Fermentation of mixed substrates by Clostridium pasteurianum and its physiological, metabolic and proteomic characterizations
© The Author(s) 2016
Received: 3 February 2016
Accepted: 27 May 2016
Published: 21 June 2016
Clostridium pasteurianum is becoming increasingly attractive for the production of chemicals and fuels such as n-butanol and 1,3-propanediol. Previously we have shown that dual substrate fermentation using glucose and glycerol enhanced the cell growth and butanol production significantly. Although C. pasteurianum can grow efficiently with either glucose or glycerol alone, under certain conditions, glucose limitation in the mixed substrate fermentation leads to growth cessation. To understand this phenomenon and for process optimization, fermentation experiments were performed in the presence of excess glycerol but with varied initial concentrations of glucose which were followed by physiological, metabolic and proteomic analyses.
Physiological characterization showed that the observed cease of growth is not due to the toxicity of n-butanol. Furthermore, the growth can be resumed by addition of glucose or the intermediate oxaloacetate. Proteomic analysis shed more light on the system-level regulation of many proteins directly or indirectly associated with this phenomenon. Surprisingly, it is found that the specific growth rate of C. pasteurianum in the different growth phases (e.g. before and after glucose limitation) correlated well with the expression level of the ATP dependent pyruvate carboxylase and with the expression level of biotin synthase which provides the cofactor biotin for the formation of oxaloacetate from pyruvate. Bioenergetic analysis based on the formation rates of metabolites further show that ATP supply is not a limiting factor for the pyruvate carboxylation to oxaloacetate.
The results of physiological and proteomic analyses clearly show that the anaplerotic synthesis of oxaloacetate plays a key role in determining the growth behaviour of C. pasteurianum in fermentations with mixed substrates of glucose and glycerol. This study provides interesting targets for metabolic engineering of this emerging industrial microorganism.
Biological production of chemicals and fuel from renewable resources is an attractive approach to a sustainable future industry. In particular, n-butanol has received increased attention as a potential fuel substitute and an important chemical feedstock. Previously, biofuels have been predominantly produced from crop biomass, resulting in competition with limited food resources and land. Therefore, bacterial fermentation of non-food biomass has been considered a possible answer to this problem . Recent interest in the fermentative route of n-butanol production has led to a large number of studies on the metabolism and genetics of solventogenic clostridia, and on the improvement of fermentation and product recovery technologies [1–6]. Currently, there are still three major hurdles for fermentative n-butanol production to compete with the petroleum-based one [7, 8]. These include (a) high cost of substrates, (b) low final product concentrations due to limited bacterial tolerance and therefore, and (c) high product recovery costs. Significant energy savings can be achieved if the concentration of n-butanol in the fermentation broth is increased.
Clostridium pasteurianum can produce n-butanol and 1,3-propanediol (1,3-PDO) with completely different patterns from the well-studied C. acetobutylicum in the classic aceton-butanol-ethanol (ABE) process [9, 10]. In C. acetobutylicum, the metabolic pathway of ABE fermentation comprises two characteristic phases: acidogenesis and solventogenesis, whereas in C. pasteurianum DSMZ 525, n-butanol together with 1,3-PDO is produced in the culture medium from the beginning, and only one phase is detected when grown on glycerol as sole carbon source . Acids (and to a lesser extent n-butanol) are the major products produced if glucose is used as the main carbon source. Mixed-substrate fermentation using glucose and glycerol was shown to be superior for n-butanol production by C. pasteurianum [10, 11]. However, limitation of either substrate led to decreased n-butanol formation significantly . The growth pattern was also affected by the nature of substrate used. The highest biomass concentrations were found in experiments with higher glucose concentration (as mono substrate or in blend), followed by with glycerol as mono substrate. Moreover, in mixed substrate fermentation, despite the presence of excess glycerol, limitation of glucose stopped cell growth and limited n-butanol production significantly . The mechanisms behind such phenomenon are not well understood.
In this study we performed mixed substrate fermentations at different initial glucose concentrations. In addition to physiological characterization of cell growth and product formation, comparative proteomic analysis of cultures from different growth phases was performed. Proteomic analysis indicated the importance of the anaplerotic synthesis of oxaloacetate. The ATP and biotin dependent pyruvate carboxylase enzyme is down regulated in the glucose limited phase, and hence may explain the observed growth limitation. Biotin synthesis together with several important enzymes needed for growth was also down regulated in the glucose limited phase. These results provided interesting target for optimization of the growth of C. pasteurianum on mixed substrates of glucose and glycerol.
Results and discussion
Growth patterns of C. pasteurianum in mixed substrate fermentation
Comparison of key parameters of C. pasteurianum cultures grown on mono and dual substrates
Dual substrate fermentation with
5 g/L glucose
10 g/L glucose
10 g/L glucose (GS)
Butanol concentration at the onset of stationary phase (g/L)
8.01 ± 2.10
4.61 ± 1.50
2.23 ± 0.20
3.2 ± 0.40
14.08 ± 0.90
Total acids at the onset of stationary phase (g/L)
2.20 ± 0.15
4.50 ± 0.50
4.41 ± 0.40
25.3 ± 2.90
0.5 ± 0.12
Maximum biomass concentrations at the onset of glucose limitation (g/L)
2.13 ± 0.12
4.49 ± 0.28
3.97 ± 0.21
Maximum biomass concentrations in batch phase (g/L). (maximum biomass values after re-addition of glucose)
4.79 ± 0.12 (5.01 ± 0.08)
4.49 ± 0.28 (6.5 ± 0.21)
3.97 ± 0.21 (6.29 ± 0.15)
11.53 ± 0.57
4.88 ± 0.25
Growth rate before glucose limitation (h−1)
0.37 ± 0.15
0.29 ± 0.01
0.29 ± 0.03
0.25 ± 0.01a
0.23 ± 0.03a
Growth rate after glucose limitation (h−1)
0.15 ± 0.08
0.03 ± 0.01
0.02 ± 0.03
Specific uptake rate of glucose before glucose limitation (g/g/h)
0.91 ± 0.05
0.76 ± 0.07
0.72 ± 0.31
1.7 ± 0.21a
Specific uptake rate of glycerol before glucose limitation (g/g/h)
1.45 ± 0.28
1.47 ± 0.11
1.62 ± 0.25
3.4 ± 0.35a
Comparative proteome analysis
List of proteins whose levels showed at least twofold change during the course of fermentation with 5 and 10 g/L initial glucose without gas stripping, before and after glucose limitation
Function or description
Fold change (II/I) (5 g/L)
Fold change (II/I) (10 g/L)
Serine protein kinase
18.0, 12.8, 4.7
12.3, 5.4, 2.7
Peptidoglycan-binding protein LysM
Stage V sporulation protein T
Single-stranded DNA-binding protein
Nitrogen regulatory protein P-II
Pyruvate phosphate dikinase
Stage IV sporulation protein A
NADP-dependent glyceraldehyde-3-phosphate dehydrogenase
Gene_glgA glycogen synthase
SpoVR family protein
Putative phosphate starvation-inducible protein PhoH
Phosphocarrier protein (HPr)
7.4, 4.8, 4.0
Bifunctional acetaldehyde-CoA/alcohol dehydrogenase
Thiamine pyrophosphate protein central region
Chaperone protein clpb
30S Ribosomal protein S2
Transcription accessory protein TEX, RNA-binding protein containing S1 domain
Pyridoxal biosynthesis lyase PdxS
Cell division protein FtsZ
Cell division protein DivIVA
DTPD-d-glucose 4,6 -dehydratase
Gene_pyrG CTP synthetase
NifU related domain containing protein
Ribosomal protein L7/L12
DNA-directed RNA polymerase subunit beta
Gene_guaA GMP synthase
Pyruvate:ferredoxin (flavodoxin) oxidoreductase
−2.1, −2.3, −3.0
Ferredoxin-NADP(+) reductase subunit alpha
As shown in Table 2, in response to glucose limitation and with glycerol as the remaining carbon source (phase II), several proteins, namely pyruvate phosphate dikinase (PPdK), NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase (Eno), involved in the interconversion of the intermediates of the glycolysis/gluconeogenesis pathway were found to be up-regulated in both fermentations. Pyruvate phosphate dikinase, known to catalyze the interconversion between pyruvate and PEP has been found to be induced on the transfer of glucose-grown cells of Acetobacter xylinum to succinate- or pyruvate-containing media , serving thereby a gluconeogenic function. However, in C. symbiosum, PPdK substitutes for the absent pyruvate kinase and thus fulfils a glycolytic function . In Thermoproteus tenax PPdK shows a bidirectional activity with a preference for the catabolic reaction, and it is suggested that PPdK serve as a ‘stand-by’ enzyme and thus allows for quick adaptation to changing intracellular conditions . The NADP-dependent GAPDH is a multifunctional enzyme capable of catalyzing among others the interconversion between glyceraldehyde-3-phosphate and 3-phosphoglycerate as well as the conversion of 1,3-bisphosphoglycerate to glyceraldehydes-3-phosphate. Whether these enzymes serve a glycolytic or gluconeogenic function is not clear, however, their up-regulated expression highlighted their importance in the central carbon metabolism of C. pasteurianum in response to glucose limitation conditions, and they might all serve as ‘stand-by’ enzymes for a rapid adjustment of the central metabolic fluxes to changes in nutrient availability.
Proteins which also showed increased levels in phase II in both fermentations also include those involved predominantly in stress sensing and stress responses. Examples of these proteins include serine protein kinase, rubrerythrin and rubredoxin/flavodoxin/oxidoreductase related to oxidative stress or heat shock, sporulation proteins, like stage IV sporulation protein A and SpoVR family protein, the phosphate starvation-inducible PhoH family protein, and the signaling protein nitrogen regulatory protein P-II involved in the regulation of nitrogen metabolism.
In fact, serine protein kinase was one of the mostly up-regulated proteins after glucose limitation (Fig. 4). Compared to phase I, the three identified spots of serine protein kinase were 12.3-, 5.4- and 2.7-fold up-regulated in phase II in the fermentation with 10 g/L initial glucose concentration. Expression of serine protein kinase has been reported to be positively controlled by guanosine pentaphosphate [(p)ppGpp] and involved in (p)ppGpp-induced stringent response upon nutritional deprivation. For example, it is strongly up-regulated during stationary phase and involved in metabolic adaptation in Rhizobium etli  or in sporulation of Bacillus subtilis . Therefore, the strong upregulation clearly indicates the stringent response and the metabolic switching of C. pasteurianum upon glucose depletion. After the re-addition of glucose (phase III) no further change on the expression levels of these protein spots was observed (Additional file 1: Table S1). The up-regulation of serine protein kinase was even stronger in the fermentation with 5 g/L L initial glucose concentration after entering glucose limitation. Compared to phase I the expression levels of three spots showed 18.0-, 12.8- and 4.7-fold of increases in phase II, despite the fact that cells had not yet entered the stationary phase but were still growing on glycerol in phase II. Therefore, the (p)ppGpp-induced stringent response shown by serine protein kinase was triggered by glucose limitation, though limitation of other nutrients could not be totally ruled out. This glucose limitation-caused stringent response might further trigger a set of cellular reactions to glucose limitation, including the expression of sporulation proteins. Indeed, in the fermentation with 5 g/L initial glucose concentration, except the above mentioned two spore proteins, an additional spore protein, the stage V sporulation protein T, was found to be strongly up-regulated by 12.2-fold (Table 2).
The decrease in the specific growth rate observed after glucose limitation at both initial glucose concentrations (Table 1), may explain the down regulation of some cell growth associated proteins, such as cell division proteins FtsZ and DivIVA, pyridoxal biosynthesis lyase PdxS for the synthesis of vitamin B6). Two ribosomal proteins, 30S ribosomal protein S2 and 50S ribosomal protein L7/L12, were found to be down-regulated by −4.5- and −2-fold, respectively, but only in the fermentation with 10 g/L initial glucose concentration. Previous studies showed the linear correlation of the ribosomes concentration in bacterial cells with their growth rate, the ribosomes concentration is drastically reduced compared to logarithmic growth phase . Thus, the reduced expression levels of the ribosomal proteins could correlate to the cessation of growth at phase II in the fermentation with 10 g/L initial glucose concentration, whereas to the sampling time point at phase II in the fermentation with 5 g/L initial glucose concentration, cells did not enter stationary phase after glucose limitation but continued to grow on glycerol.
In general, H2 is generated in Clostridia in a ferrodoxin-dependent reaction, and till recently the reactions used to regenerate the reduced ferrodoxin in vivo were not known . In C. ljungdahli, it has been recently shown that Rnf complex in the cell membrane is a proton-translocating ferredoxin:NAD+ oxidoreductase which contributes to ATP synthesis by an proton-translocating ATPase . From the genome of C. pasteunianum DSM 525 studied in this work, C. pasteunianum DSM 525 possesses an arsenal of hydrogenases but not all the components comprising the Rnf complex. Among the hydrogenases are three ferrodoxin-dependent hydrogenases encoded by the genes F502_14390, F502_17487 and F502_13920, as well as two non-ferrodoxin-dependent hydrogenases, namely the hydrogene dehydorgenase and the hydrogenase-1, which are 100 % identical in protein sequence but encoded by two adjacent genes F502_18292 and F502_18287, respectively (http://www.biocyc.org/organism-summary?object=CPAS1262449). Interestingly, none of the ferrodoxin-dependent hydrogenases were among the significantly regulated proteins, whereas the non-ferrodoxin-dependent hydrogenases (hydrogene dehydorgenase and/or hydrogenase-1) were found to be highly down-regulated after glucose limitation, however, only in the fermentation with 10 g/l initial glucose concentration (Table 2). Hydrogenase-1 is annotated as a menaquinone-dependent proton-translocating enzyme in C. pasteurianum DSM 525 by translocating a pair of protons outside the cell membrane with concomitant consumption of H2 present outside the cell membrane (http://www.biocyc.org/gene?orgid=CPAS1262449&id=G10RN-3668#). Therefore, hydrogenase-1 might be involved in the ATP synthesis and its down-regulation after glucose limitation might negatively affect the ATP production. The shift from dual substrate fermentation with glucose and glycerol to mono substrate fermentation with glycerol, a more reduced substrate than glucose, will certainly alter cellular response to achieve a new redox balance. The differences in the regulation of the menaquinone-dependent proton-translocating enzyme between the two fermentations with different initial glucose concentrations are interesting but still elusive and deserve further investigation.
Bioeneregtics and cellular metabolism
ATP is the energy currency of the cell, providing energy for cell growth and maintenance. It also serves as a substrate for RNA synthesis, and regulates a variety of biological processes . Intracellular ATP concentration is also important for the in vivo regulation of many metabolic pathways at the enzyme level . In C. pasteurianum, substrate level phosphorylation and ATP formation are generally coupled to acids production. In fact, the relatively higher acids production with glucose as C-source, and hence the increase in the ATP production rate, together with the lower butanol formation, may explain the relatively high biomass concentration obtained with glucose (Fig. 2; Table 1). Additionally, with glucose as the sole C-source, the relative tolerance towards butanol inhibition was enhanced (compared to glycerol as the sole C-source, Additional file 1: Figure S2). To examine whether the bioenergetics status of the cells after glucose limitation was the reason for cell growth cessation at 10 g/L initial glucose concentration, ATP formation rate after glucose limitation at both initial glucose concentrations were here studied. Compared to the dual substrate utilization (Phase I), the calculated specific ATP production rate (mmol/gbiomass/h) decreased by about 45 and 30 % after glucose limitation (Phase II) for the two cultures with 5 and 10 g/L initial glucose concentration, respectively, and decreased further in phase III by about 55 and 40 %, respectively (Fig. 6). Obviously, the decrease in the ATP production owing to glucose limitation cannot alone explain the phenomenon observed in the dual substrate fermentation of C. pasteurianum caused by the difference in initial glucose concentration, but it may contribute to the inhibition of the pyruvate carboxylase reaction and the overall cellular responses to glucose limitation.
Carbon sources affect the kinetics of C. pasteurianum growth through a cellular response which distributes the product formation to achieve a balance of oxidation and reduction reactions. In a medium with excess carbon source, two distinct conditions exist under which the growth may cease in batch culture of C. pasteurianum: (i) butanol overproduction which challenge the cells to increase the percentage of saturated fatty acids and the formation of more rigid or stable membranes in the stationary phase to counteract n-butanol fluidization  and (ii) metabolic shift after glucose limitation in the dual substrate fermentation which leads to abrupt decrease in acid production and the down regulation of pyruvate carboxylase. The analysis of fatty acids performed for samples before and after glucose limitation at 10 g/L initial glucose concentration indicated no significant difference in the saturated to unsaturated ratio, which also explain the revival of growth after either glucose or oxaloacetate addition. Our working hypothesis is thus that the growth limitation despite the presence of excess glycerol is partly due to the lack of precursor supply important for growth and to a less extent a decrease in cell energy balance.
To develop a commercial process for the production of n-butanol by C. pasteurianum, it is necessary to better understand the primary metabolism and its regulation. Whereas glucose utilization supports biomass and acid production, glycerol utilization leads to significantly lower biomass formation but higher alcohol productions. The highest butanol production by C. pasteurianum was recorded in fermentations using a dual substrate of glucose and glycerol rather than in any of the mono-substrate fermentations. However, in the presence of excess amount of glycerol, the initial glucose concentration affects the cell growth after glucose limitation. Compared to the fermentation with 5 g/L initial glucose concentration, glucose limitation in the fermentation with 10 g/L initial glucose concentration leads to cease of cell growth even in the presence of excess glycerol in the medium, and hence affects negatively the butanol production. The physiological analyses in this work indicated that the cessation of cell growth is not primarily due to n-butanol toxicity. Moreover, cell membrane fluidity as indicated by fatty acid compositions did not change significantly before and after glucose limitation. The, to our best knowledge, first proteomic analysis of C. pasteurianum has provided some clues on the metabolic responses of C. pasteurianum to the changing growth conditions. Among others, the proteomic analysis has revealed the down-regulation of pyruvate carboxylase and biotin synthase as one of the major cellular responses which limit the anaplerotic formation of oxaloacetate and consequently the cell growth. Addition of oxaloacetate to the glucose limited culture has revived cell growth and thus confirms the limited availability of this intermediate as a key determinate of growth of C. pasteurianum. Thus, in order to improve the growth of C. pasteurianum and the productivity of n-butanol and eventually also other products from this bacterium efforts should be made to ensure the availability of glucose and oxaloacetate, i.e., through fed-batch fermentation or overexpression of genes related to the formation of oxaloacetate, especially pyruvate carboxylase and biotin synthase.
Bacteria strain, culture medium and growth conditions
Clostridium pasteurianum DSMZ 525 was cultivated anaerobically at 35 °C without shaking. The strain was maintained in Reinforced Clostridial Medium (RCM, Oxoid Deutschland GmbH, Wesel, Germany) and preserved using glycerin 20 % (v/v) at −80 °C.
RCM medium inoculated from a cryoculture was left to grow at 35 °C for 18–20 h and then used as inocula for the production medium. The standard production medium for batch cultures contained the following ingredients in 1 L of distilled water (modified from Biebl, ). Glycerol, varied; glucose, varied; K2HPO4, 0.5; KH2PO4, 0.5, MgSO4·7H20, 0.2 g; (NH4)2SO4, 3 g; CaCl2 2H2O, 0.02 g, FeSO4·7H2O, 0.01 g; cysteine HCL, 0.3; resarzurin, 0,005; trace element solution SL7, 2 mL and 1 g yeast extract.
Batch cultivations were carried out in a pH-controlled 2 L stirred tank bioreactor (bioengineering) with a working volume of 1.5 L. After sterilization, the medium in the fermenter was flushed with sterile O2-free N2 until room temperature was reached. Filter sterile cystein HCl and FeSO4 solution was then added and inoculated immediately. Glucose was autoclaved separately. Flushing with nitrogen was stopped after inoculation and the bacteria were grown under their own produced gases. The pH was adjusted to six by the automatic addition of 5 N KOH. Carbon dioxide and hydrogen signals were measured online by CO2 and H2 sensors connected in series according to the manufacturers instructions (BluSens GmBH, Germany).
To determine the effect of carbon source on the relative tolerance toward butanol, anaerobic bottles with production media containing either glucose or glycerol as the sole C-source and with different concentration of butanol were incubated and the initial growth rate after 8 h were calculated (equation below).
Since n-butanol concentration above about 5 g/L can negatively affect the bacterial metabolism, batch fermentation with mixed substrates and in situ removal of butanol by gas stripping was done according to Jensen et al. . Briefly, own produced fermentation gases (mainly CO2 and H2) collected in the bioreactor headspace are pumped at a flow rate of 7 VVM through the fermentation broth and then to a condenser cooled at 1 °C (with a cooling surface of 0.34 m2). Condensates containing mainly butanol and water are collected in a cooled bottom separate vessel.
Cell concentration was measured optically, at 600 nm and correlated with cell dry weight determined directly. The concentrations of glucose, glycerol, butanol, 1,3 propanediol, ethanol, acetic, butyric, formic and lactic acids in supernatant were determined by HPLC using an Aminex HPX-87H column (300 × 7.8 mm) and the detection was assessed by refractive index and ultraviolet detectors. The operating conditions were as follows: mobile phase, H2SO4 0.005 M; flow rate, 0.6 ml min−1; temperature, 60 °C.
For the determination of the cell wall fatty acid composition, GC analysis of the fatty acid methyl esters was performed as previously reported  with a Varian 3900 gas chromatograph equipped with a flame ionization detector (FID) and a TR-FAME column (Thermo Scientific, Germany, 50 m × 0.22 mm × 0.25 µm).
Comparative proteomic analysis was carried out by separating intracellular proteins using a two-dimensional gel electrophoretic method (2-DE) established in our lab . Briefly, cells samples re-suspended in a lysis/rehydration buffer containing 7 M urea, 2 M thiourea, 4 %w/v CHAPS, 100 mM DTT, 0.5 % IPG buffer 3–10 and protease inhibitors cocktail (Roche) were disrupted with Lysis Matrix B silica beads in a FastPrep-24 high-speed homogenizer (MP Biomedicals) at 6.0 m/s for 8 cycles with 5 min intervals between each cycle. Protein extracts obtained were purified by phenol precipitation, reconstituted in the lysis/rehydration buffer and, after determining protein concentrations, subjected to 2-DE separation. The first dimension isoelectric focusing (IEF) was conducted using 18 cm IPG strips (pH 4–7) in a Ettan IPGPhor 3 IEF system with the following voltage program: 30 V for 6 h, 60 V for 6 h, 200 V for 1 h, 500 V for 1 h, 1000 V for 1 h, gradient to 8000 V within 30 min and 8000 V for 8 h. Subsequently, the focused IPG strips were equilibrated in two steps of 15 min each with 15 mL of equilibration buffer (50 mM Tris–HCl, pH 8.8, 6 M urea, 30 % w/v glycerol, 2 % w/v SDS) supplemented with 1 % w/v DTT in the first and 2.5 % w/v iodoacetamide in the second step, respectively. The second dimension SDS-PAGE was carried out using 12.5 % polyacrylamide gels with the following running conditions: 1.5 W/gel for 1 h and then 10 W/gel until the bromophenol blue dye front reached the bottom of the gels. After staining with self-made ruthenium II bathophenanthroline disulfonate chelate (RuBPS) fluorescent dye, gels were scanned with a molecular imager (VersaDoc MP4000, Bio-Rad), and gel images were analyzed using the Progenesis SameSpots software v3.3 (Nonlinear dynamic, UK) to detect protein spots showing statistically significant changes in their expression levels before (phase I) and after (phase II) glucose limitation.
Identification of protein spots with significantly changed expression levels was done by nanoLC–ESI–MS/MS analysis using a Ultimate 3000 RSLCnano HPLC system (ThermoFisher Scientific) coupled to an amaZon ETD ion-trap mass spectrometer (Bruker Daltonics). Briefly, after overnight in-gel tryptic digestion at 37 °C, extraction of tryptic peptides and purification with reversed-phased C18 ZipTips (Millipore), tryptic peptides dissolved in 0.1 % TFA were pre-concentrated on a Acclaim PepMap100 C18 (100 µm × 2 cm, 5 µm) column and then separated on a Acclaim PepMap RSLC C18 (75 µm × 15 cm, 2 µm) column. The mobile phases used were A: 0.1 % formic acid in water and B: 10.1 % formic acid in acetonitrile/water (90:10). Peptide were separated using a 30 min linear gradient from 2 to 45 % B delivered at a flow rate of 300 nL/min. Tryptic peptides eluted from the C18 analytical column were introduced into the mass spectrometer through a CaptiveSpray nano-ESI source (Bruker Daltonics) operating at positive mode controlled by using the trapControl acquisition software (version 4.0). The following tuning parameters were used: capillary voltage −1500 V, flow rate and temperature of the drying gas 3 L/min and 160 °C, respectively. The scan range was 300–1500 m/z for MS and 100–2400 m/z for MS/MS. The MS/MS experiments were carried out in data-dependent auto MS/MS mode using a 4 Da window for precursor ion selection and an absolute threshold of 25,000. After the acquisition of 2 MS/MS spectra from the same precursor ion the m/z is excluded from the precursor selection for 1 min. Data acquired from the nanLC–ESI–MS/MS analysis were processed using the Compass DataAnalysis software (version 4.1) to generate XML files, by which only the 300 most intense MS/MS spectra per MS/MS analysis were converted into compounds and used for protein database search. For protein identification the XML files were imported into the ProteinScape software for search against a specific protein database of C. pasteurianum DSMZ 525 installed in-house on a licensed Mascot server. The following parameters were used for protein identification: allow up to 1 missed cleavage, 0.6 Da tolerance both for peptide and MSMS, 1+, 2+ and 3+ peptide charges, carbamidomethyl (C) as fixed moidification, oxidation (M) as variable modification, only accept protein identified by at least 2 peptides with false positive rate <1 %. Samples from two biological replicates [each sample with three technical replicates (3 gels)] were used for proteomic analysis.
Stoichiometric analysis for energy (ATP), reducing equivalents and product balances
Glucose → 2 Acetate + 2 CO2 + 4 ATP + 2 NADH2 + 2 FdH2
Glucose + 2 NADH2 → 2 Ethanol + 2 CO2 + 2 ATP +2 FdH2
Glucose → Butyrate + 2 CO2 + 3 ATP + 2 FdH2
Glucose + 2 NADH2 → Butanol + 2 CO2 + 2 ATP + 2 FdH2
Butyrate + ATP + 2 NADH2 → Butanol
Glycerol → Acetate + CO2 + 2 ATP + 2 NADH2 + FdH2
Glycerol → Ethanol + CO2 + ATP + FdH2
2 Glycerol → Butyrate + 2 CO2 + 3 ATP + 2 FdH2 + 2 NADH2
2 Glycerol → Butanol + 2 CO2 + 2 ATP + 2 FdH2
Glycerol + NADH2 → 1,3-propanediol
WS designed and performed the bioreactor experiments, interpret the results and wrote the manuscript. WW carried out the proteomic studies, helped in results interpretation and draft the manuscript. SS helped in bioreactor and proteomic experiments. CG participated in the gas stripping experiments. APZ supervised the research. All authors read and approved the final manuscript.
This work has received funding from the European Union Seventh Framework Program (FP7/2007 2013) through the EuroBioRef (Grant agreement Nr. 241718). The authors also thank the help of Jan Bomnüter for his excellent assistance with the two dimensional electrophoresis of proteomic analysis.
The authors declare that they have no competing interests.
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