The document analyzes the carbohydrate and redox metabolism of the thermophilic anaerobe Caldicellulosiruptor bescii. It finds that C. bescii utilizes the non-oxidative pentose phosphate pathway but lacks key enzymes for the oxidative branch. Enzyme activity assays showed low or no activity for glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, supporting that C. bescii does not use the oxidative pentose phosphate pathway to regenerate NADPH. A better understanding of C. bescii metabolism is needed to engineer it for biofuel production.

1. 1
Analysis of Carbohydrate and Redox Metabolism in the
Thermophilic Anaerobe Caldicellulosiruptor bescii:
Utilization of the Non-Oxidative Pentose Phosphate
Pathway
Ryan Sanders, Amanda Rhaesa, and Gerrit Schut
University of Georgia, Department of Biochemistry and Molecular Biology
BCMB 4970L
Michael W. W. Adams
27 April, 2015

2. 2
SUMMARY
Thermophiles are promising candidates for bioprocessing because at the high growth
temperatures risk of contamination is minimized, rate of metabolism is high, and part of plant
biomass degrades spontaneously (3). One such cellulolytic thermophile, Caldicellulosiruptor
bescii, has a high optimal growth temperature of 80°C and offers relevant advantages for
application in bioindustry. However, there are many components of their metabolism that are not
well understood and must be studied further in order to potentially utilize this organism in
biofuel production.
Studies have elucidated metabolic pathways in C. bescii that could be manipulated to
produce biofuels (3). C. bescii has been shown to grow efficiently on high loads of crystalline
cellulose and unpretreated plant biomass, further illustrating its applicability in bioindustry (1).
Another thermophile, Thermotoga maritima, is well studied and appears to have very similar
metabolism to C. bescii. We compared expression data of C. bescii enzymes with those
annotated in T. maritima. Next, Subsequent growth experiments carried out on xylose, gluconate,
and cellobiose substrates revealed differential utilization of the two branches of the Pentose
Phosphate Pathway (PPP) to regenerate redox substrates and interconvert hexose and pentose
sugars.
This study examines the activity of two oxidative enzymes of the PPP through UV/Vis
spectrophotometry - Glucose-6-Phosphate Dehydrogenase (G6PDH) and 6-Phosphogluconate
Dehydrogenase (6PGDH). Enzyme activity assays of C. bescii extracts supports the hypothesis
that C. bescii lacks activity of integral enzymes of the oxidative branch of the PPP and therefore
does not utilize that branch to regenerate NADPH. Further growth studies and genomic analysis

3. 3
of C. bescii to ferment different carbon substrates is needed to construct a better understanding of
the metabolic pathways that can be engineered to produce biofuels in this organism.
INTRODUCTION
Environments with extreme physiological conditions, such as those in hot springs or near
hydrothermal vents, are not suitable for many organisms. However, there exists a number of
archea, fungi, and bacteria that are capable of thriving in such high temperature environments
known as ‘thermophiles’. Previous research has elucidated the integral role these organisms play
in ecology and the evolution of global ecosystems, as thermophiles are believed to be among the
oldest organisms on the planet. Over time, thermophiles have evolved to metabolize a wide range
of carbon sources with novel pathways that co-utilize pentose and hexose sugars (8). These
characteristic pathways, among other temperature-dependent advantages, make thermophiles
promising candidates for use in bioprocessing. For example, operating bioprocesses at the high
temperatures required by thermophiles (≥50°C) provides industry-relevant advantages that most
mesophilic organisms (i.e. optimal growth temperature of 24-40°C) cannot. Operating at higher
temperatures increases the rate of metabolic activity as compared to lower temperatures, reduces
the risk of contamination by other organisms, and partially degrades organic substrates to
promote further degradation by the organism’s metabolic machinery (14). Because of these
advantages, many thermophiles are promising candidates for biofuel production.
One anaerobic thermophile, Caldicellulosiruptor bescii, grows at an optimal temperature
of 80°C and utilizes distinct metabolic pathways to degrade plant biomass (e.g. cellulose,
hemicellulose, lignin) and ferment the released carbohydrates into biofuel products (1). C. bescii
ferments carbohydrates derived from plant biomass and therefore is a good candidate for a
process known as Consolidated Bioprocessing (CBP). Traditional CBP requires a costly

4. 4
pretreatment of plant biomass to prevent recalcitrance, but C. bescii has the enzymatic activity to
degrade plant cell walls without pretreatment (1). Currently, this organism does not directly
make a biofuel, but has proven to be suitable for potential bioengineering of an ethanol pathway.
However, there exist many unknown elements in C. bescii metabolism including both enzyme
and redox specificities. In order to successfully implement a metabolic pathway for biofuel
production in C. bescii, a better understanding of its redox metabolism is necessary.
In order to further understand the metabolic redox networks present in C. bescii, other
organisms utilizing similar pathways should be examined. Previous genome analyses of the
thermophilic organism, Thermotoga maritima, have shown its metabolic similarities to C. bescii
and therefore, illustrate the organism as a good model for examining C. bescii metabolism (2).
Side-by side genomic and bioinformatic analysis of these two organisms may identify suitable
potential targets for metabolic engineering strategies for biofuel optimization. Specifically, both
organisms lack the presence of acetaldehyde dehydrogenase and bifunctional
alcohol/acetaldehyde dehydrogenase activity and therefore must utilize other enzymes present in
the Pentose Phosphate Pathway (PPP) for regenerating reducing equivalents of NADPH and
pentose/hexose interconversions (2, 12). Further examination of PPP enzyme expression in both
organisms reveals distinct utilization of the oxidative or non-oxidative branches of the pathway
and results in differential gene annotation in each individual organism. Both organisms have
been shown to utilize the non-oxidative branch to metabolize glycolytic intermediates for the
synthesis of nucleic and amino acids (11), however their implementation of the oxidative branch
to maintain redox balance through NAD(P)H production and recycling is not as well understood.
Two integral enzymes catalyzing key redox recycling steps of the oxidative PPP include
Glucose-6-Phosphate Dehydrogenase (G6PDH) and 6-Phosphogluconate Dehydrogenase

5. 5
(6PGDH). G6PDH is an NADP+ dependent oxidoreductase catalyzing the rate limiting
production of NADPH and yielding 6-phosphogluconolactone in the first step of the oxidative
PPP (13). 6PGDH is an oxidative carboxylase that catalyzes the decarboxylating reduction of 6-
phosphogluconate into ribulose 5-phosphate in the presence of NADP producing NADPH and
CO2. Together, these enzymes play a role in NADP+ to NADPH recycling and redox balance
and thus have been studied in depth as biomarkers for oxidative PPP utilization (13). Comparing
the activities on these enzymes in both organisms will allow an accurate metabolic pathway of C.
bescii to be constructed and engineered to produce biofuel in high yields.
In this study, we aim to present differential enzyme expression and activity profiles of
two thermophilic organisms to better understand which branches of the PPP are crucial for
overall C. bescii metabolism. The absence of annotated genes integral to the oxidative PPP and
low cell growth on substrates feeding the oxidative branch suggests that C. bescii does not utilize
this branch. Additionally, measurement of enzyme activities showed low to no activity for
substrates of this pathway. Thus, C. bescii must utilize an alternate NADPH generation system,
such as a bifurcating transhydrogenase NfnAB (12), and its glycolytic intermediates feed the
non-oxidative branch of the Pentose Phosphate Pathway.
EXPERIMENTAL METHODS
Caldicellulosiruptor bescii Growth
C. bescii strain DSM 6725 was obtained from the DSMZ culture library(Braunschweig,
Germany). It was grown in modified DSMZ 516 medium as published (6) with the following
modification: 1𝜇M sodium tungstate and 1 𝜇M ammonium molybdate were added. The final pH
was adjusted to 7.2. The medium was then filter-sterilized using a 0.22 mm pore filter. All
substrates were used at a final concentration of 0.5 % (w/v) and were added directly to sterilized

6. 6
culture bottles followed by the addition of the filter-sterilized medium. Carbon sources for this
study included cellobiose, xylose, and gluconate purchased from Sigma Aldrich. To investigate
substrate utilization, cultures were grown at 75 °C. Growth was determined after 24 and 48h by
measuring cell counts (phase-contrast microscope with a Petroff-Hausser counting chamber) and
total cell protein (Bradford assay).
Thermotoga maritima Growth
Cultures of T. maritima were prepared in complex medium containing 1× base salts, 1×
trace minerals, 10 μM sodium tungstate, and 0.25 mg/ml resazurin, with added cysteine at 0.5
g/liter, sodium sulfide at 0.5 g/liter, sodium bicarbonate at 1 g/liter, and 1 mM sodium phosphate
buffer (pH 6.8), and for complex medium, containing combinations of 0.05% (wt/vol) yeast
extract, 0.5% (wt/vol) carbon substrate. The 200× vitamin stock solution contained (per liter) 10
mg each of niacin, pantothenate, lipoic acid, p-aminobenzoic acid, thiamine (B1), riboflavin (B2),
pyridoxine (B6), and cobalamin (B12) and 4 mg each of biotin and folic acid (7). The final pH was
adjusted to 6.8 using 1M HCl or NaOH. The medium was then filter-sterilized using a 0.22 mm
pore filter. All substrates were used at a final concentration of 0.5 % (w/v) and were added
directly to sterilized culture bottles followed by the addition of the filter-sterilized medium. To
investigate substrate utilization, cultures were grown at 75 °C. Growth was determined after 24
and 48h by measuring cell counts (phase-contrast microscope with a Petroff-Hausser counting
chamber) and total cell protein (Bradford assay).
E. coli Growth

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The E. coli extracts used for positive controls were cultured in LB media containing
(grams per liter): 5g yeast extract, 10g casein hydrolysate, and 10g NaCl. Substrates were
sterilized separately and added at a level of 0.02 M (4). The cultures were incubated at 37 °C and
allowed to grow aerobically for 24 h.
Extract Preparation
Cultures were collected after 24hrs of shaking in the incubator at 75°C and harvested by
centrifugation at 6,000 X g for 10min and washed twice with 100mM phosphate buffer, (pH 7.5)
C. bescii and T. maritima extracts were lysed anaerobically by sonication in a chamber with 5%
H2 and 95% Ar. E. coli extracts were lysed aerobically by sonication (4).
Protein Concentration Calculations
1 mL samples from each extract were taken from each time point and centrifuged, the
pellet was then taken for analysis. The pellet was resuspended in distilled water to give a 10x
concentration of cells. Cell lysis was performed by sonication. Protein was determined for all
time points and for concentrated extracellular protein by Bradford assays using the 96 well
plates.
RNAseqData
RNA sequencing data was performed previously in collaboration with Steve Brown at
Oak Ridge National Laboratory. For this study, average reads per gene for C. bescii grown on
xylose was used to establish expression levels for genes of interest. The overall average for all
genes grown on xylose was 454 and was used to establish expression level of other genes. Low
expression include total read averages that fell in the range of 0-125, average expression fell in
the range of 125-800 reads, and high expression was represented by average reads exceeding
800.

8. 8
Enzyme Activity Assays
Enzyme activity for G6PDH and 6PGDH was measured by examining the increase in
absorbance at 340 nm on a Cary WinUV/Vis spectrophotometer, equipped with a temperature
controller. The reaction mixture was allowed to reach the desired temperature, and the reaction
was then initiated by injecting the substrate. The standard assay (total volume, 2.1 ml) contained
100 mM phosphate buffer (pH 7.5), 1.0 mM substrate, 2.0 mM NAD(P), pH 7.0, and an
appropriate amount of cell extract (5). The enzyme activity was determined from the initial
velocity of the reaction. Glucose-6-Phosphate (G6P), 6-Phosphogluconate (6PG), NAD, and
NADP were confirmed as being stable at temperatures up to 85°C for at least the time period of
the assay by variable temperature NMR studies (5). Appropriate amounts of E. coli extract were
added to each assay for positive controls and to determine assay efficacy.
RESULTS
In order to visualize an accurate map of the C. bescii PPP, we integrated bioinformatic
data (Table 1) with metabolic pathways generated by the KEGG database. This allowed us to
determine the presence and activities of certain annotated genes in both organisms and construct
an accurate PPP for C. bescii (Figure 1). After consulting the KEGG-generated pentose
phosphate pathways for both C. bescii and T. maritima, it is apparent that C. bescii lacks an
annotated G6PDH gene in the oxidative branch but does contain a 6PGDH enzyme of the same
branch (Athe_1982). The expression of 6PGDH in C. bescii but not G6PDH begs the question if
C. bescii contains a novel gene for the G6PDH enzyme, or that C. bescii does not contain the
enzyme capable to utilize that part of the oxidative branch. T. maritima, on the other hand, has an

9. 9
annotated G6PDH enzyme (TM1155) and a highly expressed 6PGDH (TM0438) and therefore
can be used as a reference to determine enzyme activity in C. bescii when grown on the same
carbon substrate.
In order to support the known utilization of non-oxidative branch in C. bescii,
bioinformatic data of C. bescii grown on xylose were examined as xylose feeds the non-
oxidative branch of the PPP in C. bescii (13, Figure 1) and can be used to visualize gene
expression relating to this branch (Table 1). The enzyme catalyzing the first step of the oxidative
branch (G6PDH) is not currently annotated in C. bescii and when grown on xylose, genes
encoding subsequent enzymes of the oxidative branch of the PPP (6PGDH) are expressed at low
levels. Additionally, genes encoding enzymes resident to the non-oxidative branch (XK,
transaldolase, transketolase) are expressed at average or high levels (Figure 1,Table 1).
To further determine if C. bescii utilizes the oxidative branch of the PPP, a substrate
known to feed that branch in T. maritima was utilized in growth experiments and to subsequently
generate cell extracts for enzyme activity assays. This substrate, gluconate, feeds the oxidative
branch of the PPP (13, Figure 1) and thus was used in conducting growth experiments (Figure 2).
In order to establish a baseline to determine effective growth on other substrates C. bescii was
grown on media containing only yeast extract (YE) as the carbon substrate. T. maritima was
unable to grow in media lacking carbon substrates other than YE. Cellobiose was used as a
substrate to illustrate. After 48 hrs the culture of C. bescii grown only with YE grew to a cell
density of 5.12x107 cell/ml. When grown for 48 hrs on media supplemented with 5g/L cellobiose
substrate, C. bescii is shown to achieve a final cell density of 1.03x108 cell/ml. However, C.
bescii shows poor growth in media containing 5g/L gluconate as it achieved cell density of
2.8x107 cell/ml after 48 hrs. T. maritima, is able to utilize the same concentration of cellobiose to

10. 10
reach a cell density of 6.8x108 after 48 hrs and reaches a cell density of 1.5x108 when grown in
the presence gluconate (Figure 2).
The cell extracts were prepared for enzyme activity assays to examine G6PDH and
6PGDH redox activity. G6PDH activity assays carried out with the C. bescii and T. maritima
extracts and E. coli extracts as a positive control. Assays carried out with NAD as the redox
substrate yielded little enzymatic activity (Figure 2). Assays containing T. maritima extracts and
NADP as a redox substrate revealed a specific activity of 0.02 U/mg and assays containing C.
bescii extracts exhibited no G6PDH enzyme activity (Table 2). The 6PGDH activity assay
carried out with T. maritima extract exhibited a specific activity of 0.02 U/mg and assays with C.
bescii extracts revealed no specific activity when NADP was used as the redox substrate In order
to establish a positive control for the assays, the enzyme activities were assayed in E. coli. We
first tested the activity in the E. coli extracts alone and recorded activities of 0.36 U/mg with
NADP and G6P as substrates in the first assay and 0.34 U/mg with NADP and 6PG substrates in
the second assay. E. coli was added to cuvettes exhibiting low activity after decreasing the
reaction temperature to 37°C as a positive control to verify the low or non-detectable activity.
The C. bescii assay mixture with NADP and G6P exhibiting no specific activity was
supplemented with E. coli extract and a resulting specific activity of 0.36 U/mg was recorded
(Figure 3, Table 2). . Additionally, when E. coli extracts were added to the assay with NADP and
6PG, an increase in specific activity to 0.34 U/mg was recorded (Figure 4, Table 2).
DISCUSSION
The relative C. bescii gene expression of PPP enzymes in both branches supports the data
presented by the growth experiments and enzyme activity assays. When grown on xylose, C.

11. 11
bescii enzymes feeding the non-oxidative branch of the PPP are expressed at average or above
average levels in contrast to lower expression of the oxidative branch enzymes. This suggests C.
bescii’s inability to utilize the oxidative branch. Additionally, poor growth of C. bescii, relative
to T. maritima on gluconate gives further insight into which branch of the PPP C. bescii utilizes
to ferment carbon substrates into potential biofuel products. In T. maritima, gluconate feeds the
oxidative branch and results in higher organismal growth (Figure 2). C. bescii grown on the same
carbon substrate however, revealed lower growth potentially caused by a decrease in enzyme
activity. C. bescii’s inability to grow well on gluconate supports the lack of key enzymes innate
to the oxidative branch of the PPP in C. bescii and therefore confirms its inability to utilize the
oxidative branch of the PPP to recycle redox substrates.
In order to further support the proposed C. bescii non utilization of the oxidative branch
of the PPP, more positive controls for assays and extract viability are needed as little activity was
recorded in enzymes that were previously annotated to be active in both organisms. 6PGDH
activity is annotated in C. bescii and T. maritima (KEGG) and the fact that we were unable to
measure similar activity in both organisms in our investigation suggests either a misannotation in
the database or an assay that is in need of optimization. One suggestion for future investigation
would be to grow T. maritima on gluconate and assay the 6PGDH enzyme activity with those
extracts as gluconate incorporation into the oxidative pathway occurs immediately upstream of
the 6PGDH enzyme (Figure 1). Additionally, a phosphate release assay in C. bescii could be
carried out to measure the ATP-dependent activity of xylulokinase - which produces xylulose-5-
phosphate feeding the non-oxidative branch. If this assay produces high activity, it can be further
proposed that this branch is the primary pathway used to interconvert hexose and pentose sugars.
This work suggests that C. bescii may contain the presence of a bifurcating transhydrogenase,

12. 12
similar to NfnAB in T. maritima (12), is the main pathway for C. bescii to regenerate NADPH.
These areas for future study should further confirm the utilization of another pathway besides the
oxidative PPP for redox recycling in C. bescii and will allow for novel metabolic engineering of
this organism for future application in biofuel production.
Thermophiles are useful organisms in bioindustry because of their metabolic ability and a
better understanding of their metabolism can lead to higher yields of biofuel production. Two
thermophiles, Caldicellulosiruptor bescii and Thermotoga maritima, have related metabolic
capabilities and can be compared in order to gain more insight into the redox and carbohydrate
metabolism in these high-temperature dwelling organisms. C. bescii’s ability to grow on plant
biomass whose components feed the Pentose Phosphate Pathway (PPP) suggests that it utilizes
this pathway for recycling of NADPH and pentose/hexose sugar interconversions. When grown
on xylose, bioinformatic data illustrates the higher activity of C. bescii enzymes comprising the
non-oxidative branch of the PPP compared to enzymes in the oxidative branch. Gluconate feeds
the oxidative branch of the PPP in T. maritima and was used as a growth substrate to analyze
activity of C. bescii enzymes in this branch. Poor growth of C. bescii on this substrate coupled
with little to no measured activity of the oxidative enzymes G6PDH and 6PGDH, reveals that C.
bescii must be using another pathway to regenerate NADPH. The presence of a bifurcating
hydrogenase in C. bescii is one proposal for how it accomplishes redox recycling without the
oxidative PPP and is an area to be investigated in the future. Continual investigation of redox
metabolism in C. bescii will allow the bioengineering of high yield biofuel pathways in this
thermophilic organism.

13. 13
FIGURES AND TABLES
Figure 1. Pentose phosphate pathway in C. bescii (generated from KEGG database)with relative enzyme expression levels included. C. bescii Xylose and T.
maritima gluconate utilization pathways are shown with red and blue arrows respectively. C. bescii enzymes are annotated by Athe_ gene numbers and T.
maritima with TM_.

14. 14
Table 1. RNAseq expression data of key enzymes of C. bescii PPP grown on xylose.
Figure 2. Growth profiles of C. bescii and T. maritima over 48 hrs. C. bescii grown in media containing 5g/L
gluconate (Blue), 5g/L cellobiose (Red), and media with only Yeast Extract as the sole carbon substrate (YE, dotted
Blue). T. Maritima grown in medias containing 5g/L gluconate (Green) and 5g/L cellobiose (Purple).
1.00E+06
1.00E+07
1.00E+08
1.00E+09
0 10 20 30 40 50 60
CellDensity(cells/ml)
Time (hrs)
Growth of Organisms on 5g/L Substrates
Cb gluconate
Cb Cellobiose
Tm gluconate
Tm Cellobiose
Cb only YE
Athe # Enzyme
Average
Reads
Expression
Level PPP Branch
Athe_0567 Xylulokinase 239 Average Non-Ox
Athe_0603 Xylose Isomerase 2590 High Non-Ox
Athe_0632
Ribulose 5-Phosphate
isomerase 260 Average Non-Ox
Athe_1047
Ribulose-phosphate 3-
epimerase 631 Average Non-Ox
Athe_1489 Putative transaldolase 3879 High Non-Ox
Athe_2059 Transketolase 1025 High Non-Ox
Athe_1982
6-phos6phogluconate
6PGDH 301 Average Ox

15. 15
Specific Activity (U/mg)
Glucose-6-Phosphate
(1mM)
6-Phosphogluconate
(1mM)
C. bescii
NAD 0.01 ±0.01 0.00 ±0.00
NADP 0.01±0.01 0.00 ±0.00
T.
maritima
NAD 0.00 ±0.00 0.00 ±0.00
NADP 0.04 ±0.01 0.07 ±0.02
E. coli
NAD 0.00 ±0.00 0.00 ±0.00
NADP 0.36 ±0.02 0.34 ±0.04
Table 2. Specific enzyme activities (w/ standard deviations) of C. bescii, T. maritima, and E. coli whole cell extracts
assayed with 1mM carbon substrates G6P and 6PG and 2mM redox substrates NADand NADP.
Figure 3. Specific activity (U/mg) of G6PDH in T. maritima, C. bescii, and E. coli grown in media with gluconate
as carbon source. Assays were carried out with G6P as the carbon substrate and NADP as redox substrate.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
G6P
SpecificActivity(U/mg)
Substrate (1mM)
Enzyme Activity w/ NADP Redox Substrate
T. maritima
C. bescii
E. coli

16. 16
Figure 4. Specific activity (U/mg) of 6PGDH in T. maritima, C. bescii, and E. coli grown in media with gluconate
as carbon source. Assays were carried out with 6PG as the carbon substrate and NADP as redox substrate.
REFERENCES
1. Basen, M., Rhaesa, A. M., Kataeva, I., Prybol, C. J., Scott, I. M., Poole, F. L., & Adams,
M. W. (2014). Degradation of high loads of crystalline cellulose and of unpretreated plant
biomass by the thermophilic bacterium Caldicellulosiruptor bescii. Bioresource
technology, 152, 384-392.
2. Carere, C. R., Rydzak, T., Verbeke, T. J., Cicek, N., Levin, D. B., & Sparling, R. (2012).
Linking genome content to biofuel production yields: a meta-analysis of major catabolic
pathways among select H2 and ethanol-producing bacteria. BMC microbiology, 12(1),
295.
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
6PG
SpecificActivity(U/mg)
Substrate (1mM)
Enzyme Activity w/ NADP Redox Substrate
T. maritima
C. bescii
E. coli

17. 17
3. Cha M, Chung D, Elkins JG, Guss AM, Westpheling J. Metabolic engineering
Caldicellulosiruptor bescii yields increased hydrogen production from lignocellulosic
biomass. Biotechnology for Biofuels. 2013. 6:85.
4. Eisenberg, R. C., & Dobrogosz, W. J. (1967). Gluconate metabolism in Escherichia
coli. Journal of bacteriology, 93(3), 941-949.
5. Iyer, R. B., Wang, J., & Bachas, L. G. (2002). Cloning, expression, and characterization
of the gsdA gene encoding thermophilic glucose-6-phosphate dehydrogenase from
Aquifex aeolicus. Extremophiles, 6(4), 283-289.
6. Kataeva I.A., Yang S.J., Dam P., Poole F.L., Yin Y., Zhou F.F., Chou W.C., Xu Y.,
Goodwin L., Sims D.R., et al. Genome sequence of the anaerobic, thermophilic, and
cellulolytic bacterium “Anaerocellum thermophilum” DSM 6725. J. Bacteriol. 2009.
191:3760–3761.
7. Lipscomb GL, Stirrett K, Schut GJ, et al. Natural Competence in the Hyperthermophilic
Archaeon Pyrococcus furiosus Facilitates Genetic Manipulation: Construction of
Markerless Deletions of Genes Encoding the Two Cytoplasmic Hydrogenases . Applied
and Environmental Microbiology. 2011. 77(7):2232-2238.
8. Lu Lin, Jian Xu. Dissecting and engineering metabolic and regulatory networks of
thermophilic bacteria for biofuel production. Biotechnology Advances. 2013. 31:827-
837.
9. Lynd, L. R., W. H. van Zyl, et al. (2005). "Consolidated bioprocessing of cellulosic
biomass: an update." Curr Opin Biotechnol. 16(5): 577-83.

18. 18
10. Pickl, A., Schönheit, P. The oxidative pentose phosphate pathway in the haloarchaeon
Haloferax volcanii involves a novel type of glucose-6-phosphate dehydrogenase – The
archaeal Zwischenferment. FEBS Letters. 2015.
11. Rydzak, Thomas, et al. "Proteomic analysis of Clostridium thermocellum core
metabolism: relative protein expression profiles and growth phase-dependent changes in
protein expression." BMC microbiology 12.1 (2012): 214.
12. Schut, G. J., & Adams, M. W. (2009). The iron-hydrogenase of Thermotoga maritima
utilizes ferredoxin and NADH synergistically: a new perspective on anaerobic hydrogen
production. Journal of bacteriology, 191(13), 4451-4457.
13. Stincone, Anna, et al. "The return of metabolism: biochemistry and physiology of the
pentose phosphate pathway." Biological Reviews (2014).
14. Taylor, M. P., Eley, K. L., Martin, S., Tuffin, M. I., Burton, S. G., & Cowan, D. A.
(2009). Thermophilic ethanologenesis: future prospects for second-generation bioethanol
production. Trends in biotechnology, 27(7), 398-405.
15. Yang, S, et al. Classification of ‘Anaerocellum thermophilum’ strain DSM 6725 as
Caldicellulosiruptor bescii. Int J Syst Evol Microbiol. 2010. 60: 2011-2015.