyrate (85.0–98%) was most abundant in fermentor liquid for

mesophilic condition. Increase of pH from 4.5 to 7.5 resulted in a decrease of acetate

but in an increase of propionate (2–13%). Although acetate (0.3–15%) and butyrate

Table 1 Comparison of hydrogen yield and hydrogen production rate.

Conditions Initial

pH

ml H2/g

COD

mmol H2/g

COD

ml H2 /

(g VSS h)

H2 Yield

(mol/mol lactose)

Mesophilic

(36 � 1�C)

4.5 94 3.7 2.1 1.3

5.5 233 9.2 5.1 2.3

6.5 135 5.4 12.0 2.5

7.5 138 5.5 9.2 2.5

Thermophilic

(55 � 1�C)

4.5 220 8.1 1.1 3.1

5.5 169 6.3 1.7 2.4

6.5 149 5.4 2.6 2.1

7.5 194 6.7 1.0 2.6

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(0.2–0.8%) were detected for mesophilic condition, other tested VFAs were not detected

at significant amounts. For thermophilic condition, the reactor mixture was composed of

acetate (20– 46%); isobutyrate (31–55%), butyrate (4–10%), and propionate (11–17%).

While increase of pH from 4.5 to 7.5 resulted in higher production of propionate, this

caused lower acetate concentrations. Isobutyrate was the dominant VFA in both experi-

mental conditions (mesophilic and thermophilic). In general, it is common to see a shift

from VFAs to alcohol production at low pHs, especially ethanol below 4.5 during

fermentation reactions (Byung 1985; Khanal 2003); however, in this study, no alcohol

production was detected.

The percentage of COD utilization conversion for mesophilic condition (between

43–90%) was higher than thermophilic condition (27–56%) except for the case at pH 5.5, as

shown in Figure 4. For mesophilic conditions, percentage of COD conversion was

increased with increasing pH values; on the other hand, percentage of COD removal peaked

at pH 6.5 and then decreased with increasing pH values. These findings are in parallel with

some literature indicating high COD removal during hydrogen production as is the case in

Zhang (2006). They reported glucose conversion rates between 77–99%.

H2H2

H2H2

CO2

CO2CO2

CO2

0

20

40

60

80

100

4,5 5,5 6,5 7,5

Gas

Con

tent

(%)

36±1 ºC

H2H2

H2

CH4 CH4 CH4

CH4

CO2 CO2

CO2

CO2

H2

0

20

40

60

80

100

4,5 5,5 6,5 7,5

Gas

Con

tent

(%)

55±1 ºC

Figure 2 Relationship between the initial pH and % gas content.

3,5

4,5

5,5

6,5

7,5

8,5

4,5 5,5 6,5 7,5pH

pH

36±1 ºC 55±1 ºC Initial pH

Figure 3 Relationship between the initial pH and the final pH.

BIO-HYDROGEN PRODUCTION FROM CHEESE WHEY WASTEWATER 197

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Degradation of one mole carbohydrate into acetate should yield formation of four moles

hydrogen, if no ethanol is produced. Thus lactose fermentation could be written as follows:

C12H22O11 þ 5H2O! 8H2 þ 4CO2 þ 4CH3COOH :

Based on the above equation, the theoretical yield of hydrogen on lactose should be 8 mol

per mole of lactose. However, the highest yield found in the literature was 3.0 mol hydrogen

per mole lactose, which was reported by Collet (2004). In this study, highest H2 yields

(2.5 and 3.1 mol H2 /mollactose) were obtained at pH 5.5 for mesophilic and pH 4.5 for

thermophilic conditions, respectively (see Table 1).

Results of hydrogen productivity by other Clostridium species are available in

literature. Most of the studies were carried out on glucose as a substrate. However it

has been reported that many other organic substrates, such as inulin (Sridhar 2000),

sucrose (Lee, 2002), acetylglucosamine and chitin (Evvyernie 2001), waste streams

containing xylose (Taguchi 1995), lignocellulosic waste (Sparling 1997), and even

wastewater sludge (Wang 2003) are potential sources for hydrogen production.

According to our knowledge, cheese whey wastewater, which is rich in lactose, as the

0102030405060708090

100

4.5 5.5 6.5 7.5Iinitial pH

CO

D re

mov

al (%

)

36±1 ºC 55±1 ºC

Figure 4 pH effect on COD removal.

Table 2 Comparison of VFA production and COD conversion for mesophilic and thermophilic conditions.

Conditions Initial

pH

%

CODused

Total

VFA

(mg/L)

Hac

(mg/l)

HPr

(mg/l)

HBu

(mg/l)

I-HBu

(mg/l)

I-HVal

(mg/l)

HVal

(mg/l)

I-I-

HCap

(mg/l)

HCap

(mg/l)

HHep

(mg/l)

Mesophilic

(36 � 1�C)

4.5 43 659 97 18 5.3 561 1.4 0.0 0.0 0.3 1.2

5.5 43 1417 3.7 24 9.2 1390 4.0 0.2 0.4 0.1 2.2

6.5 90 2398 1.0 85 11 2293 6.9 1.2 0.0 0.9 0.0

7.5 83 2045 0.0 268 3.1 1766 7.4 0.3 0.0 0.6 0.0

Thermophilic

(55 � 1�C)

4.5 27 4290 1979 463 413 1340 24 6.0 0.0 0.7 64

5.5 56 6229 2042 865 329 2913 57 0.0 0.0 2.0 21

6.5 47 7135 3081 868 299 2814 50 0.0 0.0 1.9 21

7.5 31 6631 1305 1096 469 3623 115 0.0 0.0 1.4 22

CODused: Chemical Oxygen Demand-Used; VFA: Volatile Fatty Acids; HAc:Acetic acid; HPr: Propionic acid;

HBu: Butyric acid; I-HBu: Iso-Butyric acid; HVal: Valeric acid; HCap: Caproci acid, I-HCap: Iso caproic acid;

HHep: Heptanoic acid.

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sole carbon source for hydrogen production has never been reported in a way to compare

the effect of both temperature and initial pH values on H2 production using granular

anaerobic seed material.

CONCLUSIONS

Cheese whey wastewater, which is rich in lactose, is amenable for bio-hydrogen

production using mixed anaerobic microflora at both mesophilic and thermophilic

conditions. The H2 yield peaked at an initial pH 4.5 in thermophilic conditions and pH

5.5 in mesophilic conditions, but the highest substrate conversion took place when the

initial pH was 5.5 for thermophilic conditions and pH 6.5 for mesophilic conditions. The

highest hydrogen yield from cheese whey effluent were found to be 9.2 mmol H2/g COD

and 8.1 mmol H2/g COD for mesophilic and thermophilic conditions, respectively. All final

pHs were acidic at the end of the fermentation of both conditions and the reaction mixture

was mostly composed of isobutyrate (85–98% for mesophilic and 31–55% for thermo-

philic). Thermophilic conditions resulted in higher production of VFAs, especially increase

in both acetic acid and propionic acid were observed. Alcohol production was not detected

for two conditions.

This opens new perspectives for the valorization of huge amounts of wastewater

formed by the cheese industry, containing a valuable substrate as a free of charge substrate.

This waste stream could be used for production of both valuable fatty acids and cheap

bio-hydrogen.

ACKNOWLEDGMENTS

The authors wish to thank TUBITAK-CAYDAG for the financial support of this study under the grant

No 104Y298. The data presented in this article was produced within the project above, however, it is

only the authors of this article who are responsible for the results and discussions made herein.

REFERENCES

APHA, AWWA, WPCF. 2001. Standard Methods for the Examination of Water and Wastewater, 21st

Ed. Washington, D.C.: American Public Health Association.

Byung, H. K., and J. G. Zeikus. 1985. Importance of hydrogen metabolism inregulation of solventogenesis

by Clostridium acetobutylicum continuous culture system of hydrogen-producing anaerobic

bacteria. Proceedings of the 8th International Conference on Anaerobic Digestion 2: 383–390.

Collet, C., N. Adler, J.P. Schwitzgulebel, and P. Pleringer. 2004. Hydrogen production by Clostridium

thermolacticum during continuous fermentation of lactose. International Journal of Hydrogen

Energy 29:1479–1485.

Dubois, M., K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorometric method for

determination of sugars and related substances. Analytical Chemistry 28(3): 351–356

Evvyernie, D., K. Morimoto, S. Karita, T. Kimura, K. Sakka, and K. Ohmiya. 2001. Conversion of

chitinous wastes to hydrogen gas by Clostridium paraputriscum M2–1. Journal of Bioscience

and Bioengineering 91(4): 339–343.

Fabiano, B., and P. Perego. 2002. Thermodynamic study and optimization of hydrogen production by

Enterobacter aerogenes. International Journal of Hydrogen Energy 27: 149–156.

Ferchichi, M., E. Crabbe, G. H. Gil, W. Hintz, and A. Almadidy. 2005a. Influence of culture

parameters on biological hydrogen production by Clostridium saccharoperbutylacetonicum

ATCC 27021. World Journal of Microbiology and Biotechnology 21 (6–7): 855–862.

BIO-HYDROGEN PRODUCTION FROM CHEESE WHEY WASTEWATER 199

Dow

nloa

ded

by [

DT

U L

ibra

ry]

at 1

0:31

19

Oct

ober

201

2

Ferchichi, M., E. Crabbe, G. H. Gil, and W. Hintz. 2005. Influence of initial pH on hydrogen

production from cheese whey. Journal of Biotechnology 120:402–409.

Gahly, A. E., D. R. Ramkumar, S. S. Sadaka, and J. D. Rochon. 2000. Effect of reseeding and pH

control on the performance of a two stage mesophilic anaerobic digester operating on acid

cheese whey. Canadian Agricultural Engineering 42: 173–183.

Hawkes, F. R., R. Dinsdale, D. L. Hawkes, and I. Hussy. 2002. Sustainable fermentative hydrogen

production: challenges for process optimization. International Journal of Hydrogen Energy

27: 1339–1347.

Kalia, V. C., S. R. Jain, A. Kumar, and A. P. Joshi. 1994. Fermentation of bio-waste to H2 by Bacillus

licheniformis. World Journal of Microbiology and Biotechnology 10: 224–227.

Khanal, S. K., W. H. Chen, L. Li, and S. Sung. 2004. Biological hydrogen production: effects of pH

and intermediate products. International Journal of Hydrogen Energy 29: 1123–11131.

Lay, J. J., Y. J. Lee, and T. Noike. 1999. Feasibility of biological hydrogen production from organic

fraction of municipal solid waste. Water Resources 33(11): 2579–2586.

Lee, Y. J., T. Miyahara, and T. Noike. 2002. Effect of pH on microbial hydrogen fermentation.

Journal of Chemical Technology and Biotechnology 77(6): 694–698.

Mizuno, O., R. Dinsdale, F. R. Hawkes, D. L. Hawkes, and T. Noike. 2000. Enhancement of hydrogen

production from glucose by nitrogen gas sparging. Bioresource Technology 73(1):59–65.

Noike, T., H. Takabatake, O. Mizuno, and M. Ohba. 2002. Inhibition of hydrogen fermentation

of organic wastes by lactic acid bacteria. International Journal of Hydrogen Energy

27: 1367—1371.

Owen, W. F., D. C. Stuckey, J. B. Herly, L. Y. Young, and P.L. McCarty. 1979. Bioassay

for monitoring biochemical methane potential and anaerobic toxicity. Water Resources

13: 485–492.

Sparling, R., D. Risbey, and H. M. Poggi-Varaldo. 1997. Hydrogen production from inhibited

anaerobic composters. International Journal of Hydrogen Energy 22(6): 563–566.

Sridhar, J., M. A. Eiteman, and J. W. Wiegel. 2000. Elucidation of enzymes in fermentation pathways

used by Clostridium thermosuccinogenes growing on inulin. Applied Environmental

Microbiology 66(1): 246–251.

Taguchi, F., N. Mizukami, T. S. Taki, and K. Hasegawa. 1995. Hydrogen production from continuous

fermentation of xylose during growth of Clostridium sp. strain 2. Canadian Journal of

Microbiology 41: 536–540.

Wang, C. C., C. W. Chang, C. P. Chu, D. J. Lee, B. V. Chang, and C. S. Liao. 2003. Producing

hydrogen from wastewater sludge by Clostridium bifermentans. Journalof Biotechnology

102(1): 83–92.

Zhang, Z., K. Y. Show, J. H. Tay, D. T. Liang, D.-J. Lee, and W. J. Jiang. 2006. Effect of hydraulic

retention time on biohydrogen production and anaerobic microbial community. Process

Biochemistry 41: 2118–2123.

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