Differential Solvent Production by Clostridium beijerinckii NRRL-B593 Grown on Carbohydrates and Peptides
Sarah Danielle Kim, Xiao Zhu, Yutong Jin, Thuy
Nikki Nguyen, Kate Johnston, Annie Li, Xiang Zhang, Jun Zhao, Kesen Ma*
Department of Biology, University of
Waterloo, Waterloo, Ontario, Canada
*Corresponding author: Kesen Ma, Department of Biology, University of
Waterloo, 200 University Avenue West, Waterloo, Canada. Tel: 1-519888-4567
x33562; Fax: 1-519-746-0614; Email: kma@uwaterloo.ca
1. Introduction
Solvents, which are mainly petroleum
based [1], are commonly used substances for a plethora of applications ranging
from house-hold uses (ie. detergents) to more industrial purposes (ie. chemical
synthesis). With the growing concerns of utilizing petrochemicals and the
consequences they put onto the environment, more research efforts have been
focused on using sustainable renewable sources, namely, microbial based sources
to combat such impacts [1]. The use of microorganisms is quite versatile in
that the type of end-products that are produced, including solvents and
biofuel, can vary depending on the microorganism and type of substrate
used [2]. Most microbes can readily convert sugars, starches, and fats to
solvents. Escherichia coli, for example, is used for
the production of solvents for biofuel from biomaterials, with reports showing
30 and 25 g/L of butanol and ethanol produced, respectively [2]. Substrates can
range from a variety of sources including plant- and animal-based biomass. The
metabolic pathway(s) involved are also important in determining which
end-product(s) will be produced. Saccharomyces cerevisiae is a
commonly used ethanol-producing eukaryotic microorganism, whose pathway
involves the decarboxylation of pyruvate to produce ethanol [3]. Clostridia are
a group of anaerobic bacteria capable of producing solvents in large quantities
consisting of a few representative species including Clostridium
acetobutylicum, Clostridium pasteuranium, and Clostridium
beijerinckii, among others [4]. This group is known for their solvent
producing capabilities particularly in acetone, butanol, and ethanol production
using the butyrate and butanol-acetone fermentation pathway (a pathway which is
well-studied in C. acetobutylicum [5]). These solventogenic
clostridia are able to utilize carbohydrates as their main carbon and energy
sources to grow and produce solvents, however, it is unclear whether
proteinaceous materials can be used as substrates for solvent production or
not.
C. beijerinckii NRRL-B593 is a particular solvent
producing bacterium, as it is able to produce isopropanol in addition to
butanol and ethanol (IBE) - where most clostridia are unable to produce
isopropanol but rather acetone [6]. For industrial biofuel production,
isopropanol is more desired as acetone is corrosive to parts of engines made up
of rubber or plastic [7]. C. beijerinckii NRRL-B593 can
produce solvents from various carbohydrate sources, however, it is unclear
whether it is able to utilize peptides to produce solvent using the same
butyrate and butanol-acetone fermentation pathway.
In this study, C.
beijerinckii NRRL-B593 was grown on both carbohydrates and
peptides. C. beijerinckii NRRL-B593’s growth, production of
solvents, and production of hydrogen were compared to determine whether it is
able to produce solvents using peptides or not.
2. Materials and Methods
2.1. Organism and Chemicals
C. beijerinckii NRRL-B593 from Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Germany was used for this
study. Spore suspensions of C. beijerinckii NRRL-B593 were
also prepared, and stored at - 80 °C until use (Kasap 2002).
All chemicals were commercially available.
2.2. Growth Conditions
C. beijerinckii NRRL-B593 was cultured in media
adjusted to pH 7.0 and grown in an anaerobic serum bottle at 37 °C.
All media contained a salt solution, which was prepared according to DSMZ
(Germany). The salt solution contained (in 1 L unless otherwise specified):
0.01 g calcium chloride, 0.02 g magnesium sulfate, 0.04 g dipotassium
phosphate, 0.16 g sodium bicarbonate, 0.08 g sodium chloride. The basal media
contained (in 1 L unless otherwise specified): 5.0 g yeast extract, 40 mL salt
solution (as described above), 0.001 g resazurin, 4.0 g ammonium acetate, 0.5 g
L-cysteine-HCl, 0.01 g biotin, and 0.01 g p-aminobenzoic acid. Peptide sources
added to the basal media included: beef extract, beef peptone, wheat peptone,
casein, tryptone powder (to a final concentration of 5 g/L).
Four different media compositions
were used for this experiment (in 1 L unless otherwise specified): 1) mixed
media: 5 g tryptone, 10 g yeast extract, 5 g dextrose (or sucrose), 40 ml salt
solution, 1 mg resazurin, 0.5 g L-cysteine-HCl; 2) tryptone media: 20 g
tryptone, 40 ml salt solution, 1 mg resazurin, 0.5 g L-cysteine-HCl, 4 g
ammonium acetate, 0.268 g L-alanine, 0.162 g L-isoleucine, 0.189 g L-valine, 45
mg vitamin B1, 45 mg vitamin B6, 0.2 mg Folic acid; 3)
sucrose media: 60 g sucrose, 40 ml salt solution, 1 mg resazurin, 0.5 g
L-cysteine-HCl, 4 g ammonium acetate, 0.268 g L-alanine, 0.162 g L-isoleucine,
0.189 g L-valine, 45 mg vitamin B1, 45 mg vitamin B6, 0.2
mg folic acid; and 4) Dextrose media: 60 g dextrose, 40 ml salt solution, 1 mg
resazurin, 0.5 g L-cysteine-HCl, 4 g ammonium acetate, 0.268 g L-alanine, 0.162
g L-isoleucine, 0.189 g L-valine, 45 mg vitamin B1, 45 mg vitamin B6,
0.2 mg folic acid.
2.3. Batch Fermentation
After autoclaving, serum bottles
containing media were sealed with a sterile gray butyl stopper (20 mm, Fisher
Scientific, Ontario) and crimped with an aluminum seal (20 mm CS/M, Fisher
Scientific, Ontario), and following were made anaerobic using a manifold system.
Batch fermentations were carried out in 160 mL serum bottles with an
overpressure of 3 psi nitrogen gas without agitation. Culture bottles were
inoculated with either a C. beijerinckii NRRL-B593 seed
culture (grown to late log-phase) or a C. beijerinckii NRRL-B593
spore suspension.
2.4. Analyses of fermentation
products
Fermentation products including
butanol, ethanol, acetone, and isopropanol were measured using a Shimadzu
GC-14A Gas Chromatograph (GC) equipped with two connected capillary columns
(DB-624 and DB-5) and a Flame Ionization Detector (FID). GC analyses were
performed under the following conditions: helium, 50 psi; hydrogen, 30 psi;
air, 10 psi; injector temperature, 200 °C; detector
temperature: 250 °C. The temperature program the following:
initial temperature of 35 °C, held for 10 minutes; program
rate: 2 °C/min to 45 °C and 40 °C/min
to 150 °C (hold for 1 min). The sample (1 µl) was directly
applied to the injector for GC analyses. Peaks were identified using external
standards and peak areas were calculated by integration using Peak Simple
software (SRI Instruments, California, USA).
2.5. Hydrogen Detection
The GC-Buck Model 910 GC equipped
with a Supelco 60/80 molecular sieve 5A column and a Thermal Conductivity
Detector (TCD) was used for hydrogen detection. The carrier gas was nitrogen,
pressure was 11 psi (~76 kPa), and flow rate was 16 ml min–1. The
injector, TCD and column temperatures were set to 110, 100 and 60 °C,
respectively. A standard curve was prepared for the determination of hydrogen
produced. Peak Simple was used as the integrating software (SRI Instruments,
USA).
2.6. Preparation of Cell-Free Extract
Frozen cells of C.
beijerinckii NRRL-B593 (1 g) were re-suspended anaerobically in 5 ml
of 6 mM Tris/HCl buffer (pH 7.5) containing 2 mM Sodium Dithionite (SDT), 2 mM
Dithiothreitol (DTT), and 5% (v/v) glycerol. French press was used to lyse
cells in lysing buffer then the supernatant was collected as cell-free extract
after 10 min of centrifugation (10,000 x g).
2.7. Protein and Enzyme Assays
Protein content was determined using
the Bradford method [8] and bovine serum albumin was used as the standard.
Alcohol dehydrogenase activity was measured by monitoring the acetone-dependent
absorbance change of NADPH at 340 nm at 45 °C (ε340 =
6.3 mM-1cm-1, acetone + NADPH + H+ → NADP+ +
H2O + 2-propanol). The assay mixture (2 ml) contained 100 mM EPPS
(pH 8.5) and 30 mM acetone and the reaction started by the addition of enzyme.
One unit of alcohol dehydrogenase activity is defined as 1 μmol of NADPH formed
or oxidized per min.
3. Results and Discussion
C. beijerinckii NRRL-B593 was able to grow on
all types of media prepared (Figure 1). Either dextrose or sucrose (data not
shown) supported the highest growth, followed by mixed media, and lastly
tryptone. Similar growth curve patterns were shown on mixed and tryptone media.
Both mixed and tryptone media supported a much faster growth compared to that
on dextrose (or sucrose) media (Figure 1). The slowest growth on either
dextrose or sucrose may be a result from the supplement with vitamins only.
There are likely other factors present in yeast extract or tryptone that are
required for its fast growth. The result also showed that peptides alone could
only support its growth to a lower cell density. This observation is similar to
what was noted by Mead, which was that saccharolytic clostridia showed little
to no growth on 3% casein hydrolysate [9]. It was also observed in those
studies that there was an increase in growth proportional to the concentration
of glucose added [9].
Growth of C. beijerinckii NRRL-B593
on tryptone and mixed media, respectively, was further extended to determine
whether varying substrate concentrations influenced growth. In samples that
varied dextrose concentrations in mix media (30 g/L - Mix30 media, 60 g/L -
Mix60 media), no significant variation in growth was visible (Figure 2). It is
possible that C. beijerinckii NRRL-B593 cells did not utilize
all the glucose provided, suggesting possible repressive effects by a component
in the media causing cells to reach stationary phase and eventually
sporulating. A similar trend was observed for cells grown on tryptone. In
samples with 15, 20, and 25 g/L tryptone concentrations respectively, C.
beijerinckii NRRL-B593 displayed similar growth patterns (Figure 2).
The results indicate growth is not proportional to the amount of substrate added
under the growth conditions specified.
Solvent production by C.
beijerinckii NRRL-B593 was determined using gas chromatography. In
nearly all the solvents tested (with the exception of ethanol), C.
beijerinckii NRRL-B593 grown in mixed media resulted in the highest
solvent production overall (Figure 3). Ethanol was the only solvent produced in
all three media conditions (Figure 3b), while butanol (Figure 3a), acetone
(Figure 3c), and isopropanol (Figure 3d) were only produced in mixed and
sucrose media, indicating that tryptone could not be used to support the
production of butanol, acetone and isopropanol. Other peptide substrates
including beef extract, beef peptone, yeast extract, wheat peptone, and casein
were tested, however, results show that they also could not support production
of butanol, acetone and isopropanol. Solvent production in C.
beijerinckii NRRL-B593 grown in a “peptone-yeast extract-glucose”
medium has been previously reported to be 8 mM and 61.7 mM of isopropanol and
butanol, respectively [4]. A more recent study reported butanol and
isopropanol production in C. beijerinckii NRRL-B593 batch
culture grown in glucose to be approximately 30 mM and 60 mM, respectively
[10].
Hydrogen production by C.
beijerinckii NRRL-B593 grown on various substrates were measured
(Figure 4). Growth on dextrose media resulted in the highest hydrogen
production while growth on all other peptide sources tested resulted in similar
hydrogen concentrations (Figure 4). Hydrogen production in cultures grown on the
basal medium and peptide supplemented media were similar. Out of the different
peptides tested, wheat peptone supplemented media had cultures that produced
the most hydrogen (2.4 ± 0.14 mM; Figure 4). This data correlates to the
solvent production data obtained from GC analyses (Figure 3), where little to
no solvent production was observed for culture grown on “peptide only” media.
For culture grown on peptides, hydrogen production was lower compared to
culture grown solely on carbohydrates (Figure 4), which is expected as it has
been previously reported that the major source of hydrogen produced by
fermentation is from carbohydrates [11]. Butanol production detected on samples
grown on dextrose had similar concentrations as reported in literature (74 mM by
[12]; 93 mM by [13]). Isopropanol on dextrose, however, had lower
concentrations than reported in literature (50 mM by [12]; 53 mM by [13]).
Alcohol Dehydrogenase (ADH)
activities in C. beijerinckii NRRL-B593 grown on three
different media were determined (Table 1). Cells grown on mixed media had the
highest specific activity (0.209 ± 0.052 U/mg), followed by tryptone, then
glucose media (0.150 ± 0.018 U/mg and 0.109 ± 0.003 U/mg, respectively). The
specific activity measurements taken from C. beijerinckii NRRL-B593
cells grown in carbohydrate media and peptide media suggests that metabolic
processes may be dependent on substrate availability. Specific
activity is higher for cells grown in peptide media compared to glucose
media, further suggesting that the expression of solvent-producing genes may be
partially dependent on the carbon source available for C.
beijerinckii NRRL-B593 [14].
It has been
speculated that only one enzyme is present that catalyzes the production of
butanol and isopropanol in C. beijerinckii NRRL-B593
[14]. C. acetobutylicum ATCC 824, contrastingly, is known to
produce the same solvents with the exception of isopropanol (it instead
produces acetone) [4]. ADH activity of C. beijerinckii NRRL-B593
grown on mixed media resulted in the highest specific activity, followed by
tryptone, then glucose with the lowest activity (Table 1). Similar results were
obtained for both non-spore culture and spore cultures of C.
beijerinckii NRRL-B593, however, only non-spore culture data is
presented. With supporting data from the solvent production (Figure 3) and
enzymatic assay data (Table 1), it is evident that C. beijerinckii NRRL-B593
is unable to utilize peptide sources to produce butanol, isopropanol, and
acetone. Although this organism can utilize peptides to support its growth
(Figure 1), peptides that are metabolized into keto-acids (such as pyruvate)
cannot be utilized to support the production of solvents (other than ethanol).
Ethanol can still be produced by C. beijerinckii NRRL-B593 as pyruvate
(a product of peptide metabolism) is a precursor to its production, which is
catalyzed by a three-enzyme pathway [15].
Although the pathway for butanol
production from the fermentation of peptides has not yet been elucidated
in C. beijercinkii NRRL-B593, there are pathways known to
produce butanol in other microorganisms. In clostridia, the butanol production
pathway and key enzymes involved have been extensively studied in C.
acetobutylicum ATCC 824, an Acetone-Butanol-Ethanol (ABE) producing
bacterium that grows on sugars [16]. It is thought that C. beijercinkii NRRL-B593
may share such a similar pathway [17]. In C. acetobutylicum ATCC
824, fermentation of sugars is biphasic – with the first phase being
acidogenesis and the second phase being solventogenesis [18]. Its solvent
production is pH-dependent where a lower pH resulted from an accumulation of
organic acids (ie. butyric and acetic acid) signals the pathway to initiate the
solventogenesis phase and begin converting these acids to their corresponding
alcohols (ie. butanol and acetone) [18]. Contrastingly, reports have shown
solvent production by C. beijerinckii NRRL-B593 to be
independent of pH changes [19,20], suggesting a possible rerouting of carbon
and electron flow in the conversion of acid to solvent.
Acetoacetyl-CoA:butyrate:CoA transferase, a key enzyme in C.
acetobutylicum ATCC 824, allows for the production of acetone and the
conversion of acetate and butyrate to ethanol and butanol, respectively [7].
The complete genome of C. beijerinckii NRRL-B593 has yet to be
sequenced, however, its pH-independent solvent production suggests that its
butyrate and butanol-acetone fermentation pathway may be regulated differently.
In non-clostridial species,
alternative butanol production pathways also exist, such as the two-step
amino-acid pathway present in S. cerevisiae which involves the
catabolism of amino acids [21]. In this pathway, amino acids are converted into
a keto acid (ie. 2-ketovalerate), which is then further decarboxylated into its
corresponding aldehyde (ie. butanal) and ultimately its corresponding alcohol
(ie. butanol) [21]. A similar keto-acid pathway was successfully engineered
into E. coli by Atsumi, et al. [22] for the production of
long-chained alcohols (ie. butanol). It is unlikely that any of such butanol
production pathways of non-clostridial species would be present in C.
beijerinckii NRRL-B593.
4. Conclusion
The
lack of solvent production by C. beijerinckii NRRL-B593 grown
on peptides sources indicates the inability for the bacterium to utilize these
sources for significant growth, and furthermore solvent and hydrogen
production. It appears that it is only favourable for C. beijerinckii NRRL-B593
to utilize peptide sources for the production of ethanol explaining the inability
of butanol, acetone, and isopropanol production. This study provides clear data
in helping understand the metabolism and growth of C.
beijerinckii NRRL-B593 on peptides. Further investigation is required
to elucidate its regulatory metabolic mechanism when grown on different
substrates.
5. Acknowledgements
This work was supported by
research grants from the Natural Sciences and Engineering Research Council
(Canada) and the Canada Foundation for Innovation to K.M.
Figure 1: Growth of C. beijerinckii NRRL-B593 on three different media types (Dextrose, Tryptone, and Mix, respectively). Growth was monitored and measured using a Genesys 10UV Spectrophotometer at 600 nm. Similar results were obtained for both non-spore culture and spore cultures of C. beijerinckii NRRL-B593 however, only non-spore culture data is presented here.
Figure 2: Growth of C. beijerinckii NRRL-B593
on two different media (Mix and Tryptone, respectively) at various
concentrations. Growth was monitored and measured using a Genesys
10UV Spectrophotometer at 600 nm. Both dextrose and tryptone media sources
supported growth of C. beijerinckii
NRRL-B593, with an increase in concentration resulting in an increase in
growth. Both concentrations of mixed media (tryptone, sucrose, yeast extract)
(30, 60 g/L) tested resulted in higher growth as compared to all concentrations
of tryptone (15, 20, 25 g/L).
Figure 3: Production of butanol, acetone, isopropanol, and ethanol by C. beijerinckii NRRL-B593 on three
different media types (Dextrose, Tryptone, and Mix, respectively). Production of (a) 1-butanol, (b) ethanol, (c)
acetone, (d) isopropanol (mM) detected using a Shimadzu GC 14-a equipped with
an FID. Similar results were obtained for both non-spore culture
and spore cultures of C. beijerinckii
NRRL-B593, however, only non-spore culture data is presented.
Figure 4: Hydrogen production by C.
beijerinckii NRRL-B593 grown on various substrates. Error bars are in ± SD.
Cells
grown on media |
Specific
Activity (U/mg)* |
Mixed |
0.209 ± 0.052 |
Tryptone |
0.150 ± 0.018 |
Sucrose |
0.109 ± 0.003 |
*(value ± SD) |
Table 1: Alcohol dehydrogenase activity of C. beijerinckii NRRL-B593 grown on three
different media types (mixed, tryptone, and sucrose, respectively).
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