Stable coexistence of two Caldicellulosiruptor species in a de novo constructed hydrogen-producing co-culture
© Zeidan et al; licensee BioMed Central Ltd. 2010
Received: 28 November 2010
Accepted: 30 December 2010
Published: 30 December 2010
Mixed culture enrichments have been used frequently for biohydrogen production from different feedstock. In spite of the several advantages offered by those cultures, they suffer poor H2 yield. Constructing defined co-cultures of known H2 producers may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved.
The extreme thermophiles Caldicellulosiruptor saccharolyticus DSM 8903 and C. kristjanssonii DSM 12137 were combined in a co-culture for H2 production from glucose and xylose in a continuous-flow stirred tank reactor. The co-culture exhibited a remarkable stability over a period of 70 days under carbon-sufficient conditions, with both strains coexisting in the system at steady states of different dilution rates, as revealed by species-specific quantitative PCR assays. The two strains retained their ability to stably coexist in the reactor even when glucose was used as the sole growth-limiting substrate. Furthermore, H2 yields on glucose exceeded those of either organism alone under the same conditions, alluding to a synergistic effect of the two strains on H2 production. A maximum H2 yield of 3.7 mol (mol glucose)-1 was obtained by the co-culture at a dilution rate of 0.06 h-1; a higher yield than that reported for any mixed culture to date. A reproducible pattern of population dynamics was observed in the co-culture under both carbon and non-carbon limited conditions, with C. kristjanssonii outgrowing C. saccharolyticus during the batch start-up phase and prevailing at higher dilution rates. A basic continuous culture model assuming the ability of C. saccharolyticus to enhance the growth of C. kristjanssonii could mimic the pattern of population dynamics observed experimentally and provide clues to the nature of interaction between the two strains. As a proof, the cell-free growth supernatant of C. saccharolyticus was found able to enhance the growth of C. kristjanssonii in batch culture through shortening its lag phase and increasing its maximum biomass concentration by ca. 18%.
This study provides experimental evidence on the stable coexistence of two closely related organisms isolated from geographically-distant habitats under continuous operation conditions, with the production of H2 at high yields. An interspecies interaction is proposed as the reason behind the remarkable ability of the two Caldicellulosiruptor strains to coexist in the system rather than only competing for the growth-limiting substrate.
In natural ecosystems, mixed microbial populations are the rule rather than the exception. Distinct microbial populations frequently interact with each other in a variety of ways giving rise to many beneficial effects and, therefore, it is not surprising that mixed cultures possess broader metabolic capabilities and show more robustness to environmental fluctuations than individual populations . In biotechnology, continuous enrichment techniques have been used for obtaining stable microbial consortia adapted to continuous culture conditions, which is especially valuable in a continuous-flow commercial process . The use of such enrichments has indeed been proven useful in several applications, such as anaerobic digestion and biopolymer and solvent production .
Alternatively, microbial communities can be built, where the diversity and metabolic capabilities of the community can be tuned by careful selection of the individual members for achieving improved or new structures and functions . This approach has been widely adapted in food fermentations [5, 6] and successfully employed for lab-scale bioethanol [7–9], acetic acid  and lactic acid  production. It also provides an essential tool for fundamental studies on the different mechanisms of microbial interactions [12–16]. Although the approach appears interesting, the stability of these artificial communities is remarkably difficult to achieve for various reasons, as discussed by Weibel . To date, there appears to be only few studies that report on the construction of co-cultures consisting of two or more bacterial species that can stably coexist over a long period of time [17–21].
Owing to its high economic value and wide range of applications in the chemical industry, H2 has been a subject of increasing interest in recent years. Considerable research efforts have been dedicated to producing H2 by biological conversion of biomass via dark fermentation, with a notable emphasis on the use of mixed-culture enrichments [22–25]. The starting culture is usually derived from samples of compost or anaerobic treatment sludge  and in spite of the high volumetric H2 production rates achieved by these cultures, H2 yields usually do not exceed two moles per mole of hexose sugar converted . Since the process is always coupled to biowaste treatment, high yields may not be viewed as essential. However, dark fermentation allows the release of up to 4 moles of H2 per mole of hexose sugar , and therefore, if H2 is to be produced from energy crops, this maximum yield should be aimed for.
Construction of 'designer' H2-producing communities can be foreseen as a possible way to overcome some of the issues inherent to the use of undefined consortia, most notably the low H2 yields [22, 28]. In a previous study, the extreme thermophiles Caldicellulosiruptor saccharolyticus and C. kristjanssonii have been combined in a H2-producing co-culture with the primary aim of improving the rate of glucose consumption during co-fermentation with xylose . A more interesting outcome of this co-culture was its ability to produce H2 at a higher yield than that of either organism alone. However, the fermentations were carried out in batch mode and the stability of the co-culture in a continuous system remained questionable. This is particularly important since in practical application chemostat conditions are more likely to be used. In the present work, we demonstrate H2 production at high yields in a continuously-stirred tank reactor (CSTR) by a stable co-culture of C. saccharolyticus and C. kristjanssonii under chemostat conditions. Due to the lack of morphological differences between the two species, a quantitative PCR (qPCR) assay for monitoring the growth of each organism in the co-culture was developed. In addition, a proof of the ability of one or more products of C. saccharolyticus cells to enhance the growth of C. kristjanssonii could be obtained. The remarkable potential of the two, closely related, strains to stably coexist under nutrient-limited conditions, as could be captured in a simple mathematical model describing the system, is discussed.
Materials and methods
C. saccharolyticus DSM 8903 (also, ATCC 43494) and C. kristjanssonii strain I77R1B (DSM 12137; ATCC 700853) were purchased from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany).
C. saccharolyticus and C. kristjanssonii were individually subcultured twice at 70°C in 50 mL modified DSM medium 640  containing the same sugar(s) used in the subsequent fermentation (10 g L-1) under N2 atmosphere in 250-mL crimped-seal bottles. After the optical density at 620 nm (OD620) of the second subculture has reached 0.3 - 0.4, it was used to inoculate the bioreactor at a level of 15% (v v-1). For initiating the co-culture, an equal volume of each organism was used.
The organisms were grown in a jacketed, 3-L stirred-tank bioreactor (Applikon, Schiedam, The Netherlands) at 70 ± 1°C. The bioreactor was equipped with an ADI 1025 Bio-Console and an ADI 1010 Bio-Controller (Applikon, Schiedam, The Netherlands). A modified DSM medium 640 was used for all cultivations at a working volume of 1 L. A sterile solution of glucose and/or xylose was added to the medium after sterilization to the required concentration. The medium was continuously sparged with N2 gas containing less than 5 ppm O2 (AGA Gas AB, Sundbyberg, Sweden) at 100 mL min-1 and stirred at 300 rpm. The pH was maintained automatically at 6.7 ± 0.1 at the operating temperature, using 3 M NaOH as a titrant. The medium was rendered completely anaerobic by the addition of cysteine-HCl at a final concentration of 1 g L-1 prior to inoculation. After an initial growth in batch mode, the bioreactor was fed at the end of the exponential growth phase with a fresh medium, having a similar composition to the start-up medium except for the omission of cysteine-HCL, at 0.04 h-1. The feed bottle was continuously sparged with N2 to avoid medium displacement by air. After a steady state has been attained, a stepwise increase in the dilution rate (D) was carried out. Steady states were assessed after at least 5 volume changes through constant H2 and CO2 production rates and constant biomass concentration.
Gas samples from the bioreactor's headspace were regularly analyzed for H2 and CO2 composition. Culture samples were regularly withdrawn for monitoring the biomass concentration. In addition, 10 mL culture samples were aseptically withdrawn and cells were harvested by centrifugation at 5,000 × g and 4°C for 12 min, washed twice with 1× phosphate-buffered saline (PBS) by centrifugation and stored at -20°C for genomic DNA extraction. The supernatant was further clarified by passing through a 0.2-μm cellulose acetate filter (Advantec, Tokyo, Japan) and analyzed for sugar consumption and metabolite formation.
Effect of C. saccharolyticus cell-free growth supernatant on C. kristjanssonii
The growth supernatant of C. saccharolyticus was evaluated for its ability to enhance the growth of C. kristjanssonii in batch cultures. Growth supernatants from 50-mL cultures of each organism were collected by centrifugation at 12,000 × g and 4°C for 6 min. The supernatant of C. kristjanssonii culture was discarded and the cell pellet was retained. The supernatant of C. saccharolyticus culture was filtered through a sterile, 0.2-μm-pore-size filter (Sarstedt, Nümbrecht, Germany) to obtain a cell-free spent culture broth in which C. kristjanssonii cells were resuspended. All manipulations were carried out inside an anaerobic glove box (Plas Labs Inc., MI, USA) under an atmosphere of N2-H2-CO2 (0.85:0.10:0.05). The 50-mL resuspended culture of C. kristjanssonii was combined with either 50 or 100 mL of untreated C. kristjanssonii inoculum to inoculate 1 L modified DSM 640 medium, containing 10 g L-1 glucose as a substrate, in the bioreactor. The fermentations were conducted in batch mode under the same conditions described above and biomass concentration was monitored at regular intervals.
The concentrations of H2 and CO2 were determined by gas chromatography, using a dual-channel CP 4900 Micro-GC (Varian, Middelburg, The Netherlands). H2 was analyzed on a molecular sieve column (CP-MolSieve 5Å PLOT) with the injector and column temperatures at 80 and 100°C, respectively, whereas CO2 was analyzed on a CP-PoraPLOT Q column with the temperature of both the injector and the column at 80°C. The carrier gases for the MolSieve and the PoraPLOT Q columns were N2 and He, respectively, at 150 kPa. Each channel was equipped with a micro-machined thermal conductivity detector. Sugars, organic acids and ethanol were separated by high-performance liquid chromatography (HPLC; Waters Corporation, Milford, MA) on an Aminex HPX-87H column (Bio-Rad, Hercules, CA) at 45°C. Sulfuric acid (5 mM) was used as the mobile phase at a flow rate of 0.6 mL min-1. The analytes were detected on a refractive index detector (RID-6A; Shimadzu, Tokyo, Japan).
A U-1800 spectrophotometer (Hitachi, Tokyo, Japan) was used for regular monitoring of the OD620. For cell dry weight (CDW) determination, 10 ml of culture were transferred to 15-mL dried, pre-weighed Falcon tubes and centrifuged at 5,000 × g for 15 min, washed with deionized H2O and dried at 70°C to a constant weight. Enumeration of C. saccharolyticus and C. kristjanssonii in their pure cultures was performed by direct microscopic count, using a Bürker-pattern counting chamber (Marienfeld, Lauda-Königshofen, Germany).
Genomic DNA was extracted from washed, frozen cell pellets using the Easy-DNA Kit (Invitrogen, San Diego, CA), according to the manufacturer's protocol. For complete RNA degradation, DNA preparations in Tris-EDTA buffer (TE buffer; pH 8) were incubated with RNase A (40 μg mL-1) for 30 min at 37°C. The concentration and purity of DNA were analyzed spectrophotometrically in an Eppendorf Biophotometer (Hamburg, Germany). Aliquots of DNA in TE buffer were stored at -20°C until use.
Real-time PCR assays
Real-time PCR primers for quantification of C. saccharolyticus and C. kristjanssonii in their co-culture.
Product size (bp)
Quantification was achieved by determining the crossing point (Cp) of the sample - that is, the fractional cycle number that corresponds to the maximum of the second derivative of the amplification curve - and comparing to standards. For preparing the standards, pure cultures of C. saccharolyticus and C. kristjanssonii were individually grown on glucose in 3-L bioreactors under the same conditions described above, albeit in batch mode. Culture samples of each organism were collected at different time intervals for cell count and genomic DNA extraction. A first standard curve between DNA concentration and cell number was plotted to validate the DNA extraction protocol and the capacity of the extraction kit. DNA preparations from samples of varying cell numbers were then used, after appropriate dilution, as templates for qPCR. The mean Cp of two independent real-time PCR replicates was plotted against the logarithm of the corresponding cell count to obtain a second standard curve. For quantitative determination C. saccharolyticus and C. kristjanssonii in different samples of their co-culture, the second standard was regenerated for each species with every run in the LightCycler to overcome any variability in the amplification efficiency.
where x1 is the biomass concentration of C. saccharolyticus (gCDW L-1), x2 is the biomass concentration of C. kristjanssonii (gCDW L-1), s0 is the glucose concentration in the feed (mmol L-1), s is the residual glucose concentration (mmol L-1), and t is the fermentation time (h). The biomass yield coefficient [gCDW (mmol glucose-1)], is considered to be constant, whereas and the growth rates (μ1 and μ2) are each a function of several variables as explained below.
in which μmax1 and μmax2 are the maximum specific growth rates (h-1) of C. saccharolyticus and C. kristjanssonii, respectively, and and are the Monod constants for glucose (mmol L-1) for C. saccharolyticus and C. kristjanssonii, respectively.
with and as the maximum specific growth rates in the absence of compound E and in its presence at the critical concentration (EC), respectively. Likewise, and are the biomass yield factors in the absence of compound E and in its presence at the critical concentration, respectively.
Values of the different parameters included in the model are listed in Additional file 1 (Table S1). The set of differential equations was solved using Matlab (R2009a; The MathWorks, Inc.).
Hydrogen production and stability of the co-culture under non-carbon limitation
H2 production and biomass formation by the co-culture on a mixture of glucose (5 g L-1; 27.8 mM) and xylose (5 g L-1; 33.3 mM) at steady states of different dilution rates (yeast extract, 1 g L-1; pH 6.7; 70°C).
[mol (mol C6)-1]
Biomass concentration (gCDW L-1)
Ysx[gCDW (mol C6)-1]
Residual sugar (mM)
Both the volumetric and specific H2 production rates ( and , respectively) markedly increased with the dilution rate (Table 2). The maximum volumetric hydrogen production rate obtained was 11.6 mmol L-1 h-1 at 0.25 and 0.3 h-1. On the other hand, H2 yield () was inversely proportional to the dilution rate, with the maximum of 3.6 mol H2 per mol of hexose equivalent obtained at 0.06 h-1 (Table 2). This yield represents 90% of the maximum theoretical yield that can be obtained from glucose during dark fermentative hydrogen production. The biomass yield (Ysx) increased sharply at D > 0.15 h-1 (Table 2), which could be due to the accumulation of a storage material, e.g., glycogen.
Carbon limitation with two sugars
Performance of the co-culture in a carbon-limited chemostat at steady states of two different dilution rates with glucose and xylose (4 g L-1; 1:1) as the growth limiting substrates (yeast extract, 1 g L-1; pH 6.7; 70°C).
[mol (mol C6)-1]
Biomass concentration (gCDW L-1)
Ysx[gCDW (mol C6)-1]
Residual sugar (mM)
3.6 ± 0.1
4.5 ± 0.2
10.2 ± 0.3
0.44 ± 0.03
21.0 ± 0.9
3.2 ± 0
8.6 ± 0.4
24.7 ± 1.1
0.35 ± 0.03
19.4 ± 1.0
1.7 ± 0.1
1.4 ± 0.1
Competition for one growth-limiting substrate
Comparison of steady-state H2 and biomass production by C. saccharolyticus, C. kristjanssonii and their co-culture in glucose-limited chemostat cultivations at two different dilution rates (yeast extract, 1 g L-1; pH 6.7; 70°C).
[mol (mol C6)-1]
Biomass concentration (gCDW L-1)
Ysx[gCDW (mol C6)-1]
Residual glucose (mM)
3.5 ± 0.1
4.9 ± 0.14
13.2 ± 0.1
0.38 ± 0.01
15.8 ± 0.2
3.1 ± 0.2
7 ± 0.3
24.4 ± 1
0.29 ± 0.03
19.3 ± 1.1
8.6 ± 0.6
3.5 ± 0.1
4.8 ± 0.1
17.8 ± 0.5
0.27 ± 0.01
11.7 ± 0.0
3.0 ± 0.1
10.3 ± 0.16
34.6 ± 1.6
0.3 ± 0.02
13.0 ± 0.1
0.2 ± 0.0
3.7 ± 0.0
4.8 ± 0.2
14.8 ± 0.8
0.33 ± 0.03
15.4 ± 0.8
3.5 ± 0.0
10.4 ± 0.14
21.4 ± 0.8
0.49 ± 0.01
24.6 ± 0.8
Steady-state H2 and biomass production by the co-culture on xylose (4 g L-1) in the presence of yeast extract (1 g L-1) at two different dilution rates (pH 6.7; 70°C).
[mol (mol C6)-1]
Biomass concentration (gCDW L-1)
Ysx[gCDW (mol C6)-1]
Residual xylose (mM)
2.7 ± 0.1
3.0 ± 0.2
22.6 ± 4.1
0.13 ± 0.04
7.4 ± 1.7
6.9 ± 0.9
2.7 ± 0.0
8.5 ± 0.2
33.0 ± 8.9
0.27 ± 0.08
13.0 ± 3.7
3.7 ± 1.4
Capturing the population dynamics in the co-culture in silico
Effect of C. saccharolyticus culture supernatant on growth of C. kristjanssonii
Hydrogen-producing co-cultures constructed de novo may offer a better performance than mixed-population enrichments, while overcoming some of the limitations of pure cultures based on synergies among the microorganisms involved. In several previous studies on defined co-cultures, it was a common strategy to proceed by isolating the dominant members of a mixed enrichment culture and then recombining the isolates in order to reproduce the stability and function of the original microflora [17, 21, 29, 30]. Here, we combined two closely-related strains from geographically distant, albeit similar, habitats, i.e., C. saccharolyticus DSM 8903 and C. kristjanssonii DSM 12137. The former bacterium was isolated from a thermal pool in New Zealand [31, 32], whereas the latter was isolated from a biomat sample of an Icelandic hot spring . Both organisms are strictly anaerobic, extreme thermophilic heterotrophs with the ability to degrade complex polysaccharides and ferment both hexose and pentose sugars [28, 31, 33, 34].
A CSTR seeded with C. saccharolyticus and C. kristjanssonii could be successfully operated over a period of 70 days at different dilution rates with both species coexisting in the system, when glucose and xylose (10 g L-1) were used as the main carbon and energy sources. According to steady-state residual sugar analysis, the culture was carbon-sufficient at all dilution rates, indicating that another nutrient must have been limiting the growth. Based on the standard biomass formula, i.e., CH1.8O0.5N0.2, and the maximum biomass concentration obtained by either organism in batch fermentations (ca. 1 gCDW L-1), the amount of inorganic nitrogen in the feed, present as NH4Cl, is considered to be in excess. A component in yeast extract (YE), e.g., a growth factor, is most likely to be responsible for growth limitation and incomplete sugar utilization by the co-culture, since the YE-to-sugar ratio in the feed was only 1/10. This kind of limitation has been previously suggested for C. saccharolyticus and for Thermoanaerobacter ethanolicus during continuous fermentation of glucose and xylose, respectively. In our study, this was verified by increasing the YE-to-sugar ratio in the feed to 1/4, where the co-culture could be carbon limited when grown on a mixture of glucose and xylose (Table 3) or glucose only (Table 4).
In general, the H2 yields obtained in C. saccharolyticus-C. kristjanssonii co-cultures in this study under both carbon and non-carbon limited conditions are significantly higher than the yields previously reported for defined co-cultures [29, 38–40] or mixed-culture enrichments [3, 23, 41], regardless of the type of reactor employed. The decrease in H2 yield with the dilution rate observed in the co-culture fermentations as well as in the pure cultures of C. saccharolyticus and C. kristjanssonii was, in part, a result of increased lactate formation (see Additional file 2), which drains some of the electrons required for H2 production. The highest H2 yield of the co-culture was 3.7 mol per mol of hexose sugar (i.e., 92.5% of the maximum theoretical H2 yield that can be achieved via dark fermentation). This yield could only be achieved by limiting the co-culture on glucose at low D (i.e., 0.06 h-1) and was higher than that obtained by either organism in pure culture under the same experimental conditions (Table 4), alluding to a possible synergistic effect of the two strains on H2 production. This also indicates that carbon limitation can be a successful strategy for improving the sugar-conversion efficiency and increasing H2 yield of the co-culture. Although it remains unclear why limiting the cells for glucose leads to what appears to be more efficient substrate utilization and increased H2 production, Bisaillon et al  suggested the possibility of carbon flow to secondary metabolic pathways, for example, glycogen synthesis, being restricted at low glucose concentrations thereby shunting most of the carbon to the catabolic, H2-generating, pathways.
In contrast to H2 yield, the volumetric H2 production rate of the co-culture constructed in this study increased by increasing the dilution rate, which agrees with data from previous reports on fermentative H2 production in chemostat cultures [36, 43]. The maximum H2 production rate achieved by the co-culture at the highest D tested in this study (Table 2) is equivalent to 0.28 LH2 Lculture-1 h-1, which lies within the upper range of previously reported productivities of mixed culture-fermentations in CSTRs [41, 44–46]. The optimum balance between yield and productivity was observed under glucose limitation at steady state of the higher D (Table 4), implying that both carbon limitation and the dilution rate are critical factors for optimizing H2-production efficiency by the co-culture. Under those conditions, the volumetric H2 production rate of the co-culture was equivalent to that of C. kristjanssonii, which indeed was the predominant organism in the co-culture. H2 yield, however, was significantly higher than that of C. kristjanssonii, pointing again at a possible synergistic effect of the two strains on H2 production.
An interesting outcome of the present study is the ability of the two closely related Caldicellulosiruptor species to stably coexist over the whole range of conditions tested. Since both species occupy the same trophic level [31, 33], they are expected to compete for the same growth-limiting substrate in a chemostat culture. Based on the competitive exclusion principle , this competition should result in one strain in the co-culture completely displacing the other in the development towards a steady state under a given condition. Since YE, which is a complex nutrient, was suspected for growth limitation in the co-culture under carbon-sufficient conditions, the coexistence could be explained in that each species was limited on a different component in YE. However, the stable coexistence of the two strains observed at steady-state conditions with glucose as the sole growth-limiting substrate cannot be explained on that basis. As discussed below, the reproducible pattern of population dynamics observed under different conditions in the present study provides a clue on the occurrence of interspecies interaction in the co-culture, which could be the reason behind the stable coexistence of C. saccharolyticus and C. kristjanssonii.
The prevalence of an organism in a batch culture depends on its maximum specific growth rate compared with that of the other organisms capable of growth in the inoculum . Since C. kristjanssonii exhibits a 40% lower maximum specific growth rate than that of C. saccharolyticus during batch growth on glucose and xylose , the prevalence of the former in their co-culture during the batch start-up phase and at higher dilution rates implies that it acquires a higher specific growth rate in presence of the latter. In addition, the biomass yield of the C. kristjanssonii-dominated co-culture in glucose-limited chemostat cultivations at a D of 0.15 h-1 was significantly higher than that of C. kristjanssonii alone under otherwise the same conditions (Table 4), which points to an enhancing effect of C. saccharolyticus on the biomass yield of C. kristjanssonii. Indeed, the fact that none of the strains was completely displaced by the other at any of the steady states evaluated supports the hypothesis of the occurrence of a beneficial sort of interspecies interaction in the co-culture rather than only competition for the same growth-limiting nutrient. The simple mathematical model we developed to describe this proposed interaction, in terms of compound E that has a growth-enhancing effect on C. kristjanssonii, could successfully mimic the stable coexistence and predict the population dynamics in the co-culture, ruling out any other required mechanisms (Figure 5). Furthermore, it is the combination of enhancing the growth rate and biomass yield of C. kristjanssonii that creates the condition for stable co-existence as observed in the chemostat-cultivations. This condition only remains valid as long as C. saccharomyces stays in the system to provide the proposed growth-enhancing compound. The reduction in the lag phase and the increase in maximum biomass concentration of C. kristjanssonii in the presence of C. saccharolyticus growth supernatant (Figure 6) provided experimental evidence on the presence of such a compound or signaling molecule that can enhance the growth of C. kristjanssonii. Although the maximum specific growth rate was not affected under these conditions, this could be due to the absence of C. saccharolyticus cells in the reactor, thus preventing the continuous supply of the growth-enhancing compound. It cannot also be ruled out that this compound is actually a product of cell lysis rather than a secretory product since C. saccharolyticus cells are prone to lyse significantly [48, 49].
In light of the social evolution theory of microorganisms , the ability of C. saccharolyticus to enhance the growth of C. kristjanssonii might be classified as an altruistic cooperation. An explanation for altruistic cooperation between close relatives is best provided by the kin selection theory, where an individual species can still pass on its own genes to the next generation indirectly by helping a closely related species to reproduce. Unraveling the precise mechanism of this interaction or the nature of the involved molecules, however, remains a conceptual and methodological challenge.
It is well known that each population or individual detects and responds to the presence of others in a consortium by trading metabolites or by exchanging dedicated molecular signals [1, 51]. Growth enhancement as a consequence of interspecies interactions in bacteria may occur in response to some signaling molecules . Processed oligopeptides are known quorum sensing molecules, or autoinducers, that are used by Gram-positive bacteria for communication . Nichols et al  presented evidence that short peptides may be essential factors for initiating growth of 'uncultivable' cells. The existence of a peptide-based quorum sensing in hyperthermophilic bacteria was also previously demonstrated in a co-culture of Thermotoga maritima and Methanococcus jannaschii and was responsible for inducing exopolysaccharide production and enhancing biofilm formation by the former organism . Moreover, it has been recently suggested that the addition of a second microbe, viz. Tm. maritima, to a pure culture of C. saccharolyticus triggers events causing the presence, absence and differential expression of protein species within the system . Although the genome of C. saccharolyticus has been fully sequenced and annotated , it is difficult to examine for the presence of putative small peptide signaling molecules since signaling peptides are often products of genes encoding proteins less than 100 amino acids in length. In C. saccharolyticus genome, there are 496 of such genes, around 55% of which encode for hypothetical proteins (NCBI Entrez; http://www.ncbi.nlm.nih.gov/sites/entrez).
While genomic information did not prove useful, other approaches, such as microarray-based functional genomics approaches [12, 14] or bioassay-directed fractionation and analysis of the growth supernatant , can be adopted for the identification of candidate signaling molecules and understanding the molecular mechanisms behind the interactions in the co-culture. This is, however, beyond the scope of the current study and may become more feasible by the availability of the complete genome sequence of C. kristjanssonii.
The present study provides essential evidence on the possibility of stable co-existence of two closely related bacteria isolated from distant habitats in a continuous-flow system under steady-state conditions. This augments the suggestion of de novo constructed or 'designer' co-cultures as potential alternatives for several biotechnological applications that are, otherwise, carried out using mixed culture enrichments. Although the increase in H2 yield by the co-culture constructed in this study was not dramatic, as compared with that of the individual strains, it is still higher than the H2 yield reported for any mixed culture to date. Generally, benefits of the use of the co-culture other than improving product yield may include enhanced resistance to invasion by other species and increased chance of biofilm formation ; the latter being a desirable feature in several industrial fermentation systems. Extending the range of substrate utilization is another advantage that can be gained by combining C. saccharolyticus and C. kristjanssonii in a co-culture. For example, C. saccharolyticus ferments L-arabinose and L-rhamnose, whereas C. kristjanssonii does not grow on these sugars  and probably the latter would be able to utilize some substrates not readily consumed by the former, which merits further investigation.
AAZ acknowledges support from the Ministry of Higher Education in Egypt and the Swedish Energy Agency (Energimyndigheten; nr. 31090-1). This study was financially supported by the Commission of European Communities, Sixth Framework Program, Priority 6, Sustainable Energy Systems (019825 HYVOLUTION).
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