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

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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

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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

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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

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(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 1μM 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

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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.

RNAseq Data

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.

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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

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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

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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.

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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,

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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.

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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_.

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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).

Specific Activity (U/mg)

Glucose-6-Phosphate(1mM)

6-Phosphogluconate(1mM)

C. besciiNAD 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. coliNAD 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 NAD and NADP.

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Athe # EnzymeAverage Reads

Expression Level PPP Branch

Athe_0567 Xylulokinase 239 Average Non-OxAthe_0603 Xylose Isomerase 2590 High Non-Ox

Athe_0632Ribulose 5-Phosphate

isomerase 260 Average Non-Ox

Athe_1047Ribulose-phosphate 3-

epimerase 631 Average Non-OxAthe_1489 Putative transaldolase 3879 High Non-OxAthe_2059 Transketolase 1025 High Non-Ox

Athe_19826-phos6phogluconate

6PGDH 301 Average Ox

_x0003_G6P0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Enzyme Activity w/ NADP Redox Substrate

T. maritimaC. besciiE. coli

Substrate (1mM)

Spec

ific

Act

ivity

(U/m

g)

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.

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6PG0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

Enzyme Activity w/ NADP Redox Substrate

T. maritimaC. besciiE. coli

Substrate (1mM)

Spec

ific

Act

ivity

(U/m

g)

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.

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