Ethanol and Hydrogen Production from Lignocellulosic
Biomass by Thermophilic Bacteria
Arnheidur Ran Almarsdottir1, Margret Audur Sigurbjornsdottir1, Johann Orlygsson1,2
1)
Faculty of Natural Resource Science, University of Akureyri, Akureyri, Iceland
2)
RES, The School for Renewable Science, Akureyri, Iceland
e-mail: [email protected]
KEY-WORDS: THERMOPHILIC, BACTERIA, ANAEROBIC, ETHANOL,
LIGNOCELLULOSIC
ABSTRACT
Strain AK67, a thermophilic ethanol and hydrogen producing bacteria was isolated from
hot spring in SW–Iceland. Growth for strain AK67 was observed at pH’s between 4.0 to
7.5 and temperatures between 50.0 to 70.0°C; optimal growth conditions were at pH 6.0
and 65°C. As determined by full 16S rRNA gene sequence analysis, strain AK67 belongs
to the genus Thermoanaerobacterium with closest relation to T. islandicum and T.
xylanolyticum. Kinetics of glucose degradation revealed that the main flow of carbon
ended in ethanol but other products were acetate, H2 and CO2. At low initial substrate
concentrations, ethanol production rate and yield were 4.5 mM h-1 and 1.25
molEtOH/mol glucose, respectively. At loadings of ≥ 50 mM, a clear inhibition in both
end product formation and glucose degradation were observed. Apart from glucose, the
strain degraded xylose, ribose, glucose, fructose, mannose, galactose, lactose, sucrose,
serine, xylan and pectin. The major end products in all cases were ethanol, acetate,
hydrogen and carbon dioxide. Ethanol and hydrogen production from hydrolysates (5.0 g
L-1 (dw)) made of lignocellulosic biomass (cellulose, newspaper, grass (Phleum
pratense), barley straw (Hordeum vulgare), and hemp (stem and leaves (Cannabis sativa
L), was investigated. The biomass was pretreated with acid or base as well as enzymes
(Celluclast® and Novozymes 188) and heat (autoclaved for 30 min). The strain produced
most ethanol (31.0 mM) and hydrogen (14.5 mmol L-1; 4.64 mol H2/g VS cellulose) from
cellulose hydrolysates. Ethanol and hydrogen formation from untreated lignocellulosic
biomass was considerable less as compared to cellulose; ranging from 1.5 mM ethanol
and 0.6 mmol L-1 hydrogen (hemp leaves) to 9.2 mM ethanol and 9.1 mmol L-1 hydrogen
(hemp stem). Chemical pretreatment of lignocellulosic biomass always resulted in higher
end product formation, especially ethanol. This was most profound in the case of hemp
leaves where ethanol production increased from 1.5 mM to 10.1 mM by acid
pretreatment. Base pretreatment of biomass also increased ethanol yields but to a lesser
extent as compared to acid pretreatment. This was however not the case with acetate and
hydrogen production where pretreatment of biomass led to similar or even less hydrogen
yields.
1. INTRODUCTION
Today, most of the world’s energy demands are met by non-renewable energy
sources, causing depletion, environmental deterioration and public health problems.
Energy consumption is growing at rising rates at the same time, demanding novel
renewable energy sources [1]. Ethanol and hydrogen are two renewable energy sources
that have gained increased interest in recent years.
Hydrogen has a great potential as a clean, renewable energy carrier since when
combusted no CO2 emissions occurs [2,3]. Additionally H2 energy yields are almost three
times higher compared to most hydrocarbon fuels [3].
Today, more than 51 billion liters of bioethanol are produced from biomass
worldwide [1,4]. Brazil and USA are the largest ethanol producers with 87% of global
production [1]. Currently, ethanol production in Brazil, India and South Africa is mainly
based on sugar cane but in USA from corn (first generation ethanol) [5]. This production
has been strongly debated the last few years because it is competing with the food and
feed application, leading to increased interest towards more complex biomass resources.
Fermentation from lignocellulosic biomass (e.g. wood, straw and grasses) is an
interesting alternative for the production of second generation bioethanol and
biohydrogen [6]. Both hydrogen and ethanol can be produced microbiologically through
fermentation from various starch- and sugar-based materials [7], there among
lignocellulosic biomass. Attention in both ethanol and hydrogen production through dark
fermentation has increased in the past few years and high production rates has been
achieved [8,9,10]. Thermophiles have many advantages compared to mesophilic
microorganisms in hydrogen and ethanol production concerning fast growth rates and
their ability to degrade a broad variety of substrates, as well as higher hydrogen yields
[11,12]. Additionally, many thermophiles have narrower spectrum of fermentation end
products compared to mesophiles.
Hot springs are a potential source for hydrogen and ethanol producing
microorganisms [8,13]. In this study a thermophilic fermentative bacterium efficient
ethanol production is studied. Various monosugars, polymetric carbohydrates and
hydrolysates from various complex biomass were used to test the performance of the
bacterium. Optimal conditions for hydrogen production in terms of temperature and pH
were investigated. Kinetic parameters from glucose degradation were identified.
2. MATERIALS AND METHODS
2.1. Media, isolation and identification of isolate
The medium composition and preparation was as described by Orlygsson &
Baldursson [14]. Inoculation was 1% and all experiments were done in duplicate. Strain
AK67 was isolated from a hot spring (66°C, pH = 7.7) in Graendalur in the SW-Iceland.
The methodology concerning isolation procedure is described in Sveinsdottir et al. [13].
The sample was incubated at 60.0°C and pH 6.0 on pectin (2 g L-1) as carbon substrate.
The medium (BM) used is described by Orlygsson and Baldursson [14]. For 16S rRNA
analysis 16S rRNA genes were amplified from DNA with primers F9 and R1544, specific
for bacterial genes [15]. For alignment of sequences with similar sequences was done in
the program BioEdit and Clustal X [16,17].
2.2. Determination of growth
Cell concentration was determined by measuring absorbance at 600 nm by Perkin
Elmer spectrophotometer. Maximum (specific) growth rate (μmax ) for each growth
experiment was derived from the absorbance data (OD600) using the equation: ln(x/x0) =
(µ)(t), where x is the measurement of optical density of the culture, x 0 is the initial optical
density of the culture, t is the elapsed time and µ denotes the maximum growth rate.
Growth kinetic experiment was done for the strain using glucose (20 mM) as the sole
carbon source. Growth was measured on time and end product analyzed to determine
generation time and the production of end products. Experiments were done in 120 mL
bottles with 50 mL liquid medium. Experiments were done in 120 mL serum bottles with
50 mL liquid medium.
2.3. Determination of pHopt and Topt
To determine the strain’s optimum pH for growth the pH was set to various levels in
the range of 3.0 to 9.0 with increments of 1.0 pH unit. The experimental bottles were
supplemented with acid (HCl) or base (NaOH) to set the pH accordingly. To determine
the optimum temperature for growth the incubation temperature varied from 30°C to
75°C. For the pH optimum determination the strain was cultivated at 60°C and for the
temperature optimum determination the pH was 5.0. Control samples did not contain
glucose. Optimal pH and temperature were thereafter used in all experiments performed.
Experiments were done in 120 mL serum bottles with 50 mL liquid medium.
2.4. Effect of substrate concentration on growth
Effect of increased initial glucose concentration was tested on the strain. Initial
glucose concentration varied between 5 to 400 mM. Control samples did not contain
glucose. Optical density was measured at beginning and at the end of incubation time (5
days) to determine growth. Glucose, hydrogen, ethanol and acetate were measured as
well. Experiments were done in 120 mL serum bottles with 50 mL liquid medium.
2.5. Substrate utilization
The ability of strain AK67 to utilize different substrates was tested using the BM
medium supplemented with various filter sterilized substrates (20 mM or 2 g L -1).
Substrates tested were glucose, fructose, galactose, mannose, xylose, ribose, arabinose,
sucrose, lactose, lactate, formate, succinate, malate, pyruvate, oxalate, crotonate,
glycerol, inositol, starch, cellulose, xylan, sorbitol, pectin, casamino acids, peptone, beef
extract, tryptone, alanine, aspartate, glycine, glutamate, serine, threonine, histidine and
cysteine. Growth was observed by increase in optical density which was measured at the
beginning and at the end of incubation time (5 days). Where growth was detected,
hydrogen, acetate and ethanol were analyzed. Experiments were done in 23 mL serum
bottles with 10 mL liquid medium.
2.6. Effect of gas/liquid volume ratio on hydrogen production
The influence of partial pressure of hydrogen (ρH2) on hydrogen production was
investigated with different ratios of liquid and gas phase. The liquid phase varied from 5
– 90 mL in serum bottles with total volume of 120 mL, thus, the liquid/gas volume ratio
varied from 0.043 to 3.0. After 5 days of incubation, optical density was measured as
well as end product formation (hydrogen, acetate, ethanol). Glucose was measured at the
end of incubation time.
2.7. Pretreatment of biomass and hydrolysate preparation
Hydrolysates (HL) were made from different biomass: Whatman filter paper
(cellulose), hemp (Cannabis Sativa L.) – leafs and stem fibers, newspaper with ink,
barley straw (Hordeum vulgare) and grass (Phleum pratense). The Whatman paper
consists of 99% cellulose and was therefore used as control sample. The preparation of
the hydrolysates was according to Sveinsdottir et al. [13] yielding a final dry weight of 25
g L-1. Pretreatment of biomass consisted of acid (0.75% H2SO4) or base (0.75% NaOH)
(control was without chemical pretreatment) before heating for 30 minutes at 121°C.
After heating, the bottles were cooled down to room temperature and the pH adjusted to
5.0 by adding either HCl or NaOH. Two enzymes were added to each experimental
bottle, Celluclast® and Novozymes 188 (1 mL of each; 0.25% vol/vol), and incubated in
water bath at 45°C for 68h. After the enzyme treatment the pH was measured again and
adjusted to the pH optimum of the strain. The hydrolysates were then filtered into sterile
bottles to collect the hydrolysates.
Fermentation of hydrolysates by strain AK67 was done in 23 mL serum bottles. Eight
mL of BM medium was supplemented with 2 mL of hydrolysate (total liquid volume 10
mL) giving a final HL-concentration of 5.0 g L-1. Control sample contained no
hydrolysate. The concentration of salts, vitamins and trace elements were kept the same
as in medium without hydrolysate additions.
2.8. Analytical methods
Hydrogen ethanol and volatile fatty acids were measured by gas chromatograph as
previously described [14]. Determination of glucose was performed according to the
method from Laurentin and Edwards [18].
3. RESULTS AND DISCUSSION
3.1. Isolation, identification and phenotypic characterization
The strain was isolated from a hot spring in Grensdalur in SW-Iceland. The
temperature and pH of the hot spring it originates from were 66.0 °C and 7.7,
respectively. The carbon source that was used for isolations was pectine (2 g L-1). The
isolate have a rod shape, with a length from 2.0 to 2.5 μm. They occurred singly or in
pairs. The cells stained gram-positive. The cells did not produce spores under any oft the
culture conditions used.16S rRNA analysis revealed that strain AK67 is a member of the
genus Thermoanaerobacterium. The
closest phylogenetic relative was
Thermoanaerobacterium xylanolyticum and “Thermoanaerobacterium islandicum” with
99.1% similarity of the 1425 bp analysed.
3.2. Temperature and pH ranges
The strain grows well between 50.0°C to 70.0°C with optimal temperature being
65.0°C. The maximum growth rate (µmas) was 0.63 h-1 which corresponds to generation
time of 1.1 h. No growth was observed below 50.0°C or above 70.0°C. The pH optimum
was between 6.0 to 7.0 with maximum growth rate of 0.59 h-1 but growth occurred
between pH 4.0 to 7.5.
3.3. Kinetics of glucose degradation
Kinetics of glucose degradation to various end products was investigated (Fig. 1).
Glucose was completely degraded in 15 hours. The doubling time was 1.01 h (µ max =
0.684 h-1). The main end products produced were ethanol (25.0 mM), acetate (10.5 mM)
and hydrogen (15.6 mmol L-1). During exponential growth the ethanol production rate
was 4.5 mM EtOH h-1 and hydrogen 2.8 mmol H2 L-1h-1.
Figure 1. Degradation of glucose (20 mM) by strain AK67 and end product formation.
Thus, end product formation from glucose is as follows:
20 mM Glucose  25.3 mM EtOH + 10.5 mM Acetate + 15.5 mmol L-1 H2 + 35.8 mM CO2
The carbon balance was calculated as 100% assuming that 11% of the carbon is
assembled into biomass.
3.4. Effect of substrate concentrations
Different initial glucose concentrations were used to investigate its effect on end
product formation for the strain (Table 1).
Table 1. End product formation from different initial glucose concentrations. Also shown
are glucose concentration after fermentation. Values represent mean of two replicates.
Initial
glucose
(mM)
0
5
10
20
50
100
200
400
End product formation (mmol/L)
Ethanol
3.7
5.7
13.3
22.9
28.2
28.3
28.7
26.7
Acetate
2.2
2.0
4.5
7.4
8.9
9.3
8.6
8.9
Hydrogen
1.2
3.9
6.5
11.2
15.2
15.5
12.5
14.3
End glucose
(mM)
0.0
0.3
0.3
0.4
10.3
55.9
159.5
357.5
High initial substrate concentration may play an important role in end product
production rates and yields [11, 19, 20, 21]. This has been investigated for hydrogen
producing bacteria e.g. Citrobacter species where glucose loadings of 1, 5 and 25 g L-1
resulted in 2.5, 1.2 and 0.8 mol-H2/mol-glucose degraded [22]. However, fermenting
microorganisms can also have limited tolerance towards increased substrate loadings
[23,24]. This was observed in present study when strain AK 67 was cultivated on different
initial concentrations of glucose varying from 5 – 400 mM. More than 90% of glucose
were degraded at low (5, 10 and 20 mM) concentrations but much less at higher (≥ 50
mM) concentrations. This is also reflected in the relatively low concentrations of end
products at high initial glucose concentrations compared to the lower substrate loadings.
Additionally, although the strain can growt at low pH values, the inhibition at high
substrate loadings may simply be caused be very low pH’s in these cultures. This is
however not very likely because of relatively little amounts of acetate formed compared
to ethanol and because the buffer capacity in the medium used was above 30 mM.
Unfortunately, pH was not measured in the experiments after fermentation. The
phenotypic properties of AK67 were consisted with those of many saccharolytic species
within Thermoanaerbacterium; capable of the degradation of various carbohydrates to
volatile fatty acids, ethanol and H2 plus CO2.
3.5. Substrate utilization
One of the major issues of utilizing thermophilic bacteria for biofuel production from
lignocellulosic material is their ability to degrade a broad range of carbohydrates present
in the biomass. The isolate showed growth on various types of carbohydrates as the sole
carbon and energy source (Table 2). Of the three tested pentoses the strain degraded
xylose and ribose but not arabinose. All four hexoses and disaccharides tested were
degraded as well as serine, pectine and xylan. End product formation on all these
substrates were acetate, ethanol and H2 + CO2. Other substrates were not degraded
Table 2. End product formation from various substrates by strain AK 67. Values represent
mean of two replicates.
Substrates
Yeast extract
Xylose
Ribose
Glucose
Galactose
Fructose
Mannose
Lactose
Sucrose
Pectin
Xylan
Serine
End product formation (mmol/L)
Ethanol
2.7
25.4
20.6
24.6
35.4
19.9
22.4
43.2
33.7
10.5
26.9
3.2
Acetate
1.8
10.2
12.1
12.8
6.5
11.4
12.7
16.0
18.5
17.6
14.5
6.5
Hydrogen
1.2
6.8
8.0
8.4
4.3
7.5
8.4
10.6
12.2
11.6
9.5
4.3
3.6. Effect of partial pressure of hydrogen
To investigate the influence of the partial pressure of hydrogen (pH2) on hydrogen
production, the strain was cultivated using different liquid/gas volume ratios (Fig. 2).
Figure 2. Effect of liquid/gas phase ratio on end product formation on glucose (20 mM) by strain
AK67
The strain produces 35.5% of the theoretical yield (1.42 mol H2/mol glucose) of
hydrogen with the lowest liquid/gas phase ratio, dropping to 25.0% (1.0 mol H2/mol
glucose) with the highest ratio. In this case it is assumed that the theoretical yield is 4
moles of hydrogen per mole hexose degraded and acetate is the only volatile end product
[7,25]. The change in partial pressure of hydrogen is known to affect the end product
formation by anaerobic bacteria such that at high pH2 more reduced products like ethanol
and lactate are favoured but away from acetate/butyrate and H2 [7,26]. This was however
not reflected in ethanol production at various pH2 in present study. By using the
fermentation data from the lowest and highest L-G ratios the following equations are
observed:
1.0 Glucose  1.45 EtOH + 0.73 Acetate + 1.42 H2 + 2.18 CO2 (low L-G; Eq.1)
1.0 Glucose  1.24 EtOH + 0.59 Acetate + 1.00 H2 + 1.83 CO2 (high L-G; Eq.2)
Surprisingly, at high pH2 ethanol production decreases. The most likely explanation
for this is that the electrons are directed towards the reduction of pyruvate to lactate which
was unfortunately not analyzed in present investigation. This is also supported by the fact
that all glucose was degraded in all experimental bottles revealing lower carbon recoveries
from the high liquid/gas phase bottles.
3.7. End product formation from hydrolysates
Strain AK67 was inoculated into BM medium containing various types of biomass
hydrolysates (5 g L-1) either untreated or pretreated with acid (H2SO4) or base (NaOH) at
0.75% concentrations. Not surprisingly, highest end product formation was observed
from the cellulose (Whatman paper) controls. Experimental bottles containing cellulose
without chemical pretreatment showed ethanol and hydrogen yields of 31.0 mM and 17.9
mmol L-1 (4.64 mol H2/g TS cellulose), respectively. Lower yields were observed in
chemically pretreated cellulose. Of untreated lignocellulosic biomass highest ethanol
concentrations were observed on hemp stem and grass hydrolysates, or 9.2 mM 6.8 mM,
respectively. Chemical pretreatment always resulted in higher ethanol recoveries as
compared to untreated biomass. This increase was lowest on hemp stem (base) or 1.6fold compared to the untreated sample and highest on hemp leaves (acid), 6.7 fold.
Additionally, acid pretreatment of biomass always resulted in higher ethanol yields as
compared to base pretreatment. Hydrogen and acetate production was however not
influenced to the same extent in chemically pretreatment of the hydrolysates. Hydrogen
production is indeed in good correlation with acetate production which is not surprising
since no hydrogen is produced when carbon flow is directed towards ethanol [26]. The
reason for this different increase of end products is difficult to explain from the data
obtained. One explanation might be the formation of inhibitory compounds that act
directly on the enzyme involved in the formation of acetate and hydrogen but not on
alcohol dehydrogenase.
Table 3. Production of end products from hydrolysates made from different biomass.
Values represent mean of two replicates.
End product formation (mmol/L)
Biomass and
pretreatement
Cellulose
Cellulose - acid
Cellulose- base
Hemp Stem
Hemp Stem - acid
Hemp Stem - base
Hemp Leaf
Hemp Leaf - acid
Hemp Leaf - base
Grass
Grass - acid
Grass - base
Paper
Paper- acid
Paper - base
Straw
Straw - acid
Straw - base
Ethanol
Acetate
Hydrogen
31.0
16.0
26.5
9.2
15.2
14.7
1.5
10.1
5.0
6.8
16.7
15.2
3.1
8.6
5.3
3.9
13.6
7.5
14.5
7.5
11.9
4.8
5.5
7.3
1.4
3.9
3.6
3.5
4.4
6.1
2.5
4.4
4.1
3.0
4.9
5.1
17.9
7.5
12.5
9.2
5.0
7.2
0.6
4.7
3.9
5.5
5.2
7.8
4.0
5.4
4.5
4.9
3.8
5.0
Yields of ethanol from hydrolysates of lignocellulosic biomass by thermophilic
bacteria have been addressed lately. In our previous investigations yield of ethanol have
been similar or even higher from slightly higher hydrolysates loadings (7.5 g L-1) by
Thermoanaerobacter and Thermoanaerobacterium species [13]. Sommer and co-workers
[11] showed that thermophilic bacteria produced between 9.8 – 25.7 mM of ethanol from
undiluted wheat straw hydrolysate (60 g L-1). Thermoanaerobacter ethanolicus has been
reported to produce 24 mM of ethanol when cultivated in steam-exploited birch wood
hemicelluloses hydrolysate (0.8 w/v) [27] and Clostridium thermosaccharolyticum
produced 40 mM of ethanol in oak sawdust pretreated with 1% sulfuric acid [28].
Clearly, at high hydrolysate concentrations, the yield of ethanol decreases dramatically.
Microorganisms producing promising yields on pure glucose and xylose do not
necessarily do well in pretreated hydrolysate that contains inhibitory compounds like
acetate, furfural and lignin degradation products [29].
Hydrogen production from lignocellulosic biomass has got increased attention
recently. Several studies on thermophilic bacteria growing on untreated wastewater
cellulose have shown yields between 0.82 and 1.24 mol-H2/mol glucose equivalents [30,
31]. Co-culture studies of Clostridium thermocellum and Thermoanaerobacterium
saccharolyticum on hydrogen production from microcrystalline cellulose resulted in 1.8
mol-H2/mol-glucose equivalents [32]. Other studies on pretreated hydrolysates from
lignocellulosic biomass have shown higher yields. Lalaurette et al. [33] showed hydrogen
yields of 1.64 mol-H2/mol-glucose equivalent from corn stover hydrolysates (pretreated
with dilute sulfuric acid) by Clostridium thermocellum. Mixed culture studies (35 and
50°C) on the same biomass pretreated with steam explosion and dilute sulfuric acid
resulted in 2.84 mol-H2/mol-glucose equivalents [34]. Since strain AK67 directs its end
products mostly to ethanol, both at low and high pH2 the strain cannot be regarded as
good hydrogen producer.
Assuming that all glucose is released from pure cellulose during hydrolysis, an initial
glucose concentration of 30.8 mM would be available for fermentation. According to the
fact that the strain produces 1.32 mol EtOH/mol glucose theoretical yields of ethanol
should be 40.7 mM. The actual value is however 31.0 mM or 75% of theoretical yield.
Acknowledgements
This work was sponsored by the Ministry of Industry within the BioTec Net, The
Icelandic Research fund (Rannís), The University of Akureyri and The KEA fund.
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