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Comparison of mesophilic and thermophilic anaerobichydrogen production by hot spring enrichment culture

Jaakko A. Puhakka a, Dogan Karadag b,*, Marika E. Nissila a

aDepartment of Chemistry and Bioengineering, Tampere University of Technology, Tampere, FinlandbDepartment of Environmental Engineering, Yildiz Technical University, Davutpasa, Istanbul, Turkey

a r t i c l e i n f o

Article history:

Received 2 November 2011

Received in revised form

17 February 2012

Accepted 20 February 2012

Available online 20 March 2012

Keywords:

Hydrogen

Temperature

Mesophilic

Thermophilic

PCR-DGGE

Clostridium

* Corresponding author. Tel.: þ90 2123135384E-mail address: dogankaradag@gmail.com

0360-3199/$ e see front matter Copyright ªdoi:10.1016/j.ijhydene.2012.02.121

a b s t r a c t

Anaerobic hydrogen producing mesophilic and thermophilic cultures were enriched and

studied from an intermediate temperature (45 �C) hot spring sample. H2 production yields

at 37 �C and 55 �C were highest at the initial pH of 6.5 and 7.5, respectively. Optimum

glucose, iron and nickel concentrations were 9 g/l, 25 mg/l and 25 mg/l both at 37 �C and

55 �C, respectively. The highest H2 yields at 37 �C and 55 �C were 1.8 and 1.0 mol H2/mol

glucose, respectively, with the optimal pH, glucose concentration and iron addition.

Hydrogen production from glucose at 55 �C and 37 �C was associated with ethanol- and

acetateebutyrate type fermentations, respectively. Bacterial composition was analyzed by

16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE). Clostridium species

dominated at both temperatures and the microbial diversity decreased with increasing

temperature. At 55 �C, Clostridium ramosum was the dominant organism.

Copyright ª 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights

reserved.

1. Introduction temperature. Generally, temperatures of 37 �C and 55 �C have

Dark fermentation does not require a lot of energy and can be

operated at high rates in the absence of light input [1].

Hydrogen production through dark fermentation is controlled

by environmental parameters such as temperature, pH,

substrate concentration and iron concentration. Among

these, temperature is one of the most profound, since it

affects both activities of hydrogen producing bacteria and the

fermentative production mechanism [2].

Controversial results have been reported on the effects of

temperature on hydrogen fermentation. Wang and Wan

(2008) [3] reported higher H2 yields bymesophiles (31.9ml H2/h

at 35 �C) than thermophiles (8.5 ml H2/h at 35 �C). On the other

hand, Nazlina et al. (2009) [4] reported that biohydrogen

production from food waste increased with increasing

; fax: þ90 2123135358.(D. Karadag).

2012, Hydrogen Energy P

been compared. Lee et al. (2008) [5] reported more H2

production at 37 �C (5.34 mmol H2/g-starch), whereas Zhang

et al. (2003) [6] obtained higher H2 production yields at 55 �C(78 ml H2/g-starch). Koskinen et al. (2008) studied hot spring

enrichments for hydrogen production in the temperature

range of 37 �Ce70 �C and reported 45 �C was optimal for H2

production (1.67 mol H2/mol glucose) [7]. Makinen et al. (2009)

studied hydrogen production by a thermophilic strain, Ther-

movorax subterraneus, isolated from a geothermally active

underground mine and reported that optimum temperature

was 71 �C [8].

In this study, an intermediate temperature (45 �C) hot

spring sample was used to enrich hydrogen producing mes-

ophilic and thermophilic cultures. The effects of pH, glucose

concentration and addition of iron and nickel on dark

ublications, LLC. Published by Elsevier Ltd. All rights reserved.

i n t e rn 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 3 7 ( 2 0 1 2 ) 1 6 4 5 3e1 6 4 5 916454

fermentative hydrogen production were studied at 37 �C and

55 �C. Furthermore, the effect of enrichment temperature on

microbial communities was determined.

2. Materials and methods

2.1. Enrichment of hot spring culture

Two H2 producing cultures were enriched at 37 �C and 55 �Cfrom a sediment sample collected from a geothermal hot

spring located in Hisarkoy region in the Balıkesir province,

Turkey. The water temperature of the sampling site was 45 �C.The pH of a natural hot spring is near neutral values [9]. Two

ml of the sample sediment material was inoculated to serum

bottles with 50 ml nutrient solution. Enrichments were incu-

bated at 37 �C and 55 �C for 3 days and nutrient solution

contained 9 g/l glucose with initial pH 6.8. During the enrich-

ment, the presence of H2, CO2 and CH4 were monitored in

biogas. After the second transfer, the cultureswere used in the

experiments.

2.2. Experimental procedure

Experiments were performed in 120 ml serum bottles with

glucose. One liter of the nutrient solution contained NaHCO3,

4 g; NH4Cl, 0.6 g; NaH2PO4$H2O, 10.7 g; Na2HPO4, 3.2 g;

K2HPO4$3H2O, 0.125; MgCl2$6H2O, 0.1 g; CaCl2.2H2O, 0.11 g and

0.5 g iron [10]. 0.5 g/l L-cysteine was used as a reducing agent.

Two ml of the enriched culture, 48 ml nutrient solution sup-

plemented with yeast extract (2 g/l) and glucose was added.

Glucose concentration was kept constant at 9 g/l for all

conditions while it was changed from 4.5 to 27 g/l to study the

effect of substrate concentration.

The pH of the growthmediumwas 6.8. Bottleswere flushed

with nitrogen for 5 min to provide anaerobic conditions,

capped with rubber stoppers and placed in a shaker at

150 rpm. The experiments were carried out in duplicate.

Effect of initial pH was investigated in the pH range of

5.5e8.0 with 9 g/l glucose. The pH of the media was adjusted

using either 3 N HCl or NaOH. The effect of glucose concen-

tration was studied in the range from 4.5 to 27 g/l. Effects of

iron (Fe2þ) and Nickel (Ni2þ) on H2 production was studied by

stepwise increase of their concentration from 0.5 to 200 mg/l

using FeCl24H2O and NiCl26H2O chemicals. Hydrogen

production was calculated as the moles of H2 produced per

mole of glucose added and used to compare the effects of

variables.

2.3. Analyses

Syringe method was applied for measuring the volume of

biogas produced. The composition of biogas was analyzed by

a gas chromatograph (Shimadzu GC-2014) equipped with

a thermal conductivity detector and with Porapak N column

(80/100 mesh). N2 was used as carrier gas at a flow rate of

10 ml/min. The temperatures of the injector, column and

detector were 110 �C, 80 �C and 110 �C, respectively.End point glucose and soluble metabolites including

acetate, butyrate, formate, lactate and alcohols were analyzed

using a Shimadzu LC-20AD high-performance liquid chro-

matography with a Shodex Sugar SH1011 column (Showa

Denko K.K., Tokyo, Japan) and a refractive index detector

(Shimadzu, Kyoto, Japan). 0.01 N H2SO4 was used as mobile

phase. Final biomass concentration at end of the experiments

was analyzed as Volatile Suspended Solid (VSSf) as described

in Standard Methods [11].

2.4. Molecular characterization of microbial diversity

Microbial community analysis was performed using DNA

extraction and PCR-DGGE (polymerase chain reaction-

denaturing gradient gel electrophoresis) of partial 16S rRNA

genes followed by their sequencing. DNA was extracted from

the samples using MOBIO Power Soil DNA Extraction kit

(MOBIO Laboratories). Amplification of partial bacterial 16S

rRNA genes of the community DNA, DGGE and analysis of

sequence data were performed as previously described by

Koskinen et al. (2007) [12].

2.5. Kinetic modeling

The modified Logistic equation was used for kinetic modeling

of cumulative H2 production. The equationwas as follows [13]:

CH ¼ P1þ exp½4Rm$ðl� tÞ=Pþ 2� (1)

Where, CH represents the cumulative volume of H2 hydrogen

produced (ml), l is the lag time (h), P is the H2 production

potential (ml), Rm is the maximum H2 production rate (ml/h).

H2 production rate, r (ml/h), was calculated using Eq. (2) as

follows:

r ¼ Plþ P=Rm

(2)

Non-linear regression analysis was applied for kinetic

modeling of experimental data. Modeling was carried out

using solver add-in function of Microsoft Excel program.

3. Results and discussion

3.1. Effect of initial pH

Medium pH is one of the most important factors in hydrogen

production affecting Fe-hydrogenase activity, metabolic

pathways, and the duration of lag phase (Jo et al., 2008). In this

study, the effect of initial pH on H2 production was investi-

gated in the pH range from 5.0 to 8.0 and the results were as

shown in Fig. 1. No methane was detected in this or following

experiments. No H2 was produced at pH 5.0 by either meso-

philic or thermophilic cultures. At pH above 5.0, H2 was

produced cumulatively for 28 h. H2 yield increased with the

increase in initial pH. At 37 �C and pH 6.5 the highest H2 yield

was 1.25 mol H2/mol glucose, while at 55 �C and pH 7.5 it was

1.0 mol H2/mol glucose. Similarly, Lin et al. (2006) reported the

highest mesophilic H2 production at pH 6.5 [14]. The optimum

pH with a thermophilic enrichment culture from Hisaralan

hot spring was considerably different in our previous study

Fig. 1 e The effect of initial pH on H2 production by hot spring enrichment cultures at 37 �C and 55 �C.

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 3 7 ( 2 0 1 2 ) 1 6 4 5 3e1 6 4 5 9 16455

where 1.52 mol H2/mol glucose was produced at pH 6.5, while

no H2 was produced at pH 7.5 [10].

Initial pH and temperature had profound effects on cell

growth and H2 content in biogas (Table 1). Final VSS concen-

trations were in the range from 0.54 to 1.23 g/l at 37 �C and

from 0.51 to 0.70 g/l at 55 �C. Biomass concentrations at 37 �Cwere about two times higher than at 55 �C except at pH 5.0. H2

content in the biogas had similar profiles as H2 yields with

highest hydrogen percentages of 36.2% and 32.4% at 37 and

55 �C, respectively. During fermentation, the pH gradually

Table 1 e Changes in experimental parameters andkinetic constants of H2 production by hot springenrichment cultures at various pH.

pH VSSf (g/L) H2 (%) Final pH l (h) r (ml/h) R2

37 �C5.5 0.5 27.9 3.8 18.2 1.9 1.000

6.0 1.0 27.9 4.2 11.2 4.2 0.999

6.5 1.2 36.2 4.6 11.1 5.7 0.999

7.0 1.2 32.4 5.0 10.7 4.8 1.000

7.5 1.2 28.1 5.5 10.7 4.4 1.000

8.0 1.0 23.9 5.8 10.4 3.6 0.999

55 �C5.5 0.5 7.5 4.5 10.6 0.6 1.000

6.0 0.5 6.8 4.9 5.9 0.7 1.000

6.5 0.5 25.8 5.3 3.9 3.2 1.000

7.0 0.6 30.5 5.5 4.9 3.8 0.999

7.5 0.7 32.4 5.6 5.9 4.1 0.998

8.0 0.5 30.4 5.9 3.7 2.5 0.996

decreased due to the production of volatile fatty acids. The

final pH values (Table 1) were also affected by the initial pH as

previously reported [15].

Kinetic modeling was done using the modified Logistic

equation, since it was recommended for describing hydrogen

producing bacterial cultures [16]. Kinetic modeling gave an

excellent correlation with experimental data (Table 1).

According to experimental and kinetic modeling results, H2

productionwas optimal at the initial pH of pH 6.5 and pH 7.5 at

37 �C and 55 �C, respectively. Subsequent 37 and 55 �C incu-

bations were performed at pH 6.5 and pH 7.5, respectively.

3.2. Effect of glucose concentration

The effect of glucose concentration on H2 production and

biomass was studied in the range from 4.5 to 27 g/l and the

results were as shown in Fig. 2 and Table 2. At 37 �C, cumu-

lative H2 production and H2 yields increased with increasing

glucose concentration from 4.5 to 9 g/l that had the highest H2

yield of 1.23 mol H2/mol glucose. At the same time, final VSS

increased from 0.8 g/l to 1.40 g/l and cumulative H2 production

potential increased from 3.13 to 8.23 ml H2/g glucose. Further

increasing the glucose concentration decreased H2 yield while

therewere notmajor changes in cumulative H2 production, H2

content and kinetic parameters with glucose concentrations

over 9 g/l.

At 55 �C, the highest H2 yield and cumulative H2 production

were obtained with 9 g/l glucose. Higher glucose concentra-

tion resulted in higher biomass concentration while H2

production rate decreased. The results indicated that H2

production at lower glucose concentration of 4.5 g/L wasmore

Fig. 2 e The effect of glucose concentration on H2

production and yield by hot spring enrichment cultures at

37 �C and 55 �C.

i n t e rn 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 3 7 ( 2 0 1 2 ) 1 6 4 5 3e1 6 4 5 916456

favorable at 55 �C than at 37 �C. At 4.5 g/l glucose, the

temperature increase from 37 to 55 �C increased H2 yield and

H2 production rate from 0.2 to 0.71 mol H2/mol glucose and

from 1.2 to 2.1 ml/h, respectively. However, at 37 �C, H2 yield,

H2 production potential and H2 production rate were superior

at higher glucose concentrations. Similar to prior pH experi-

ments, higher microbial growth, H2 content, and lower final

pH values were obtained at 37 �C. Both at 37 �C and 55 �C, thefinal pH values decreased with the increasing of glucose

concentration due to the production of VFA’s [6].

3.3. Effect of iron

Effect of iron on H2 production was investigated at optimum

pH andwith 9 g/l glucose. The basal medium contained 0.5mg

Table 2 e Effect of glucose concentration on microbialgrowth, hydrogen percentage and kinetic parameters byhot spring enrichment cultures at 37 �C and 55 �C.

GlucoseConc. (g/L)

VSSf(g/L)

H2

(%)FinalpH

l

(h)r

(ml/h)R2

37 �C4.5 0.8 20.1 5.6 10.4 1.2 0.996

9 1.3 49.1 4.7 11.1 5.7 0.999

18 1.4 53.4 4.6 11.2 5.6 1.000

27 1.4 51.1 4.5 11.1 5.5 0.995

55 �C4.5 0.4 17.7 6.5 2.9 2.1 1.000

9 0.6 37.8 5.6 5.9 4.0 0.998

18 0.8 36.7 5.5 1.1 4.7 1.000

27 0.9 35.7 5.5 1.5 4.4 0.969

Fe2þ/l and was stepwise increased up to 200 mg Fe2þ/l and the

results were as presented in Fig. 3. Iron addition had similar

effect on mesophilic and thermophilic H2 production.

Increasing iron concentration from 0.5 to 25 mg/l increased

the H2 yield andH2 content in the biogas both at 37 �C and 55 C.

H2 production decreased from 1.7 to 1.6 mol H2/mol glucose at

37 �C and from 0.95 to 0.80 mol H2/mol glucose at 55 �C with

the increasing of iron concentration up to 50 mg/l. No signif-

icant changes were seen at higher iron concentrations.

Growth and iron concentrations did not correlate. Similarly,

kinetic parameters fluctuated and final pH values were close

to each other at all iron concentrations (Table 3). Furthermore,

higher H2 yield, biomass concentration and H2 production rate

were obtained at 37 �C than at 55 �C.

3.4. Effect of nickel

The influence of nickel concentration (from 0.02 to 200 mg/l)

on H2 production and growth was studied (Fig. 4). Increasing

Ni2þ concentration to 25 mg/l improved H2 yield both at 37 �Cand 55 �C.

Further increasing of nickel concentration inhibited H2

production at both 37 and 55 �C. H2 yield decreased at 50 mg

Ni2þ/l and H2 production stopped at 100 mg Ni2þ/l and 200 mg

Ni2þ/l at 55 �C and at 37 �C, respectively. Both at 37 �C and

55 �C, biomass growth increased slightly up to 50 mg Ni2þ/lwhile the growthwas inhibited at higher concentrations. Final

VSS results show that nickel inhibited more thermophiles

since three times higher biomass was obtained at 37 �C (Table

4) while ratio of biomass concentration at 37 �C and 55 �C was

about 2 in the previous experiments (Table 3). At elevated Ni2þ

concentrations metabolite production was inhibited and the

medium pH remained constant.

3.5. Soluble metabolites and H2 production mechanism

Distribution of solublemetabolites during the pH experiments

was as shown in Fig. 5. Production of the metabolites (Fig. 5)

had similar profiles with H2 production (Fig. 1). The lag phase

of soluble metabolites at 37 �C was longer than at 55 �C while

lower amounts of metabolites were produced at 37 �C. One of

the reasons for lower H2 production at 55 �C was associated

with the direction of glucose metabolism to VFAs and ethanol

0,0

0,4

0,8

1,2

1,6

2,0

0 50 100 150 200 250Fe

+2

concenctration (mg/l)

H2 Y

ie

ld

(m

ol H

2/m

ol g

lu

co

se

)

37 °C

55 °C

Fig. 3 e Effect of iron concentration on H2 production by hot

spring enrichment cultures at 37 �C and 55 �C.

Table 3 e Effect of iron concentration on growth, H2

production and kinetic constants of H2 production by hotspring enrichment cultures at 37 �C and 55 �C.

Feþ2

(mg/L)VSSf(g/L)

H2

(%)FinalpH

l

(h)r

(ml/h)R2

37 �C0.5 1.2 39.2 5.0 10.8 5.6 0.997

25 1.1 55.5 5.0 10.5 8.5 0.998

50 0.8 48.0 5.0 10.7 7.5 0.999

100 1.0 49.6 5.0 10.5 8.2 1.000

200 1.1 50.8 4.9 10.2 7.9 1.000

55 �C0.5 0.5 26.7 5.6 6.3 4.2 1.000

25 0.5 41.5 5.4 5.7 7.4 1.000

50 0.5 35.5 5.4 5.4 5.9 1.000

100 0.5 40.7 5.4 4.1 7.6 1.000

200 0.6 38.8 5.4 4.5 6.5 1.000

Table 4 e H2 production and kinetic constants at variousnickel concentrations on H2 production by hot springenrichment cultures at 37 �C and 55 �C.

Niþ2

(mg/L)VSS(g/L)

H2

(%)FinalpH

l

(h)r

(ml/h)R2

37 �C0.02 1.2 40.0 4.7 11.4 6.1 0.999

25 1.2 49.0 4.8 10.8 7.7 0.999

50 1.5 46.7 4.8 10.7 7.5 0.999

100 0.5 11.64 5.3 21.4 1.1 1.000

200 0.3 0.0 6.6 e e e

55 �C0.02 0.4 29.0 5.6 5.5 3.6 1.000

25 0.4 33.8 5.8 4.1 4.2 0.998

50 0.6 31.0 5.5 4.6 3.8 0.999

100 0.2 0.0 6.7 e e e

200 0.2 0.0 6.7 e e e

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 3 7 ( 2 0 1 2 ) 1 6 4 5 3e1 6 4 5 9 16457

instead of H2 production. The distribution of soluble metab-

olites was different at 37 �C and 55 �C. Formate, acetate and

ethanol were the main products in both mesophilic and

thermophilic cultures. Small amounts of butyrate and lactate

were produced at 37 �C while they were not detected at 55 �C.Ethanol and acetate production pathways exist together with

formate production during the ethanol type fermentation [17].

Further degradation of formate produces H2 and CO2 as

described in Eq. (3) [18].

C6H12O6 þH2O/2H2 þ 2CO2 þ C2H4O2 þ C2H6O (3)

In ethanol type fermentation H2 production decreases with

increasing pH and formic acid concentration [17]. The profiles

of the H2 and metabolites show that at 55 �C H2 was produced

through ethanol type fermentation.

At 37 �C, less formate and acetate was seen while H2

production increased with the production of butyrate. This

was different from thermophilic H2 production. The increase

in the H2 yield was likely related to the butyrate production.

Similar results were reported by Hwang et al. (2004) [18]. H2

production through the acetateebutyrate type fermentation

proceeds according to Eq. (4) [19].

3C6H12O6 þ 2H2O/8H2 þ 6CO2 þ 2C2H4O2 þ 2C4H8O2 (4)

0,0

0,4

0,8

1,2

1,6

2,0

0 50 100 150 200 250

Ni+2

concentration (mg/l)

H2 Y

ield

(m

ol H

2/m

ol g

luco

se)

37 °C

55 °C

Fig. 4 e Effect of nickel concentration on H2 production by

hot spring enrichment cultures at 37 �C and 55 �C.

Based on the distribution of soluble metabolites, H2

production at 37 �C was associated with the combination of

ethanol and acetateebutyrate type fermentations. H2

production through butyrate type fermentation is dependent

on pH [20].

3.6. Microbial community analysis

The DGGE band patterns of enrichment cultures at 37 and

55 �C were as shown in Fig. 6 and the closest relatives of the

bands are listed in Table 5. DGGE profiles of the two cultures

show that microbial communities were affected by the

enrichment temperature and changes in hydrogen yields and

Fig. 5 e Distribution of metabolic products from glucose

during H2 production by hot spring enrichment cultures at

37 �C and 55 �C.

Fig. 6 e DGGE profiles of hot spring enrichment cultures at

37 and 55 �C.

i n t e rn 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 3 7 ( 2 0 1 2 ) 1 6 4 5 3e1 6 4 5 916458

production mechanisms of cultures at 37 and 55 �C were

linked to the bacterial community diversity.

The enrichment temperature affected microbial commu-

nities and their diversity. Eight different bacterial specieswere

present at 37 �C, while seven of them disappeared and Clos-

tridium ramosum (band 8) dominated at 55 �C. The strains at

both 37 �C and 55 �Cwere mostly related to Clostridium species

which is typical hydrogen producing bacteria [12,21e24].

Clostridium butyricum (Bands 5 and 6) is very effective H2

producer [25]. Moreover, the butyrate production at 37 �Cattributed to the presence of Clostridium butyricum [26].

Table 5 e The closest relatives of DGGE bands inenrichment cultures.

Bandno

Phylogeneticaffiliation

Accessionno

% identity

1 Clostridium sp. DY-25 FJ848368 99

2 Uncultured bacterium

clone 822_20_pH7

GQ453637 97

3 Clostridium sartagoforme FJ384380 99

4 Uncultured bacterium

clone SP9-0

GQ167196 100

5 Clostridium butyricum

strain SD2

EU477411 100

6 Clostridium butyricum

strain CGS5

AY540109 100

7 Uncultured Clostridium

sp. clone FBR-A

DQ414795 100

8 Clostridium ramosum

strain CM-C50

EU869233 100

4. Conclusions

Mesophilic and thermophilic hydrogen producing enrichment

cultures were obtained from 45 �C Hisarkoy hot spring sample

from Turkey. Highest growth and H2 production yields were at

initial pH 6.5 and pH 7.5 at 37 �C and 55 �C, respectively.

Mesophilic and thermophilic H2 production yield was highest

with 9 g/l glucose, which also results in the highest bacterial

growth. Iron and nickel addition up to 25 mg/l increased H2

production. Nickel at concentrations over 50 mg/l inhibited H2

production. Higher biomass concentration and H2 production

yield were obtained at 37 �C than 55 �C. Differences between

mesophilic and thermophilic H2 yields and production

mechanisms were associated with changes in microbial

community during enrichment. DGGE profiles indicated that

both enrichment cultures were dominated with Clostridium

species and microbial diversity decreased with increasing

enrichment temperature. Ethanol and acetateebutyrate type

fermentations shift to ethanol type fermentation when

temperature is increased from 37 �C to 55 �C.

Acknowledgments

This research was funded by the Academy of Finland

(HYDROGENE Project, no 107425) and Nordic Energy Research

(BioH2 project 06-Hydr-C13). The authors thank Dr. Ahmet

Gunay from Balıkesir University, Turkey, for his help with

culture sampling from Hisaralan Hot Spring.

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