PRODUCTION OF HYDROGEN FROM LIGNOCELLULOSIC MATERIAL BY

THERMOPHILIC BACTERIA

SUMMARY

Alicja Tarazewicz

______________________________________________________

The Master Thesis was supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism -

Project PL0460.

PRODUCTION OF HYDROGEN FROM LIGNOCELLULOSIC MATERIAL BY

THERMOPHILIC BACTERIA

Alicja Tarazewicz

A 30 ECTS credit units Master´s thesis

Supervisors

Dr. Johann Orlygsson

M.Sc. Steinar Rafn Beck Baldursson

A Master´s thesis done at

RES | The School for Renewable Energy Science

in affiliation with

University of Iceland &

University of Akureyri

Akureyri, January 2010

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TABLE OF CONTENTS

Research Objectives ........................................................Błąd! Nie zdefiniowano zakładki.

1 Introduction ..................................................................................................................... 4

1.1 Sampling sites........................................................................................................... 4

1.2 Media ........................................................................................................................ 5

2 Results ............................................................................................................................. 5

2.1 Isolation of bacterial strain and phenotypic characteristics...................................... 5

2.2 Physiological characteristics .................................................................................... 5

2.3 Effect of substrate concentration .............................................................................. 6

2.4 Substrate utilization .................................................................................................. 7

2.5 Carbon and electron recovery.............................Błąd! Nie zdefiniowano zakładki.

2.6 Growth dynamics...................................................................................................... 8

2.7 Effect of partial pressure of hydrogen on hydrogen production............................... 9

2.8 End product formation from hydrolysates.............................................................. 11

3 Conclusions ................................................................................................................... 13

4 Perspectives of production and use of hydrogen in Poland........................................... 13

5 Bibliography .................................................................................................................. 14

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1 INTRODUCTION

The word’s energy demands are currently focused on the use of fossil fuels. The biggest

share in total fuel consumption is petroleum (43%), followed by natural gas (15.6%) and

coal (8.3%)(ELC 2009). It has been estimated that by the year 2050 the world’s oil

reservoir will be used up which makes the need for alternative fuels imperative (Saxena,

Adhikari and Goyal 2009). The biggest problem with using fossil fuels are the high sulfur,

nitrogen and metal content and its combustion results in extensive amounts of SO2 and

NOx emissions into the atmosphere; additionally the released CO2 which is considered to

have undesirable climatic consequences (Putun, et al. 2001).

In the last few decades biomass and energy derived from it, has gained a lot of attention.

The reason is that the policy in many countries is to convert to less climate-harmful energy

sources while attempting to decrease the CO2 concentration in the atmosphere. This can be

achieved by using biomass since it is a carbon neutral fuel and it does not contribute to any

net increase in atmospheric CO2 since the CO2 arising from renewable energy is originally

bound by plants. (Wheals, et al. 1999, Lin and Tanaka 2006, Byung-Hwan and Hanley

2008).

There is a whole spectrum of various types of renewable fuels obtained from biomass such

as; bioethanol, biohydrogen, biodiesel and biogas. Hydrogen is an energy carrier, like

electricity or gasoline, and can be used as a fuel although it is still in the development stage

(Schlapbach and Zuttei 2001). Currently the common source of hydrogen is fossil fuels but

other feedstocks are possible, e.g. biomass (Das and Veziroglu 2001).

Microbial fermentations are potential alternatives to producing biohydrogen. Various kinds

of biomass can be used as feedstock for the production, among them lignocellulosic

biomass (Zaldivar, Nielsen and Olsson 2001).

Hydrogen can be produced in several different methods, although the present investigation

focuses upon production by thermophilic bacteria fermentation.

All of the experiments were carried out at the University of Akureyri. The 16S rRNA

analysis was performed by Prokaria ehf, Iceland.

1.1 Sampling sites

The two strains investigated in this study were isolated from two different hot springs. One

of the hot springs is located in Graendalur, which is in the Hengill area of SW Iceland. The

other hot spring is in the Krafla area of NE Iceland. The temperature and pH level of the

hot spring where strain AK14 was found was 51oC and a pH of 7.8, respectively. The other

hot spring where the AK8 strain was found was a temperature of 70.6oC and pH of 8.2. The

temperatures were measured directly from the hot springs and the pH levels were measured

from experimental bottles upon arrival at the laboratory.

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1.2 Media

The medium was prepared according to Orlygsson and Baldursson (2007). After the

medium was boiled it was cooled down while flushing with N2. The appropriate amounts

of media were transferred to the serum bottles (23, 30 or 120 ml) and closed with butyl

rubber stoppers and aluminum caps. Before use, the bottles were flushed N2 and

autoclaved for 60 min at 121oC. Just before inoculation, solutions C1 and C2 were added

together with the carbon source (usually glucose in 20 mM concentration). Two grams of

yeast extract per liter was used in all experiments.

2 RESULTS

2.1 Isolation of bacterial strain and phenotypic characteristics

The two strains investigated in this study have been isolated from ongoing research

projects at the University of Akureyri (Beck Baldursson and Orlygsson 2006, Bakken

Eiðsdottir and Orlygsson 2009). After isolation the pure cultures were sent for full 16S

rRNA analysis. The phylogenetic trees show that strain AK8 is a member of the genus

Caldicellulosiruptor. The closest relative is Caldicellulosiruptor sp. YI5 with 97.1%

homology.

The full 16S rRNA analysis for strain AK14 showed that it is very difficult to place it to a

certain genus because of low similarity values. It is most closely related to mesophilic

species within the genera of Clostridium and Anaerobacter but has less than 95.0%

homology and should therefore be assigned as a new species within either of these genera

according to the 96.0% limits set for new species (Stackebrandt, et al. 1999).

The closest relative is Anaerobacter polyendosporus (DSM 5272) with only 94.1%

homology.

2.2 Physiological characteristics

Strain AK14, which was originally isolated from the hot spring at Grensdalur in

Hveragerdi, had a temperature of 51.0°C, with a pH of 7.8. Considerable work has been

done on the isolate concerning end product formation and physiological characteristics.

The temperature and pH optimum for the strain has been determined as 45-50°C and 8.0,

respectively. Glucose fermentation resulted in the following stoichiometry with 83.5%

carbon recovery (Beck Baldursson and Orlygsson 2006):

1.00 Glucose ���� 0.39 Acetate + 0.55 Butyrate + 0.15 Lactate + 0.90 H2 + 1.52 CO2 (1)

Additionally, hydrogen production capacity and substrate spectrum has been investigated

to some extent (Beck Baldursson and Orlygsson 2006, Orlygsson and Baldursson 2007).

Strain AK8 was originally isolated from the hot spring at Krafla, with a temperature of

70.6°C and a pH of 8.2. Some work has been done on the isolate concerning end product

formation and physiological characteristics.

1.00 Glucose ���� 0.56 Ethanol + 0.5 Acetate + 1.75 H2 + 1.06 CO2 (2)

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2.3 Effect of substrate concentration

The experiment was done to investigate the effect of various initial glucose concentrations

on end product formation and growth of bacteria.

Fig. 1 End product formation from different glucose concentration for strain AK8

Fig. 2 End product formation from different glucose concentration for strain AK14

A clear correlation is observed between substrate concentration and end product formation

for both strains (Fig. 1 and Fig. 2). The best yields of end products per mole of glucose

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degraded were obtained for strain AK8 in samples with 10 to 30 mM initial glucose

concentration but a clear inhibition of product formation in samples above 100 mM are

observed (Fig. 1). The main volatile end product is acetate, with more than 1 mol

acetate/mol glucose, formed in the lower glucose (5 and 10 mM) concentrations. Hydrogen

was also a significant end product of this strain (between 1 and 3 moles of H2 per mole of

glucose at low glucose concentrations) but ethanol was less than 3 mM in all experimental

set ups. Strain AK14 is showing lower end product formation compared to strain AK8. The

end product formation increases only to 10 mM initial glucose concentrations where it

levels off. The end products analyzed were ethanol, acetate, butyrate, hydrogen and carbon

dioxide (calculated). The hydrogen yields, at low initial glucose concentrations, were

approximately 1 mol H2/mol glucose, but much less at higher concentrations. The results

for AK14 in this experiment are a little surprising since earlier results in the laboratory at

the UA, with this strain have showed higher end product formation (Steinar Rafn Beck and

results not shown) Fig. 4). These experiments were done in the same way as described in

the data presented here. The most reasonable explanation for this is insufficient glucose

degradation by the strain even at low glucose concentrations. In both strains, the highest

glucose consumption was in samples with 20 mM of initial glucose concentration, or 61%

for AK8 and 80% for AK14.

2.4 Substrate utilization

Substrate utilization was tested on various carbon sources. The experiment was carried out

in serum bottles with LG ratio 10 ml: 13 ml.

The end product formations for strain AK8 on all substrates were ethanol, acetate and H2

(and CO2). The strain degraded two of the three pentoses investigated (ribose and xylose)

but not arabinose and three of the four hexoses (glucose, mannose and fructose) and one of

the two disaccharides (sucrose but not lactose). Finally, a slight growth was observed on

pectin. No growth occurred on puryvate, cellulose, peptone, threonine, tryptone, xylan,

serine, and beef extract. Acetate is the major volatile end product; the highest

concentration of acetate was from mannose (15.2 mM) but the ethanol produced is less

than 3 mM on all substrates. Hydrogen production was very poor in wall samples; only

glucose presents the H2 yield higher than 15 mmol/L whereas mannose gives only about

8.5 mmol H2/L.

The end product formations for strain AK14 on all substrates were ethanol, acetate, butyrate

and H2 (and CO2). The strain degraded only one of the three pentoses investigated, xylose.

However, all the hexoses and only one of the two disaccharides (sucrose but not lactose)

were undegraded. No growth occurred on arabinose, ribose, puryvate, cellulose, pectin,

peptone, threonine, tryptone, xylan, serine, and beef extract. Acetate and butyrate were the

major volatile end products together with hydrogen from most of the substrates. As in

strain AK8, hydrogen production by AK14 was very poor and only glucose give a high H2

yield (21.8 mmol/L). Degradation of sucrose resulted in the highest end product formation

of all substrates tested but glucose. Hydrogen is produced in amount of 10.7 mmol/L,

butyrate (13.8 mM) and acetate (10.6 mM) and ethanol (7.7 mM).

Overall the results show that both strains are able to utilize various mono- and

disaccharides but lack the enzymes needed for degrading most of the more complex

polymers.

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2.5 Growth dynamics

The kinetics of glucose degradation to various end products was followed for both strains

(Fig. 3, Fig. 4).

Fig. 3 Growth of strain AK8 with glucose as a carbon source; control sample was

subtracted

Strain AK8 had a doubling time of 4.95 h. The acetate and hydrogen production rates were

0.11 mM EtOH/h and 0.3 mmol H2/(L·h). Glucose was completely degraded after 38 hours

(Fig. 3).

Fig. 4 Growth of strain AK14 with glucose as a carbon source; control sample was

subtracted

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Strain AK14 grew much faster as compared to strain AK8, the doubling time was 0.53h,

which is 5 times faster than strain AK8.

End product formation was different as compared to experiments on this strain presented in

chapter 1.2. As earlier stated, acetate and butyrate were produced but much higher

concentrations of ethanol were also observed or 11.5 mM as compared to values less than 3

mM. Hydrogen was also a substantial end product and its production rate was 1.83

mmol/(L·h).

2.6 Effect of partial pressure of hydrogen on hydrogen production

Influence of partial pressure of hydrogen (pH2) was investigated by cultivating the strains

in experimental bottles with various liquid to gas (LG) ratios. Fig. 5 and Fig. 6 show the

calculated theoretical value of hydrogen production presented in percentages.

Fig. 5 Effect of liquid/gas volume ratio on hydrogen production for strain AK8

Fig. 6 Effect of liquid/gas volume ratio on hydrogen production for strain AK14

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Strain AK8 is presenting higher theoretical yield of hydrogen (80.5%) compared to AK14

(51.8%) at lowest LG ratio. The strain is severely inhibited by increasing the LG ratio; the

H2 yield drops to 14.3% (Fig. 5). This inhibition can also be observed for AK14 where the

H2 drops from 51.8% to 18.8% (Fig. 6). Volatile end products were also analyzed in all

experiments (Fig. 7, Fig. 8).

Fig. 7 Effect of liquid/gas volume ratio on end product formation for strain AK8

Clearly, the acetate and ethanol concentrations decrease and increase respectively, with the

increasing of the LG-ratio for strain AK8 (Fig. 7). Acetate decreases from 26 mM to 6 mM

but ethanol increases from 2.1 mM to 9.1 mM. The hydrogen produced increases from a

very low LG ratio to a ratio of 1.0 (60 ml liquid and 60 ml gas) but a clear inhibition is

observed at the highest LG ratio used.

Fig. 8 Effect of liquid/gas volume ratio on end product formation for strain AK14

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As for strain AK8, there is a clear correlation between the decrease in acetate and increase

in ethanol with increasing the LG-ratio (Fig. 7). However, for strain AK14 this trend is not

so much pronounced (Fig. 8). Acetate decreases from 7.5 mM to 5.5 mM but ethanol

increases from 2.5 mM to 5.0 mM. However, almost no change is observed in the

formation of butyrate at different LG ratios. Similar inhibition of hydrogen production is

observed as for strain AK8.

2.7 End product formation from hydrolysates

Both strains were inoculated in basal medium containing 20% of hydrolysates (5g/L) from

various types of biomass (Whatman paper, hemp leafs, hemp sticks, newspaper, straw, and

grass). The experiment was carried out in serum bottles with LG ratio 10 ml: 13 ml.

Results are showed on the Fig. 9 and Fig. 10.

Fig. 9 End product formation from 20% hydrolysates for strain AK8

Highest hydrogen production was observed on cellulose (19.6 mmol/L) and grass (9.7

mmol/L) when no chemical pretreatment was used. Acetate was the only other end product

analyzed in most of the hydrolysates. Acid and base pretreatment did not enhance end

product formation for the paper hydrolysates (for the newspaper there was a strong

inhibition of the hydrolysate treated with base). For most of the lignocellulosic

hydrolysates the chemical pretreatment increased the end product formation. This was

most significantly observed on hemp leafs where the end product formation increased

almost five times with base pretreatment and ten times with acid pretreatment. Acidic

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pretreatment slightly increased end product formation from grass but base pretreatment

completely inhibited growth of the strain. In general, the concentration of acetate and

hydrogen are in good correlation with end product formation observed from monosugar

degradation (see earlier results).

Fig. 10 End product formation from 20% hydrolysates for strain AK14

Highest hydrogen production was observed on cellulose (19.6 mmol/L) and hemp stalk

(8.0 mmol/L) when no chemical pretreatment was used. Fermentation of hydrolysates from

other biomass types resulted in low hydrogen production. Volatile end products were

acetate and butyrate (major end products) and ethanol (minor). Acid and base pretreatment

did not enhance end product formation for cellulose hydrolysates (a strong inhibition was

observed for hydrolysate treated with base). Acid pretreatment enhanced hydrogen

production significantly from 4.3 to 6.4 mmol/L on the newspaper hydrolysate. For most of

the lignocellulosic hydrolysates the chemical pretreatment greatly increased the end

product formation. This was most significantly observed on grass, hemp leaf and straw

hydrolysates where the increase is between 5 to 10 times more as compared with untreated

biomass. In general, the concentration of volatile end products and hydrogen are in good

correlation with end product formation observed from monosugar degradation (see earlier

results).

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3 CONCLUSIONS

The original research objective was to investigate hydrogen production from

lignocellulosic biomass by two strains of thermophilic anaerobic bacteria AK8 and AK14.

Strain AK8 is representing thermophilic bacteria related within the genus

Caldicellulosiruptor sp. and it is showing homology with Caldicellulosiruptor YI5

(97.1%). Strain AK14 however, is related to genus Clostridium sp. and the best homologue

is Anaerobacter poylyendosporus (94.1%).

Both strains were investigated for hydrogen production; in various substrate

concentrations, different LG ratio, through various substrates, and by kinetic of end

product formation. One of the experiments was investigating hydrogen production on

different lignocellulosic materials.

Strains AK8 and AK14 have proven in experiments that they are good hydrogen producers.

Strain AK8 has slower kinetics than AK14, and degrades glucose more slowly as compared

to AK14. The main end products for strain AK8 were ethanol, acetate (the major volatile

product), hydrogen and CO2. Plus it utilizes pentoses (ribose and xylose), hexoses

(glucose, mannose and fructose) and the disaccharide sucrose. Strain AK14 produces

ethanol, acetate, butyrate (the major volatile product), hydrogen and CO2. From various

types of carbon sources it can utilize pentose (xylose), hexoses (galactose, glucose,

mannose and fructose) and disaccharide (sucrose).

Experiments with various initial concentrations of substrate were investigated and both

strains show substrate inhibition starting above 20 mM of glucose when the product

formation stops increasing.

It was also shown that the partial pressure of hydrogen production strongly influenced the

production of hydrogen. With increasing partial pressure fermentation shifts towards

ethanol production instead of hydrogen.

Experiments on various types of hydrolysate from various types of biomass showed that

both strains were able to convert lignocellulosic materials with high yield of hydrogen.

However most of the types of tested biomass like straw, grass or hemp, need to be

pretreated to achieve high hydrogen yield. Thus the process may be scaled up and carried

out in a reactor feed with pretreated biomass to produce hydrogen i.e. to supply stationary

power system equipped with PEMPC.

4 PERSPECTIVES OF PRODUCTION AND USE OF HYDROGEN IN POLAND

Energy mix in Poland is focused mainly on burning coal. However, this process leads to

significant participation in emissions of GHG. Technologies gaining energy from hydrogen

are potentially stable option for the Polish energy system to provide clean energy. In

Poland, interest of hydrogen related projects has been steadily growing since the first

articles appeared in scientific journals (Murray, et al. 2008, Biuletyn Polskiego

StowaŜyszenia Wodoru i Ogniw Paliwowych. Rocznik Informacyjny 2005 2005, Przemysł

Chemiczny PRCHAB 2005, Rogut, et al. 2004, LTN 2005).

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5 BIBLIOGRAPHY

Bakken Eiðsdottir, H., og J. Orlygsson. BioEthanol: Fuel of the future? Akureyri: University of Akureyri, 2009.

Beck Baldursson, S. R., og J. Orlygsson. BioHydrogen. Bioprospecting: Thermophilic hydrogen producing anaerobes in

Icelandic hot-springs. University of Akureyri, 2006.

„Biuletyn Polskiego StowaŜyszenia Wodoru i Ogniw Paliwowych. Rocznik Informacyjny 2005.“ Biuletyn Polskiego

StowaŜyszenia Wodoru i Ogniw Paliwowych. Rocznik Informacyjny 2005 1 (2005).

Byung-Hwan, U., og T. Hanley. „High-solid enzymatic hydrolysis and fermentation of solka floc into ethanol.“ Journal

of Microbiology and Biotechnology 18, nr. 7 (2008): 1257-1265.

Das, D., og T. N. Veziroglu. „Hydrogen production by biological processes: a survey of literature.“ International Journal

of hydrogen Energy 26 (2001): 13-28.

ELC. http://enviroliteracy.org/subcategory.php/32.html. Environmental Literacy Council. 2009. (skoðað 31. January

2009).

Lin, Y., og S. Tanaka. „Ethanol fermentation from biomass resources: current state and prospects.“ Applied

Microbiological and Bioetechnology 69, nr. 6 (2006): 627-642.

LTN. „Nowe kierunki wytwarzania i wykorzystania energii.“ Lubelskie TowaŜystwo Naukowe, 2005.

Murray, M. L., E. H. Seymour, J. Rogut, og S. W. Zechowska. „Stakeholder perception towards the transition to a

hydrogen economy in Poland.“ International Journal of Hydrogen Energy 33 (2008): 20-27.

Orlygsson, J., og S. R. Baldursson. „Phylogenetic and physiological studies of four hydrogen-producing thermoanaerobes

from Icelandic geothermal areas.“ Icelandic Agricultural Sciences 20 (2007): 93-106.

„Przemysł Chemiczny PRCHAB.“ Przemysł Chemiczny PRCHAB 84, nr. 11 (2005): 789-896.

Putun, A. E., A. Ozcan, H. F. Gercel, og E. Putun. „Production of biocrudes from in a fixed-bed tubular reactor: product

yields and compositions.“ Fuel 80, nr. 10 (2001): 1371-1378.

Rogut, J., N. Howaniec, M. Ludwik, og S. W. Zechowska. „Gospodarka wodorowa szansą dla rozwoju górnictwa

węglowego.“ Przegląd Górniczy 6, nr. 980 (2004).

Saxena, R. C., D. K. Adhikari, og H. B. Goyal. „Biomass-based energy fuel through biochemical reutes: A review.“

Renewable and Sustainable Energy Reviews 13, nr. 1 (2009): 167-178.

Schlapbach, L., og A. Zuttei. „Hydrogen-stage materials for mobile applications.“ Macmillan Magazines Ltd 414 (2001):

353-358.

Stackebrandt, E., I. Kramer, J. Swiderski, og H. Hippe. „Phylogenetic basis for a taxonomic dissection of the genus

Clostridium.“ FEMS Immunology and Medical Microbiology 24 (1999): 253-258.

Wheals, A. E., L. C. Basso, D. M. Alves, og H. V. Amorin. „Fuel ethanol after 25 years.“ Trends in Biotechnology 17, nr.

12 (1999): 482-487.

Zaldivar, J., J. Nielsen, og L. Olsson. „Fuel ethanol production from lignocellulose: a change for methabolic engineering

and process integration.“ Applied Microbiology and Biotechnology 56, nr. 1-2 (2001): 17-34.