Aerobic Utilization of Xylose/Glucose Mixtures
Escherichia coli ZSC113 and E. coli ALS1008 are unable to consume glucose and xylose, respectively. The xylose-selective strain ZSC113 has mutations in the three genes involved in glucose uptake [12], rendering it unable to consume glucose: ptsG codes for the Enzyme IICBGlc of the phosphotransferase system (PTS) for carbohydrate transport [13], manZ codes for the IIDMan domain of the mannose PTS permease [14], glk codes for glucokinase [12]. We constructed strain ALS1008 which has a knockout in the xylA gene encoding for xylose isomerase, rendering ALS1008 unable to consume xylose. In a medium composed of a mixture of these two sugars, ZSC113 would be expected to consume the xylose selectively while ALS1008 should exclusively consume the glucose. We first sought to verify these expectations in three aerobic batch experiments.
In a first (control) experiment, a defined medium containing both 8 g/L xylose and 15 g/L glucose was inoculated with a single wild-type strain, MG1655, and grown aerobically (Figure 1). The glucose/xylose mixture was chosen to reflect the concentrations of glucose and xylose that are found in typical lignocellulosic hydrolysates. As expected, we observed diauxic growth as reported by many other studies when a single strain is inoculated into a medium containing two or more carbon sources. The important observations were that glucose and xylose were consumed sequentially, and that the complete consumption of this mixture required about 8.5 hours.
In a second set of aerobic experiments, the same defined medium containing two carbon sources was inoculated with one or the other of the two strains, ZSC113 or ALS1008. In the fermenter inoculated with only ZSC113 (Figure 2a), 8 g/L xylose was completely consumed in 7 h and the OD reached 10, In this case, the concentration of glucose remained unchanged. In the fermenter containing only ALS1008 (Figure 2b), we observed the complete consumption of 15 g/L glucose in 7.5 h with the OD reaching 15, while the concentration of xylose remained unchanged. As expected the two strains each consumed only one of the sugars, leaving the other carbohydrate unconsumed.
In a third aerobic batch experiment, we inoculated both ZSC113 and ALS1008 into a single fermenter containing 8 g/L xylose and 15 g/L glucose. For this co-culture, glucose was consumed in 7.5 h, and xylose was simultaneously consumed in 7.0 h (Figure 3). Moreover, the final OD of this mixed culture was about 25, identical to the sum of the ODs achieved in the fermentations in which one or the other carbohydrate was used. Thus, each strain appears to grow and consume its substrate independently. The combined process (i.e. consuming both sugars simultaneously) occurred at the same rate as the two individual processes so that each consumption rate was unaffected by the presence of the other carbohydrate. Compared to the wild-type (single organism) process, this process required about 15% less time to consume the same carbohydrate mixture aerobically, and moreover each substrate was consumed independently. The single-organism process (Figure 1) was completely different than the dual-organism process (Figure 3) in which both carbon sources were consumed simultaneously. No products were observed in these batch fermentations.
Aerobic Fed-Batch Utilization of Xylose/Glucose Mixtures
When a microorganism grows in a substrate-limited fashion (e.g. in a fed-batch process), the growth rate is controlled by the rate that the limiting substrate is supplied. Moreover, the concentration of that substrate remains at zero. In a bioprocess with two substrate-selective organisms which are both under carbon-limiting conditions, each organism should independently be controlled by and adapt to the quantity of the carbon source present that it can consume. We wished to test this hypothesis using a fed-batch process in which the two-carbohydrate feed increased exponentially at a nominal rate of 0.1 h-1, far below the maximum growth rate of either strain. Moreover, in addition to the flowrate exponentially increasing to maintain a fixed specific growth rate, the composition of the feed changed in discrete shifts in order to simulate a variable concentration that might be encountered in a real process. Specifically, for the first 20 h we maintained feed concentrations at 20 g xylose/L and 30 g glucose/L (20:30). At 20 h, this feed was replaced by feed concentrations of 30:30, at 30 h to 30:60, and then finally to 20:60 at 40 h. At 20 h, 30 h, 40 h and 50 h, we determined the fraction of the population which was the glucose-consuming strain ALS1008 (and thus by difference the fraction which was the xylose-consuming strain ZSC113).
During the entire fed-batch process, the xylose and glucose concentrations in the fermenter remained at zero (Figure 4), demonstrating that each substrate individually limited the process. Moreover, the distribution of the microbial population responded in unison with the shift in substrate concentrations. At 20 h, after the process had acclimated to a 20:30 xylose:glucose composition (g/L), the population was 35% ZSC113 (i.e., the xylose-consuming strain). Ten hours after the feed composition shifted to 30:30, the population was 50% ZSC113. Similarly, ten hours after the feed composition shifted to 30:60, the population returned to 42% ZSC113, and then ten hours after the feed had become 20:60, the population decreased to 32% ZSC113. These results demonstrate that the process adjusts the distribution of strains to match the distribution of substrates.
Anaerobic Product Formation with Xylose/Glucose Mixtures
Wild-type E. coli is a mixed acid fermenter, and generates acetate, lactate, formate, ethanol and succinate under anaerobic conditions, with the yield of each depending on the strain and carbon source [15]. ZSC113 and ALS1008 do not have any additional mutations which would cause the product distribution to be different from the wild-type parent MG1655. Three experiments were conducted under anaerobic conditions, analogous to those conducted under aerobic conditions previously.
In a first experiment, wild-type MG1655 was grown under aerobic conditions as before to consume 15 g/L glucose and 8 g/L xylose. When both substrates were nearly consumed (after about 8.5 h as shown in Figure 1), we added enough glucose and xylose into the fermenter approximately to return both sugar concentrations to their initial levels. Anaerobic conditions were initiated under an atmosphere of 100% CO2, and the products were measured during the anaerobic growth phase (Figure 5). In this culture of a single strain (having an OD of 21), 10 g/L of glucose was consumed in about 2.5 h (4 g/Lh), equivalent to a specific glucose consumption rate of 630 mg/gh. Initially xylose was consumed at a rate of 310 mg/gh (2 g/Lh). However, after 4 h of anaerobic conditions, the xylose consumption rate decreased to less than 150 mg/gh (1 g/Lh), and continued to slow. Nearly 4 g xylose/L remained after 9 h of anaerobic conditions. It must be noted that the organism consumed glucose then xylose in the aerobic phase preceding these anaerobic conditions (as shown in Figure 1), and therefore at the time of the switch to anaerobic conditions, the xylose-consuming pathways were fully induced. One explanation for the substantial decrease in xylose consumption rate is the sensitivity of xylose-degradation to the presence of acetate as previously reported for yeast [16, 17]. This explanation is supported by the observation that xylose consumption continued to slow even after glucose was depleted under anaerobic conditions.
In a second experiment, the two substrate-selective strains ALS1008 and ZSC113 were grown individually on the mixed substrate medium, the one depleted substrate added back, and then anaerobic conditions commenced. For the case of the xylose-consuming strain ZSC113, xylose was consumed at a constant rate of 1.4 g/Lh during the anaerobic phase and glucose was not consumed (Figure 6a). This single organism was present only at an OD of 9.5, so that on a specific basis the xylose consumption rate was 500 mg/gh, greater than the highest rate observed in the xylose portion of the fermentation using the wild-type MG1655. For the experiment in which the glucose-consuming strain ALS1008 was inoculated into the mixed substrate medium, glucose was exclusively consumed during the anaerobic phase at a constant rate of 3 g/Lh, and xylose was not consumed (Figure 6b). In this case, the specific glucose consumption rate was about 770 mg/gh, greater than the rate we observed for the wild-type MG1655 during the anaerobic phase (i.e., Figure 5). These two separate fermentations demonstrate that the strains will each consume only one substrate under anaerobic conditions and that they will consume this substrate slightly faster on a specific basis than the wild-type strain would under identical conditions.
In a third experiment, we first simultaneously grew both strains in the mixed substrate medium under aerobic conditions. At the end of the 7.0 h aerobic growth phase, we added enough of both carbohydrates to return them to their initial concentrations, and anaerobic conditions were initiated. In this process, both xylose and glucose were quickly consumed (Figure 7). Although we did not measure the proportion of the two strains, from previous aerobic results using one substrate (i.e., Figure 2), we estimate that the OD of ZSC113 was about 13 and the OD of ALS1008 was about 9. Over the first two hours of the anaerobic phase, the xylose consumption rate was therefore about 475 mg/gh, while the glucose consumption rate was about 1300 mg/gh. The key point in these results is that the two-strain process is much faster than an otherwise identical single-strain process.
The products formed from the two-sugar fermentation were the same as those generated during either one of the single-sugar fermentations, although the distribution of products changed slightly. For example, the succinate yield from xylose using ZSC113 was 0.48 mol/mol, while the succinate yield from glucose using ALS1008 was 0.30 mol/mol. From the sugar mixture, the observed succinate yield by two organisms was 0.41 mol/mol sugar consumed, a value between the yields of the individual strains on the two substrates. For all three cases, formate was generated with the highest yield (1.24 mol/mol glucose, 1.58 mol/mol xylose, and 1.46 mol/mol sugar mixture), and lactate was generated the least.
One limitation of this study was that high concentrations of acid products are known to inhibit growth and substrate consumption rates. This phenomenon would tend to affect the mixed culture more than either single-sugar culture, since for the former case both sugars would quickly be converted into more mixed acid products. This substrate-selective approach may perform significantly better for strains specifically designed to accumulate a single product such as ethanol which does not cause acid inhibition.