Experimental characterization of C. cellulolyticumcellulolytic activity at high cellulose concentration, addressing the pH effect
In the previous co-culture study [19], the pH effect on C. cellulolyticum growth and cellulolytic activity has not been addressed; therefore, to investigate the pH effect on C. cellulolyticum growth and cellulolytic activity in a mono-culture at high cellulose concentration of 20 g/L, two batch cultures of C. cellulolyticum were conducted: one batch at pH of 7.2, which is an optimal pH for C. cellulolyticum growth and cellulolytic activity [21], and another batch with the same pH profile as the co-culture run, i.e. initially at pH of 7.0 for 2 days followed by a pH switch to 6.0. The profiles of cellulose solubilization and biomass concentration are shown in Figure 2. In the mono-culture at pH of 7.2, TOC data showed that 1.5 g/L of carbon was solubilized after 14 days, which is equivalent to 4.9 g/L of cellulose degraded, assuming 24% of carbon flow goes towards CO2 formation; this amount of degraded cellulose was comparable to 5.1 g/L reported for the C. cellulolyticum mono-culture at the same initial cellulose concentration [19, 22]. Also, qPCR data showed that cells reached a stationary growth phase after 9 days with a maximum cell density of 5× 109 cell/mL and a specific growth rate of 1.23 day-1.
In contrast, the mono-culture run under co-culture pH profile did not show any cellulose solubilization during the batch (Figure 2), and cells had a very low growth rate of 0.16 day-1 (87% decrease) compared to the mono-culture batch at pH of 7.2. The effect of pH on C. cellulolyticum growth and cellulolytic activity at low cellulose concentration of 3.7 g/L has been addressed previously [21], and it has been shown that C. cellulolyticum is significantly affected by acidic pH, where a pH drop from 7.0 to 6.4 leads to fourfold lower biomass concentration. It has been suggested that acidic pH hampers biomass formation, likely through direct effect of pH on a cellular constituent such as an enzyme or a transport protein, rather than cellulose degradation capability in C. cellulolyticum. This argument is supported by the observation that the flux through cellulose degradation reaction remains almost unvarying, in the range of 1.69 to 1.84 mmol (g cell. h)-1, while pH varies between 6.4 to 7.0 [21]. However, regardless of the mechanism of inhibition, this pH effect must be considered when comparing cellulolytic activity and C. cellulolyticum growth and metabolism in the mono-culture and co-culture.
Experimental characterization of the co-culture metabolism
To prepare the co-culture medium for the first co-culture experiment A, all media components including the iron solution were autoclaved. The profile of cellulose degradation in this co-culture is shown in Figure 3a. At initial cellulose concentration of 20 g/L, which is equivalent to 6.08 g/L of final TOC concentration, assuming full degradation of cellulose and considering 24% of the total carbon would be used toward CO2 formation, about 82% of cellulose was degraded after 28 days in the co-culture experiment A. Compared to the mono-culture at optimal pH of 7.2, cellulose degradation showed about 82% improvement as shown in Figure 3c, whereas no cellulose degradation was observed in the mono-culture run under co-culture pH profile. These results confirm that C. acetobutylicum metabolic activity significantly improves the cellulolytic activity in the co-culture, and in fact makes it possible for C. cellulolyticum to survive under harsh co-culture conditions, which do not allow it to grow and metabolize cellulose independently.
Furthermore, to check and ensure iron sufficiency in the co-culture medium, the co-culture medium was prepared by filter sterilizing the ferrous sulphate solution (experiment B), rather than autoclaving as for co-culture experiment A, to avoid potential iron oxidation during medium preparation. The results of this experiment are shown in Figure 3b; while the cellulolytic activity was high in the co-culture experiment B, and C. cellulolyticum growth rate was still as high as experiment A, but the cellulose degradation was declined by 38% compared to experiment A, as it is presented in Figure 3c. Cellulose degradation in each batch was estimated from carbon balance and TOC measurements, taking into account that about 24% of the total carbon has been utilized for CO2 formation, as described in analysis section. These results confirmed that the co-culture was not under iron limiting condition, and the presence of more ferrous ions had an adverse effect on the co-culture cellulolytic activity.
The mechanism underlying such a synergy between the two clostridial species is not clearly understood. Hence, in order to understand the nature of interactions between C. acetobutylicum and C. cellulolyticum, that improve cellulose solubilization, we used qPCR to track the population of each species in the batch cultures. Figure 3a and b show the dynamic profiles of each species population in the co-culture batches. C. cellulolyticum biomass concentration reached to 3×109 cell/mL in the co-cultures, which was the same value for maximum biomass concentration in the mono-culture runs; however the growth dynamics was significantly faster in the co-cultures compared to the mono-culture run under the same pH profile. C. cellulolyticum growth rate in the co-culture was comparable to its growth rate in mono-culture under optimal pH condition of 7.2. Also, it could be observed from all co-culture TOC profiles that cellulose solubilization started after C. cellulolyticum had reached its late exponential growth phase.
C. acetobutylicum biomass profiles are also shown in Figure 3, where the initial decrease in biomass concentration in the co-culture could be attributed to the lack of available sugars for C. acetobutylicum to grow while C. cellulolyticum had been in the lag phase. Furthermore, a considerable growth could be observed for C. acetobutylicum, when C. cellulolyticum had reached its maximum concentration in the co-culture, where possibly more sugars became available in the co-culture to support C. acetobutylicum growth. Although C. acetobutylicum biomass concentration showed significant increases at some points over the course of co-culture, it was fluctuating and did not remain constant. It has been suggested that in the microbial communities growing on cellulosic material, where there is a competition between cellulose-adherent cellulolytic microorganisms and non-adhered microbes for cellulose hydrolysis products, cellulose-adherent cellulolytic microorganisms are possibly successful winners [2], and this phenomenon could explain the limited growth of C. acetobutylicum in the co-culture.
In addition, C. acetobutylicum has cellobiase and endoglucanase activities, but is not able to hydrolyze crystalline cellulose due to lack of the required enzymatic activities [23], although it produces cellulosome [24]; therefore, the improved cellulolytic activity in the co-culture cannot be attributed to C. acetobutylicum cellulolytic activity. However, C. acetobutylicum is able to ferment cellobiose and cellulose-derived sugars, and the improved cellulolytic activity in co-culture can be attributed to the role of C. acetobutylicum in consuming sugars and preventing the carbon overflow toward C. cellulolyticum, as C. cellulolyticum is unable to metabolize high concentrations of cellobiose [25].
Also, degree of synergism (DS), defined as the activity of a mixture of components divided by the sum of the component activities evaluated separately [26], in this co-culture can be estimated as the ratio of C. cellulolyticum growth rates in co-culture and mono-culture under pH profile, and was equal to 7.99; although the co-culture DS determined based on the cellulolytic activities in the co-culture and the mono-culture would be substantially high, which indicates the presence of a strong synergism in this clostridial co-culture. The DS value of 5 or higher is not very common in enzyme-microbe cellulose hydrolysis systems and has been observed under some conditions [27, 28]. Moreover, in this co-culture on fibrous cellulose, observed maximum cellulose degradation rate of 0.108 g/(L. h) is comparable with cellulose degradation rate of 0.15 g/(L. h) in C. thermocellum culture on crystalline cellulose, which shows one of the highest cellulose utilization rates among cellulolytic microorganisms [26].
Since cellulolytic bacteria are unable to grow at low intracellular pH, under acidic environment the pH gradient (ΔpH) across the cell membrane is high; consequently, the intracellular dissociation of fermentation acids, which are membrane permeable in undissociated form, and the intracellular accumulation of acid anions lead to anion toxicity, which is the likely reason of growth inhibition under acidic condition [29, 30]. Furthermore, it has been shown that presence of lactate and acetate ions in an acidic medium leads to a significant decline of glutamate synthesis in Clostridium sporogenes MD1, which inhibits the bacterial growth [30]. Also, for E. coli culture at pH of 6, incubation of cells with 8 mM acetate for 20 min was shown to result in intracellular accumulation of acetate anions (240 mM), and reduced level of intracellular glutamate pools [31]. Furthermore, in mildly acidic E. coli cultures (pH of 6), inhibition of methionine biosynthesis by acetate (8 mM) and the toxicity of accumulated homocysteine have been indicated as the cause of growth inhibition by acetate under weak acid stress [32]. Addition of methionine to this culture can restore E. coli growth rate to some significant extent. This effect has been also reported for other organic acids.
In this clostridial co-culture, the synergy could be attributed to the exchange of some growth precursors and biomass constituents between C. acetobutylicum and C. cellulolyticum, which potentially enables the cellulolytic organism to grow and metabolize cellulose under acidic pH condition. C. acetobutylicum is a fermentative bacterium which is able to grow well under acidic conditions in acidogenic and solventogenic growth phases. The results of co-culture experiment under low concentration of C. acetobutylicum, which are presented in the next section, also provides support for the role of C. acetobutylicum.
Experimental characterization of the co-culture metabolism with low concentration of C. acetobutylicum
To investigate if high initial concentration of C. acetobutylicum contributes to its low growth in the co-culture, and if C. acetobutylicum growth in the co-culture is affected by the ratio of cells to the cellulose hydrolysate, a co-culture experiment was conducted with 100 times lower initial concentration of C. acetobutylicum. 2 mL of C. acetobutylicum culture at exponential growth phase was centrifuged and the cell pellets were suspended in 2 mL of CGM medium and incoculated into the bioreactor. The results of this experiment are presented in Figure 4, where no cellulose degradation was observed in either biological replicates. Also, C. cellulolyticum as well as C. acetobutylicum did not grow in the co-culture. This experiment confirmed that the role of C. acetobutylicum in the metabolism of cellulose by the co-culture is associated with the population of C. acetobutylicum, and can be attributed to the exchange of some metabolites between the two species.
Furthermore, the metabolic behavior of C. acetobutylicum under the co-culture conditions was investigated (Additional file 1), where this study confirmed the metabolism of pyruvate and the released sugars by C. acetobutylicum in the clostridial co-culture, and that the observed oscillations in the C. acetobutylicum concentration in the co-cultures could be due to the slow release of sugars by C. cellulolyticum that can lead to starvation cycles for C. acetobutylicum in the co-culture.
Analysis of product formations in the co-culture
Figure 5 shows the ranges for co-culture and mono-culture product concentrations after 28 days. As it can be noted, acetate, ethanol, lactate, butyrate, and butanol were the main products of the fermentation in the co-culture. Butyrate appeared after C. acetobutylicum inoculation in the co-culture, but its concentration remained low. Neither acetate nor butyrate uptake, which are the characteristics of the solventogenic phase in C. acetobutylicum metabolism, was observed in this co-culture. At high cellulose concentration, C. cellulolyticum produces lactate as its main product along with acetate and ethanol [22]. The lactate uptake, observed in the co-culture batches, coincided with butanol formation (Additional file 1). The lactate uptake can be related to C. acetobutylicum metabolic activity, as C. acetobutylicum ATCC824 has been shown to co-ferment lactate and glucose [33].
It has been previously shown [22] that in pH controlled batch cultures of C. cellulolyticum on a defined medium, the distribution of carbon flow depends on the initial cellulose concentration. For concentrations less than 6.7 g/L of cellulose, acetate, ethanol, CO2 and H2 were shown to be the main fermentation end products and more than 91% of cellulose was observed to be degraded. At higher cellulose concentrations, from 6.7 g/L up to 29.1 g/L, carbon flow is redirected from ethanol and acetate towards lactate and extracellular pyruvate. In addition, in batch cultures of C. cellulolyticum on high cellulose concentration, it has been shown that the peak of pyruvate formation coincides with the start of lactate formation, and this pyruvate accumulation in the C. cellulolyticum culture shows that the rate of cellulose catabolism is higher than the rate of pyruvate consumption. Also it has been suggested that the cellulose hydrolysis depends on the concentration of C. cellulolyticum, which remains constant at and above 6.7 g/L of cellulose [22].
Furthermore, it has been shown that re-inoculating a fresh culture of C. cellulolyticum at high cellulose concentration of 29.1 g/L, where substrate is not fully consumed, significantly improves the cellulose solubilization and biomass yield compared to a classical batch [22]. This result indicates that the incomplete cellulose catabolism is not due to either the limitation of adhesion sites on cellulose fibers or product inhibition. At high cellulose concentrations, the likely explanation for the incomplete cellulose consumption is the lack of control on carbon uptake flow and an imbalanced metabolism leading to the accumulation of intracellular metabolites and self-intoxication of the cells, eventually resulting in a growth arrest [22, 34]. Similarly, extracellular pyruvate formation has been reported in C. thermocellum cultures at high cellulose and cellobiose concentrations, which evidences the overflow metabolism [35].
In our experiments, the maximum concentration of C. cellulolyticum in co-culture experiments was the same as the mono-culture experiment under optimal pH of 7.2, however the cellulose degradation was improved up to 82% (Figure 3c), which confirms the role of C. acetobutylicum in cellulose degradation, while C. cellulolyticum has reached the stationary growth phase. We observed pyruvate accumulation of 0.029 g/L in the mono-culture batch under the co-culture pH profile and 0.004 g/L in the mono-culture batch at pH of 7.2. In the co-culture replicates, maximum pyruvate concentration of 0.17 g/L was observed, which was taken up later during the course of experiments coinciding with butyrate formation in the co-cultures. Our previous modeling studies have suggested that limited pyruvate-ferredoxin oxidoreductase (PFO) activity, which cannot support high pyruvate flow, results in pyruvate overflow [36]. Hence, a potential explanation for pyruvate secretion in C. cellulolyticum cultures is the limitation on the pyruvate consumption rate and a comparatively higher carbon catabolism rate, and due to inefficient regulation of entering carbon flow [25]. Furthermore, intracellular pyruvate accumulation could be the explanation for the growth arrest at high cellulose concentrations [37], at which cells switch to stationary growth phase before substrate depletion.
Pyruvate uptake in the co-culture can be explained by the capability of C. acetobutylicum to metabolize pyruvate. It has been also reported that providing C. acetobutylicum with pyruvate as the sole carbon source results in the growth and production of mainly acetate and butyrate [38]. In another co-culture study, the removal of C. cellulolyticum metabolic products such as pyruvate and their associated inhibitory effects, by Rhodopseudomonas palustris in the co-culture of C. cellulolyticum and R. palustris has been reported as the underlying reason for the improved cellulose degradation and bacterial growth in this co-culture [39]. C. cellulolyticum growth on cellulose has been shown to be severely inhibited by pyruvate; where about 60% decrease in the biomass concentration in the presence of 2 mM (176 mg/L) pyruvate has been observed in C. cellulolyticum mono-culture [39]. Therefore, pyruvate removal by C. acetobutylicum and alleviating its inhibitory effect can be a contributing factor in the improved growth of C. cellulolyticum and its boosted cellulolytic activity in the co-culture.
The major products of pyruvate fermentation by C. acetobutylicum are acetate, butyrate and butanol, and neither acetate nor butyrate is reutilized. The effects of pyruvate on glucose fermentation by C. acetobutylicum have also been investigated before, and it has been shown that both substrates can be fermented simultaneously [40]. Furthermore, cellobiose and glucose were only detected at the early stage of batches, which could have been present in the pre-cultures, inoculated into the bioreactors, and were taken up in 24 hours. Cellobiose and glucose could not be detected in the course of co-cultures which indicated their immediate consumption in the co-culture. In conclusion, in this study we showed a strong synergism between the two species of clostridia in the co-culture, and found that C. acetobutylicum enables C. cellulolyticum to grow under harsh co-culture environment. This synergy can be attributed to the production of some growth pre-cursors, and future metabolomic studies of this co-culture can identify such metabolites.