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Dark fermentative biohydrogen production by mesophilicbacterial consortia isolated from riverbed sediments

Sneha Singh a, Anu K. Sudhakaran a, Priyangshu Manab Sarma a, Sanjukta Subudhi a,Ajoy Kumar Mandal a, Ganesh Gandham b, Banwari Lal a,*a Environmental and Industrial Biotechnology Division, The Energy and Resources Institute (TERI), Habitat Place, Darbari Seth Block,

Lodhi Road, New Delhi 110003, Indiab Hindustan Petroleum Corporation Limited, Mumbai Refinery, B. D. Patil Marg, Mahul, Mumbai 400074, India

a r t i c l e i n f o

Article history:

Received 5 October 2009

Received in revised form

2 February 2010

Accepted 2 March 2010

Available online 3 April 2010

Keywords:

Dark fermentation

Biohydrogen production

Riverbed sediments

Carbon sources

Clostridium

* Corresponding author. Tel.: þ91 11 2468210E-mail address: banwaril@teri.res.in (B. L

0360-3199/$ – see front matter ª 2010 Profesdoi:10.1016/j.ijhydene.2010.03.010

a b s t r a c t

Dark fermentative bacterial strains were isolated from riverbed sediments and investigated

for hydrogen production. A series of batch experiments were conducted to study the effect

of pH, substrate concentration and temperature on hydrogen production from a selected

bacterial consortium, TERI BH05. Batch experiments for fermentative conversion of

sucrose, starch, glucose, fructose, and xylose indicated that TERI BH05 effectively utilized

all the five sugars to produce fermentative hydrogen. Glucose was the most preferred

carbon source indicating highest hydrogen yields of 22.3 mmol/L. Acetic and butyric acid

were the major soluble metabolites detected. Investigation on optimization of pH,

temperature, and substrate concentration revealed that TERI BH05 produced maximum

hydrogen at 37 �C, pH 6 with 8 g/L of glucose supplementation and maximum yield of

hydrogen production observed was 2.0–2.3 mol H2/mol glucose. Characterization of TERI

BH05 revealed the presence of two different bacterial strains showing maximum homology

to Clostridium butyricum and Clostridium bifermentans.

ª 2010 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.

1. Introduction cyanobacteria through biophotolysis of water, photosynthetic

Hydrogen gas has been considered as a clean and efficient

renewable energy carrier based on the fact that it is renewable

and does not produce secondary pollutant. Since hydrogen

reacts with oxygen to form water as the by-product, hydrogen

from renewable sources is considered as the ultimate clean and

climate neutral energy system. Traditionally hydrogen can be

produced by thermo-chemical or electrolytic methods.

However, biological hydrogen production is considered as an

environmentally friendly and economically sustainable method

and has got potential advantages over other processes because

of its low energy requirements and reduced initial investment

costs [1]. Hydrogen can be obtained from algae and

0; fax: þ91 11 24682144.al).sor T. Nejat Veziroglu. Pu

bacteria throughdecomposition of organic acid (Rhodobacter sp.),

and fermentative bacteria (Enterobacter sp., Clostridium sp.,

Escherichia coli) [2,3]. Dark fermentative hydrogen production

seems to be an efficient process over other biological hydrogen

production processes since this process can make use of organic

wastes as the feeding substrate, which can be recycled at the

same time [4].

Renewable sources like biomass could be used as substrate

for production of hydrogen through dark fermentation. Wide

range of raw materials like starch, cellobiose, sucrose, or xylose

can also be used as substrate. Starch is found predominantly in

many common waste products released from agricultural and

food industry. Starch is a complex form of carbohydrate and

blished by Elsevier Ltd. All rights reserved.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 210646

can be hydrolyzed into glucose and maltose by acid or enzy-

matic hydrolysis. Further the simpler form of carbohydrates so

released can be converted into organic acids and then into

hydrogen gas [5]. The initial hydrolysis is the rate-limiting step

in microbial conversion and determines the efficiency of dark

fermentative hydrogen production by using starch as the

substrate. Glucose and sucrose also serve as interesting model

substrates due to their easy biodegradability nature [6].

Reports are also available on the use of pure cultures or

mixed consortia for biohydrogen production using sugars or

complex substrates such as organic wastes [7]. Various factors

such as temperature, initial pH, feeding substrates, substrate

concentration, and source of inoculation have also been

studied relating to fermentative biological hydrogen produc-

tion [8–11]. Though isolation of hydrogen producing microbes

is reported from various sources, only one report is available

on the isolation of hydrogen producing microbes from river

sediments [12]. Riverbed sediments particularly from urban

rivers have high organic load and can be considered ideal for

isolation of fermentative bacteria capable of producing

hydrogen from organic wastes.

Hence in this study, investigations were made to evaluate

the effect of different carbon sources, nutrient solution,

substrate concentration, pH, and temperature on fermentative

hydrogen production by using anaerobic bacterial consortia

isolated from riverbed sediments. Five different carbon sources;

sucrose, starch, glucose, xylose, and fructose, were employed as

the substrate. Attempts were also made to explore the bacterial

composition in a glucose fed bacterial community by employing

non-culture-dependent 16S rDNA sequencing approach.

2. Materials and methods

2.1. Seed inoculum

Seed inoculum was obtained from riverbed sediments from

Yamuna River, flowing below the Delhi-Noida-Direct flyway,

New Delhi, India (28�3704000N 77�1502100E). This river is highly

contaminated with organic pollutants. The sediment samples

were collected from seven different sites of Yamuna riverbed,

under anaerobic conditions as per the standard sample

collection protocols described in American Public Health

Association (APHA) guidelines. The sediment samples were

then transferred to the laboratory and refrigerated for further

experiments. Before proceeding for enrichment protocols for

hydrogen production, the sediment samples were initially

pretreated at 70 �C for 1 h to inactivate the methanogenic

microflora as per the protocols described earlier [12,13].

2.2. Enrichment of hydrogen producing bacterialconsortia from sediment samples

Following pretreatment, enrichment of the sediment samples

was done in DMI medium [13]. One liter of the DMI medium

contained 5.24 g of NH4HCO3, 6.72 g of NaHCO3, 0.125 g of

K2HPO4, 0.1 g of MgCl2, 0.015 g of MnSO4, 0.5 g of Na2S, 0.01 g of

FeSO4, 0.01 g of resazurin and 17.8 g of carbon source as

a substrate in 1 L distilled water. For the initial enrichment

protocol, two different carbon sources, sucrose and glucose

were used. The enrichments were conducted in 50 ml serum

vial with 25 ml of DMI medium supplemented with either

glucose or sucrose and 5 ml of pretreated sediment samples as

inoculum. The medium in each bottle was initially flushed

with oxygen free nitrogen till medium became completely

anaerobic. The bottles were then capped with rubber septum

stoppers and aluminum rings. The initial pH of the DMI

medium in each bottle was adjusted to 6.0 using 10 N HCl or

2 M NaOH. Incubation was done at 37 �C for a period of seven

days. After the end of enrichment cycle 10% of the enrichment

culture was inoculated in fresh DMI media supplemented

either with sucrose or glucose as per the protocol descried

earlier and the process was repeated for six cycles.

Analysis of headspace gas composition, concentration of

various volatile fatty acids and detection of sugar and ethanol

were quantified by the method described by Jayasinghear-

achchi [14].

2.3. Optimization of media for enhancement of hydrogenproduction with different carbon sources

To enhance the hydrogen yield of the bacterial consortium the

DMI medium was modified. One liter of modified DMI medium

consisted of 5.24 g of NH4NO3, 6.72 g of NaHCO3, 0.087 g of

K2HPO4, 0.5 g of MgCl2, 0.0075 g of MnSO4, 0.025 g of Na2S,

0.02 g of FeSO4, 4 g of malt extract, 4 g of yeast extract and 10 g

of carbon source. Batch experiments were initiated with five

different sugar sources, sucrose, starch, glucose, xylose and

fructose, supplemented to the modified DMI medium indi-

vidually. The initial pH of the medium was adjusted as per the

method described earlier and the individual bottles were

incubated at 37 �C for 72 h.

Gas composition in headspace as well as the volatile fatty

acids in the individual experimental bottles was analyzed

periodically by the GC as per the protocols described earlier

[14]. All the experiments were performed in triplicate.

2.4. Effect of different cultural parameters on hydrogenproduction

To optimize the culture conditions for the selected bacterial

consortia, three parameters viz pH, temperature and substrate

concentration have been studied for their effect on hydrogen

yields. All the experiments were performed in batch cultures.

To investigate the optimal initial pH, experiments were con-

ducted from pH 4.5, 5, 5.5, 6, 7 at 37 �C in the optimized

medium as described earlier with 10 g/L carbon source. For

optimal temperature studies, growth and hydrogen produc-

tion of the selected consortia were checked at 30, 37, 42 and

50 �C. The batch experiment was conducted to further inves-

tigate the optimal substrate concentration at 2, 4, 6, 8 and 10 g/

L. In all the experiments, the amount of hydrogen produced

and concentration of soluble metabolites were analyzed as per

the protocols described earlier [14].

2.5. Isolation and characterization of bacterial strainsfrom selected hydrogen producing microflora, TERI BH05

Pure cultures from hydrogen producing consortium, TERI

BH05, were obtained by serial dilution using saline solution

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 2 10647

(0.85% NaCl) and plating on modified DMI agar plates, under

anaerobic conditions. The agar plates were then incubated

overnight at 37 �C inside an anaerobic chamber. Single colo-

nies obtained on agar plates were streaked on fresh plates

thrice to ensure the purity of the bacterial strains.

Molecular characterization of selected bacterial strains

was carried out by sequencing of 16S rDNA gene. The over-

night incubated bacterial cells in DMI medium supplemented

with glucose were harvested by centrifugation for 10 min at

6000 rpm from which RNA free genomic DNA was extracted

and purified from the pellet by employing previously

described method [15]. The genomic DNA was used as

template for amplification of around 500 bp 16S rDNA frag-

ments. PCR was performed in a thermal cycler (Perklin Elmer

Gene Amp PCR system 2400, USA) using ‘Microseq 500 16S

rDNA PCR’ kit (Applied Biosystems, USA). The amplified PCR

products after analysis on agarose gel electrophoresis were

purified using montage PCR centrifuge filter device (Millipore,

USA) by following the manufacturer’s instructions. The

sequencing was done by Microseq� partial gene-16S rDNA

bacterial sequencing kit (PE Applied Biosystems, USA) and

analysis of the sequence was performed using an ABI PRISM

310 Genetic Analyzer (Applied Biosystems, USA) as per the

manufacturer’s instructions. To identify the bacterial

isolates, the partial gene sequences so obtained were

compared with the existing sequences available in NCBI

Database.

2.6. Nucleotide sequence accession numbers

The 16S rRNA gene partial sequences of the purified bacterial

isolates isolated from TERI BH05 mixed microflora in this

study have been deposited in the NCBI nucleotide sequence

database under the following accession numbers; FJ656099

and FJ656098.

0

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30

Sucrose Starch Glucose Fructose Xylose

Different carbon sources

Hyd

roge

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tion

(mm

ol/L

)

24 h

48 h

Fig. 1 – Hydrogen production by TERI BH05 bacterial

consortia from different carbon sources in DMI medium.

3. Results and discussion

3.1. Isolation of hydrogen producing microbial consortia

To isolate hydrogen producing bacterial strains, initially seven

riverbed sediment samples of Yamuna River, New Delhi, were

investigated for hydrogen production. Initially a set of four-

teen enrichment cultures was established with DMI medium

supplemented with either sucrose or glucose. The gas chro-

matography results demonstrated that among fourteen

enrichments cultures derived from seven sediment samples,

only eight enrichments showed bacterial activity and

hydrogen production. It was observed that these eight

enrichments cultures were from four sediment samples that

showed activity in medium supplemented with either glucose

or sucrose. Remaining three sediment samples failed to show

activity in either of the carbon sources. The enrichment

cultures that showed hydrogen activity were designated as

TERI BH01, TERI BH02, TERI BH03 and TERI BH05. The

hydrogen production was defined as mmol of hydrogen per

liter of medium (mmol/L). The hydrogen yield of enrichment

cultures TERI BH01 from sucrose and glucose was 0.21 and

0.19 mmol/L respectively. In case of enrichment culture TERI

BH02, 2.7 and 0.97 mmol/L of hydrogen was detected from

sucrose and glucose. While for TERI BH03 it was 0.17 and

1.11 mmol/L respectively. The preliminary analysis of the

enrichment cultures indicated that the hydrogen yield of TERI

BH05 was the highest among the enrichments as this

consortium could produce 3.4 and 1 mmol of H2/L from

sucrose and glucose respectively. Thus consortium TERI BH05

was selected for further investigation.

3.2. Hydrogen production by TERI BH05 from differentcarbon sources

Batch studies revealed that TERI BH05 was able to utilize all

the sugar sources to produce molecular hydrogen in DMI

medium (Fig. 1). However, the hydrogen production potential

of TERI BH05 varied with different carbon sources. Among the

selected carbon sources, the hydrogen production capability

on glucose showed the maximum hydrogen yield of 16 mmol/

L in 48 h. It further increased to 20 mmol/L when the incuba-

tion time was extended to 72 h. However, the hydrogen yield

with 48 h old TERI BH05 from fructose, starch, sucrose, and

xylose were comparatively lower than glucose, with the

hydrogen yield rates of 12.6 mmol, 8.2 mmol, 6.4 mmol, and

5.2 mmol/L, respectively (Fig. 1). The variation in the hydrogen

yield from different carbon sources might be attributed to

different fermentation pathways. Similar research studies

conducted on this regard report for significant amount of

biohydrogen production from simple sugars; glucose, sucrose,

xylose by anaerobic microbes [16–21]. However, reports on

xylose fermentation studies indicated that xylose fermenta-

tion yields comparatively lower amount of hydrogen [21].

3.3. Effect of media supplementation on hydrogenproduction in TERI BH05

Nutrients like nitrogen and iron are known to play significant

role for efficient hydrogen production [22–24]. Hence, to ach-

ieve optimum growth and to improve the hydrogen yield,

experiments on TERI BH05 bacterial consortium was dupli-

cated in modified DMI medium Modified DMI medium

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 210648

comprises of additional nitrogen source, yeast and malt

extract, and increased concentration of FeSO4, in addition to

the other components of DMI medium. When compared with

the original medium, TERI BH05 showed enhanced hydrogen

production with all the five carbon sources in modified DMI

medium. The hydrogen yield in modified DMI medium was

increased to 22.3 mmol/L, in contrast to 16 mmol/L in DMI

medium, in 48 h when glucose was used as a carbon source.

The similar trend was observed with all the other carbon

sources except for fructose (Figs. 1 and 2). The hydrogen yield

of TERI BH05 from sucrose, xylose, starch and fructose were

21.4, 20.4, 16.7, and 14.9 mmol/L respectively, in modified DMI

medium (Fig. 2).

Further, as observed in the original medium, among the

selected carbon sources, hydrogen yield was highest when

glucose in the modified medium (Figs. 1 and 2). However when

carbon source sucrose and xylose was used, it was observed

that in original DMI medium the hydrogen production of TERI

BH05 was comparatively low. However, after supplementa-

tion of nutrients in the modified DMI medium, the hydrogen

productions with these carbon sources were almost similar to

the yields observed with glucose (Figs. 1 and 2).

Enhancement of hydrogen production in modified DMI

medium could possibly be attributed to the effect of added

inorganic nitrogen, source, yeast and malt extract and FeSO4.

Yeast extract serves as an essential growth factor for bacteria

whereas Fe2þ is known to play key role in catalytic activity of

hydrogenase and is responsible for hydrogen production in

acidogenic bacteria like Clostridium butyricum [22,24]. Previous

studies on sucrose and xylose fermentation have shown that

C. butyricum CGS5 and Clostridium pasteurianum CH4 were the

efficient strains in converting sucrose and xylose to hydrogen

with a hydrogen production rate of 569 and 212.5 ml/l/h,

respectively [25]. Since glucose was found to be most

productive carbon source for TERI BH05, further investigations

were made to optimize the operational parameters for TERI

BH05 to improve its hydrogen yield in modified DMI medium,

using glucose as the substrate.

0

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30

Sucrose Starch Glucose Fructose Xylose

Different carbon sources

Hyd

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(mm

ol/L

)

24 h

48 h

Fig. 2 – Hydrogen production by TERI BH05 bacterial

consortia from different carbon sources in modified DMI

medium.

3.4. Effect of glucose concentration on hydrogenproduction

Few investigators have reported that the initial substrate

concentration is known to play a key role in hydrogen

production [26,27]. In this study for the initial experiments

10 g/L glucose concentration was used. However, it was

observed that under the experimental conditions used in this

study, the variation in the initial glucose concentrations did

not significantly influence the hydrogen yield. The data pre-

sented in Fig. 3 indicates that the hydrogen yield for TERI BH05

was almost similar when 2, 4 and 6 g/L glucose concentration

was used. Though there was a moderate increase observed

with higher glucose concentration, it was interesting to note

that the values were not significantly different when 2 g/L

glucose was used as the carbon source. There was also no

significant difference in the hydrogen yield when the incu-

bation time was extended to 48 h (Fig. 3).

These results can be attributed to the fact that the experi-

ments were conducted in batch mode and the fixed headspace

bottles. A set of experiment was inducted to increase in initial

glucose concentration to more than 10 g/L, but it did not reveal

significant increase in hydrogen production (data not shown).

Thus a study on sugar utilization by the selected consortium

TERI BH05 is warranted to divulge the further insights.

Investigators have earlier attributed such effects to substrate

inhibition, product inhibition, or combination of both and

osmolarity [28–30].

3.5. Effect of glucose concentration on production ofsoluble metabolites

Hydrogen production is usually accompanied with production

of soluble metabolites, which are often used to evaluate the

efficacy of hydrogen production. Moreover, soluble metabolite

production profile reflects the changes in the metabolic

pathway of the microorganisms involved. The soluble

metabolite yield was defined as the total soluble metabolites

produced in mg/L of medium. The distribution of soluble

metabolites formed during hydrogen fermentation in TERI

BH05 at different glucose concentrations was shown in Table

1. It was observed that with the increasing concentration of

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2 g/L 4 g/L 6 g/L 8 g/L 10 g/L

Different substrate concentration

Hyd

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(mm

ol/L

)

24 h

48 h

Fig. 3 – Effect of glucose concentration on hydrogen

production by TERI BH05, in modified DMI medium.

Table 1 – Effect of glucose concentration (g/L) onproduction of final soluble metabolites (mg/L) in modifiedDMI medium.

Glucose concentration Ethanol Acetic acid Butyric acid

2 325� 2 624� 17 145� 5

4 377� 2 681� 16 108� 3

6 1253� 9 841� 16 140� 4

8 1451� 7 814� 18 143� 3

10 490� 8 782� 17 106� 6

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 2 10649

glucose, the amount of soluble metabolites accumulated in

the medium also increased (Table 1). However, the increase

was not very significant. Further, the comparison between

hydrogen yield (Fig. 1) and production of total soluble

metabolites (Table 1) indicated a positive correlation.

However, as the differences in both the dataset were not

significant, an exact correlation could not be ascertained.

The results indicated that among the detected soluble

metabolites, acetic acid contributed significantly to the total

VFAs produced by the consortium (Table 1). It was also

observed that the amount of butyric acid detected in the

culture was considerably less. This finding is intrinsic as there

are several reports indicating the detrimental effect of butyric

acid in hydrogen yield [1,3].

3.6. Influence of temperature on hydrogen production

Temperature is an important environmental parameter that

plays a critical role in biological processes by affecting the

kinetics of cell growth and enzymatic reactions. Three

different temperature ranges; ambient, mesophilic and ther-

mophilic were considered for this study. The results demon-

strated that selected consortium TERI BH05 have the ability to

produce hydrogen at temperatures 30 �C and 37 �C (Fig. 4).

However, there was no significant difference in the hydrogen

yields (Fig. 4). Though hydrogen production continued when

the culture were further incubated till 48 h, considering the

difference (18.5 and 19.9–21.2 and 22.55 mmol/L for 30 �C and

0

10

20

30

30 37 45 55Different temperatures (degree celsius)

Hyd

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(mm

ol/L

)

24 h

48 h

Fig. 4 – Influence of temperature on hydrogen production

by TERI BH05 bacterial consortia, in modified DMI medium.

37 �C respectively), 24 h incubation was considered to be

optimal for the hydrogen yield by TERI BH05.

In the thermophilic temperatures selected for this set of

experiment (45 �C and 55 �C), the selected consortium neither

showed growth nor significant hydrogen production (Fig. 4)

when compared to the hydrogen yield at 30 �C and 37 �C. The

bacterial consortium TERI BH05 was enriched from sediment

samples collected from Yamuna riverbed. The ambient

temperature in the sampling site tends to be within the

temperature range of 30–40 �C. Thus it is can be presumed that

the bacterial flora thriving in the site is largely from the

mesophilic range and 37 �C can be considered as the optimum

temperature that is suitable for TERI BH05 consortium to

produce significant amount of hydrogen. This temperature

range is in consistence with the range as reported previously

[17,30,31].

3.7. Effect of temperature on production of solublemetabolites

Analysis of soluble metabolites formed at different tempera-

ture range reveal that distribution of VFA accumulated was

dependent to the incubation temperature (Table 2). The major

soluble metabolites produced by the TERI BH05 were acetic

acid and as observed with the data on glucose concentration,

it contributed to the total soluble metabolites (Tables 1 and 2).

As discussed in the previous section, as the bacterial consortia

was mesophilic, both hydrogen production and VFA accu-

mulation in the culture broth were maximum at 30 and 37 �C

(Fig. 4 and Table 2). This study thus reiterates that thermo-

philic fermentative hydrogen production from TERI BH05

bacterial consortium was not efficient.

3.8. Effect of pH on hydrogen production

pH is an important factor that influences metabolic abilities of

a microorganism. It plays a significant role in affecting the

fermentation pathways and thus influences on hydrogen

production [32,33]. Hence to identify the optimal pH for

hydrogen production with TERI BH05 fermentation studies

were conducted at five different initial pH values as described

in the previous section. As observed in Fig. 5, the hydrogen

yields of TERI BH05 consortium were around 22.5 mmol/L

when inoculated in the medium with initial pH 5.5, 6.0 and 7.0.

The variation observed in these initial pH values was not

significant (Fig. 5). However, when the initial pH of the

medium was lowered to 5, the hydrogen yield decreased

significantly (Fig. 5). Further, when consortium TERI BH05 was

incubated in the medium with pH 4.5, there was no growth as

well as hydrogen production. It was also observed that pH

Table 2 – Characteristics of final metabolite content (mg/L)at various temperatures (8C) in modified DMI medium.

Temperature Ethanol Acetic acid Butyric acid

30 290� 3 1110� 22 270� 4

37 490� 5 1064� 25 270� 5

45 Not detected 464� 8 110� 4

55 Not detected 323� 5 92� 3

0

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30

pH 4.5 pH 5.0 pH 5.5 pH 6.0 pH 7.0

Different pH values

Hyd

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tion

(mm

ol/L

)

24 h48 h

Fig. 5 – Effect of pH on hydrogen production by TERI BH05

bacterial consortia, in modified DMI medium.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 210650

over 7 also retards the hydrogen producing ability of this

consortium (data not shown).

The pH of the medium has an impact on the enzyme

hydrogenase. In the case of TERI BH05, the acidic pH values

below 5.5 and alkaline pH might negatively affect its hydrog-

enase activity. It has been reported that low pH facilitates the

formation of acidic metabolites that destabilizes the cell’s

ability to maintain internal pH and thus results in lowering

the intracellular ATP level, which inhibits the substrate

uptake and pH also plays role in influencing the enzyme

activities, particularly hydrogenase activity [34].

3.9. Effect of pH on production of soluble metabolites

The analysis of results indicating the total soluble metabolites

produced during fermentative hydrogen production at

different initial pH is summarized in Table 3. The maximum

metabolites were observed with the initial pH was kept at the

range of 5.5–7 (Table 3). A positive relation could be estab-

lished between hydrogen productions as a similar trend was

observed in this pH range (Table 3 and Fig. 5). As observed with

hydrogen production, the acidic pH (pH 5 and 4.5) did not favor

production of VFA or other soluble metabolites (Fig. 5 and

Table 3). Ethanol concentration varied between 317 and

785 mg/L between the pH ranges 5–7 with no production at pH

4.5 (Table 3). Acetic acid was produced in significant amount

Table 3 – Characteristics of final metabolite content (mg/L)at various pH in modified DMI medium.

pH condition Ethanol Acetic acid Propionic acid

4.5 Not detected 148� 6 Not detected

5.0 785� 7 554� 18 4� 1

5.5 413� 3 1077� 15 7� 1

6.0 317� 3 1135� 17 4� 1

7.0 705� 8 1126� 15 Not detected

accounting for 1077–1135 mg/L between pH 5.5–7, with

decreased concentration in the lower acidic pH (Table 3).

It was interesting to note that butyric acid was not detected

in this dataset. However, minute amount of propionic acid

was detected at pH range of 5–6 (Table 3). Literatures cited in

this regard indicate that fermentative hydrogen production

from propionic fermentation does not generate hydrogen [35].

However in the current study, no significant difference in the

hydrogen production was observed due to accumulation of

propionic acid in the medium when the initial pH of the

medium was set at 5–6 (Fig. 5 and Table 3). Few investigators

have reported that pH 5–6.5 is recommended to avoid propi-

onate accumulation [35]. Thus though propionic acid was

detected in the medium, the amount was not significant. Thus

it is presumed that it did not have an impact on the hydrogen

production. These studies suggest that it is necessary to

maintain a controlled pH that serves as a key parameter for

successful hydrogen fermentation.

3.10. Characterization of purified isolates isolated fromhydrogen producing TERI BH05 bacterial consortium

Based on the colony morphology two hydrogen producing

anaerobic strains were isolated from TERI BH05 bacterial

consortium, collected from riverbed sediments. Microscopic

examination revealed that the cells of these anaerobic bacte-

rial strains were gram-positive rods. The partial sequences of

16S rRNA gene were determined for these two isolates. Based

on the results of 16S rRNA partial gene sequence comparison

with existing database in GeneBank, TERI BH05-1 and TERI

BH05-2 strains belong to the genus Clostridium. The sequence

of TERI BH05-1 strain (FJ656099) had a 99% identity with Clos-

tridium bifermentans strain SH-C14, whereas TERI BH05-2 strain

(FJ656098) had a 99% identity with C. butyricum strain CM-C86.

Further these two purified bacterial isolates were investigated

for their hydrogen production ability.

3.11. Hydrogen production from glucose using purebacterial isolates

TERI BH05-1 and TERI BH05-2 isolates were evaluated for their

ability to produce hydrogen by using 10 g/L glucose as the

substrate. Batch experiments as indicated earlier were carried

out at 37 �C and the initial pH adjusted to 6. The results reveal

that both the strains were able to utilize glucose to produce

hydrogen (Fig. 6). Among the two strains, TERI BH05-2 strain

identified as C. butyricum, displayed the better hydrogen yield

(15.9 mmol/L) after 24 h of incubation and increased to

16.36 mmol/L at the end of 48 h incubation.

Whereas, TERI BH05-1 strain identified indicated the yield

of 11.9 mmol/L of hydrogen at 24 h and it increased to

15.34 mmol/L at the end of 48 h. TERI BH05-2, strain displayed

maximum hydrogen yield. The Clostridium strains have been

previously reported for hydrogen production [13,26].

When compared to the hydrogen production ability of

consortium TERI BH05, the individual strains isolated from

this consortium had shown lower yields (Fig. 6). This could be

attributed to the synergistic effect of the two bacterial strains

in the bacterial consortia that yields higher hydrogen

production.

0

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8442Time (h)

Hyd

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(mm

ol/L

)

TERI BH05 Clostridium buytricum Clostridium bifermentas

Fig. 6 – Comparison of fermentative hydrogen production

by TERI BH05 bacterial consortia and purified TERI BH05-1

and TERI BH05-2 strains, in modified DMI medium.

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 3 5 ( 2 0 1 0 ) 1 0 6 4 5 – 1 0 6 5 2 10651

3.12. Production of soluble metabolites during hydrogenfermentation in pure bacterial isolates

Hydrogen fermentation in purified TERI BH05-1 and TERI

BH05-2 strains accompanied with production of soluble

metabolites. Acetic acid was the major fermentation end

product produced from both the strains accounting for

production of 1031 and 817 mg/L, respectively (Table 3).

Besides acetic acid, the strains also produced butyric acid, but

the butyric acid production by strain TERI BH05-1 is signifi-

cantly less when compared with the second strains (Table 3).

These results indicate that TERI BH05-2 strain (identified as C.

butyricum) follows a butyrate type fermentation route to

produce hydrogen. In contrast, the soluble metabolite

composition resulting from TERI BH05-1 strain was very

different as it included isobutyric acid (36 mg/L), isovaleric

acid (55 mg/L) and minute amount of butyric acid (11 mg/L), in

addition to the production of acetic acid (Table 3).

4. Conclusions

The present work demonstrated the production molecular

hydrogen through dark fermentative hydrogen by anaerobic

bacterial consortia isolated from riverbed sediments. The

selected consortium TERI BH05 has the potential to produce

hydrogen by utilizing five different carbon sources, starch,

sucrose, glucose, fructose, and xylose. This indicates that this

consortium may have the ability to utilize different carbohy-

drate rich waste biomass stream to produce biohydrogen.

Presence of nitrogenous supplements and ferric ions were

found to enhanced hydrogen production from TERI BH05

when these five sugars were used. However, hydrogen

production from glucose was most preferred among all the

sugars as investigated in this study. Hydrogen production was

found to be accompanied with the production of different

soluble metabolites. This study demonstrates that TERI BH05

consortium displays maximum yield of hydrogen production

(2.0–2.3 mol H2/mol glucose) at optimum temperature, pH and

glucose concentration of 37 �C, 6 and 8 g/L, respectively. Two

bacterial strains were later isolated from the consortium and

identified as Clostridium sp.

Acknowledgement

The authors are thankful to Dr R. K. Pachauri, Director General,

TERI, New Delhi, for providing infrastructure to carry out the

present study. We gratefully acknowledge the financial support

assisted by Hindustan Petroleum Company Limited (HPCL).

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