Effect of supplementation of nitrogen sources into RM
To understand the uncoupling of cell growth and ethanol production, we evaluated factors in the medium affecting cell growth. Since the growth condition of Z. mobilis is not complicated, and the recipe of RM is relatively simple containing carbon source such as glucose, phosphate source of KH2PO4, and yeast extract as the source of nitrogen, minerals and vitamins, 10 mM NH4Cl was added into RM using the yeast extract from OXOID (RMOXOID) to increase inorganic nitrogen content. However, extra NH4Cl supplemented into RMOXOID did not reduce the time for glucose utilization and ethanol production of ZM4 (Data not shown).
The effect of supplementation of different organic nitrogen sources such as peptone and tryptone were then tested. Our results showed that the supplementation of peptone did not reduce the time of glucose utilization. It still took more than 24-h for cells to consume up all glucose in the media with 20 g/L peptone supplemented into RMOXOID, although the final biomass in terms of OD600 value did increased slightly corresponding to the increase of peptone added (Fig. 1).
Subsequently, different concentrations of tryptone were added in RMOXOID. The results showed that the addition of extra tryptone could significantly increase both cell biomass and glucose consumption of ZM4 (Fig. 2). The addition of more than 5 g/L tryptone in RMOXOID can not only enhance both cell growth and glucose utilization of ZM4, but also couple the growth and ethanol fermentation of ZM4. The final highest OD600 value of ZM4 in RMOXOID with 5 g/L tryptone increased from 1.8 to 4.7, and all glucose was completed consumed up within 12 h while the one without tryptone had only half of glucose utilized at the same time point of 12-h post-inoculation (Fig. 2).
The positive effect of adding tryptone into RMOXOID was further evaluated by comparing cell growth as well as glucose utilization and ethanol production of ZM4 in both RMOXOID and RMOXOID+5T at different temperatures of 30, 36 and 40 °C. The results demonstrated that the supplementation of tryptone increased the growth and fermentation performance of ZM4 at different temperatures including the specific growth rate, ethanol yield, and productivity (Table 1).
Table 1
Ethanol yield (Yp/s), theoretical ethanol yield (%Yp/s), ethanol productivity (QP), and specific growth rate (µ) of ZM4 in RMOXOID and RMOXOID supplemented with 5 g/L tryptone at conditions of different temperatures.
Conditions | Yp/s | %Yp/s | Qp | µ |
30 ℃, RMOXOID | 0.39 ± 0.02 | 76 ± 4 | 0.67 ± 0.03 | 0.45 ± 0.01 |
36 ℃, RMOXOID | 0.36 ± 0.02 | 70 ± 4 | 0.59 ± 0.04 | 0.45 ± 0.01 |
40 ℃, RMOXOID | 0.37 ± 0.02 | 73 ± 4 | 0.61 ± 0.03 | 0.42 ± 0.00 |
30 ℃, RMOXOID+5T | 0.45 ± 0.00 | 89 ± 1 | 1.75 ± 0.01 | 0.49 ± 0.01 |
36 ℃, RMOXOID+5T | 0.43 ± 0.04 | 83 ± 8 | 2.19 ± 0.22 | 0.55 ± 0.01 |
40 ℃, RMOXOID+5T | 0.44 ± 0.02 | 85 ± 4 | 2.23 ± 0.11 | 0.53 ± 0.01 |
It also increased cell growth, glucose consumption rate, and ethanol productivity of ZM4 at higher temperatures of 36 and 40 °C than those at the normal temperature of 30 °C in RMOXOID supplemented with 5 g/L tryptone, although ethanol yields were similar among these conditions (Table 1, Fig. 3). Compared with normal temperature 30 °C, the time that ZM4 utilized all glucose at 36 and 40 °C in RMOXOID with extra tryptone was reduced by about 3 h, while one day was not sufficient for ZM4 to completely consume glucose at 30, 36, and 40 °C in RMOXOID (Fig. 3). ZM4 took less time to completely utilize glucose with maximum ethanol production achieved in RMOXOID+5T at 36 and 40 °C (9 h) than at 30 °C (12 h) with a corresponding higher growth rate and ethanol productivity, which was increased from 1.75 ± 0.01 at 30 °C to 2.19 ± 0.22 and 2.23 ± 0.11 at 36 and 40 °C, respectively (Table 1, Fig. 3).
Effect of exchanging yeast extract from OXOID with different brand ones in RM
As briefly mentioned above that yeast extracts distributed by different companies are produced with different processes leading to difference in the amount of total nitrogen as well as trace elements such as growth factors and metal ions. Besides adding extra nitrogen source into the RMOXOID as discussed above (Fig. 1, 2, 3), we also tested the effect of yeast extract from different companies including those from Becton Dickinson (YEBD) and Sangon Biotech Co., Ltd (YESG, Shanghai, China). In addition, another industrial nitrogen source of corn steep liquid (CSL) from Macklin was also evaluated at the same time with above two yeast-based nitrogen sources. All media used in this research were list in Table 3 and Table 4.
YEOXOID in RMOXOID was replaced with different nitrogen sources including peptone (P), tryptone (T), CSL, YEBD, and YESG. Cell growth, glucose utilization, and ethanol production of ZM4 cultured in these media were then examined. Our results showed ZM4 performed differently in these media (Fig. 4). The biomass of ZM4 in RM(T) was about two times higher than that in RMOXOID, while the biomass of ZM4 in RM(P) was the lowest (Fig. 4B). It seemed that tryptone was better than YEOXOID and peptone, which was consistent with above experiments of supplementation of nitrogen source into RMOXOID (Fig. 1, 2).
Except that the specific growth rates which can be seen from the slopes of the lines in exponential phase of ZM4 in RM with 1% of peptone or CSL as the sole nitrogen source were lower, the specific growth rates of ZM4 in other RM were almost the same (Fig. 4A). Compared with YEOXOID as the sole nitrogen source, YEBD, YESG and 5%CSL all increased the final biomass of ZM4 (Fig. 4B), reduced the time of glucose consumption (Fig. 4C), and made cell growth and ethanol production coupled. For example, the biomass of ZM4 in RMBD was three times more than that in RMOXOID, the time for complete glucose utilization in RMBD was about two-thirds shorter than that in RMOXOID (Fig. 4B, C). YESG was almost as good as YEBD with lower cost, which can be used in large-scale fermentation for economic ethanol production, and appropriate nitrogen source can be chosen as needed based on above results (Fig. 1, 2, 3, 4).
Effect of exchanging (NH4)2SO4 in MM with different organic nitrogen sources
Although the RM recipe is relatively simple containing only three ingredients as discussed above, the composition of organic nitrogen sources such as yeast extract is still complicated containing nitrogen, vitamins, metal ions etc. Minimum medium (MM) was therefore selected to further examine the effect of different organic nitrogen sources on ZM4 since the only nitrogen source in MM is the inorganic nitrogen source (NH4)2SO4. The representative organic nitrogen sources used to replace (NH4)2SO4 in MM included YEBD, YEOXOID, and 5%CSL.
Our results demonstrated that the replacement of inorganic nitrogen source (NH4)2SO4 with organic nitrogen source enhanced cell growth, glucose utilization, and ethanol productivity (Fig. 5). Interestingly, ZM4 had similar growth and glucose consumption rates in two MM media with YEBD or YEOXOID as the sole nitrogen source (Fig. 5), which was different from those when YEBD or YEOXOID was used as the sole nitrogen source in RM (Fig. 4). ZM4 grew better in RM with YEBD than that with YEOXOID as the nitrogen source (Fig. 4). Therefore, some ingredients must be existing in MM that RM lacks when YEOXOID was used as the nitrogen source, which leaves to the compositions of NaCl and metal ions of Na2MoO4 and MgSO4 that are different in the ingredients of these two media (Table 3, 4).
Since metal ions of magnesium and molybdenum are the cofactors of enzymes involving in various metabolic activities, we thus investigated the effect of metal ions of MgSO4 and Na2MoO4 by removing them from both MMBD and MMOXOID to test the growth and fermentation performance of ZM4. The lack of Mg2+ and MoO42− in MMBD had no effect on the growth of ZM4, but ZM4 had a delay period in MMOXOID lacking MoO42−, and the biomass decreased in MMOXOID lacking Mg2+ (Fig. 6). Apparently, this result indicated that MoO42− and Mg2+ in YEOXOID were different from those in YEBD. MoO42− and Mg2+ played important role on cell growth of Z. mobilis, and Mg2+ probably was the factor affecting the coupling of cell growth and ethanol production.
Determination of concentrations of ions in different nitrogen sources and the impact of the Mg 2+ on fermentation performance
In order to verify this hypothesis, the concentrations of several major ion elements in different organic nitrogen sources were then measured by ICP-OES. The result showed that the concentrations of these ion elements (Fe, K, Mg, P, S) were different among different nitrogen sources with less of these ions containing in peptone and tryptone (Table 2), which probably could be one of the reasons that cell growth and fermentation performance of Z. mobilis in media using tryptone and peptone as the sole nitrogen source was not as good as in other media using yeast extract or yeast powder.
At the same concentration of nitrogen sources, the Mg2+ in the yeast extract from OXOID was 8.23 mg/L less than that from Becton Dickinson. Mg2+ is the component with major difference between these two nitrogen sources of yeast extract from OXOID and BD companies, and therefore could be the reason that cell growth and ethanol fermentation of Z. mobilis in RMOXOID was not as good as in RMBD (Table 2). Since 0.5 g/L MgSO4·7H2O containing 49 mg/L Mg2+ was provided in MM which was sufficient for cell growth (Table 2, 4), the growth difference of Z. mobilis in MMOXOID and MMBD was therefore not as obvious as that in RMOXOID and RMBD (Fig. 4, 5). The concentration of Mg2+ in peptone was the lowest, which could also be one of the reasons that ZM4 grew poorly in RM(P) compared with other nitrogen sources (Fig. 4). These results further confirmed the importance of sufficient Mg2+ concentration in the media for optimal cell growth and ethanol fermentation.
Table 2
Concentrations of different ion elements in different nitrogen sources measured by ICP-OES.
Ion(mg/L) | 1% CSL | 1% YEOXOID | 1% YEBD | 1% YESG | 1% Peptone | 1% Tryptone |
Fe | 0.48 | 0.51 | 0.42 | 0.28 | 0.13 | 0.13 |
K | 280.71 | 492.74 | 456.13 | 311.83 | 5.52 | 1.07 |
Mg | 9.72 | 2.83 | 11.07 | 18.45 | 0.06 | 2.72 |
Mo | 0.03 | 0.0009 | 0.004 | 0.0008 | 0.0089 | 0.0072 |
Na | 20.61 | 19.16 | 48.83 | 131.30 | 27.80 | 49.56 |
P | 198.13 | 159.72 | 129.10 | 278.30 | 2.67 | 16.65 |
S | 56.88 | 58.69 | 85.92 | 65.59 | 5.21 | 17.04 |
To verify the impact of Mg2+ on cell growth, different concentrations of Mg2+ were then added into the RMOXOID and MMOXOID without MgSO4 (MMOXOID△MgSO4), and the growth of ZM4 in these media was measured by the Bioscreen C (Fig. 7). Our results indicated that in both RMOXOID and MMOXOID△MgSO4, even a small amount of Mg2+ at 4 mg/L could obviously boost cell growth.
The cell growth, glucose utilization, and ethanol production were further investigated using the shake flask experiment with OD600 values, glucose and ethanol concentrations measured (Fig. 8). Consistent with the result of Bioscreen C (Fig. 7), the supplementation of at least 8 mg/L of Mg2+ in RMOXOID or MMOXOID△MgSO4 allowed ZM4 to grow as well as in RMBD or MMBD△MgSO4. At the same time, cell growth, glucose consumption, and ethanol production of ZM4 in RMOXOID with 8 mg/L Mg2+ added were coupled as in RMBD (Fig. 8), which suggested that Mg2+ is crucial for cell growth and fermentation performance of Z. mobilis, and a minimal concentration of at least 8 mg/L is needed for optimal cell growth and ethanol fermentation.
Effects of Mg 2+ on gene expression of Z. mobilis
After confirmation of the impact of Mg2+ on cell growth and ethanol fermentation of Z. mobilis (Fig. 8), next-generation sequencing (NGS)-based RNA-Seq transcriptomics was further applied to identify genes responsive for the uncoupling of cell growth and ethanol fermentation due to the difference of Mg2+ concentration in the medium.
Despite that glucose consumption, cell growth, and ethanol production exhibited apparent difference (Fig. 8), RNA-Seq result showed that only a few genes differentially expressed. There were 7 and 4 genes down-regulated when Z. mobilis was cultured in RMOXOID and RMBD (RMOXOID/RMBD, Fig. 9A, Table S1) as well as cultured in RM and RMOXOID+8 Mg (RMOXOID/RMOXOID+8 Mg, Fig. 9B, Table S2), respectively. Although genes up-regulated in RMOXOID were more than those down-regulated (Fig. 9, Table S1, S2), there were still only 11 and 10 up-regulated when Z. mobilis was cultured in RMOXOID and RMBD (RMOXOID/RMBD, Fig. 9A, Table S1) as well as cultured in RMOXOID and RMOXOID+8 Mg (RMOXOID/RMOXOID+8 Mg, Fig. 9B, Table S2), respectively. Two and eight of these down-regulated and up-regulated ones are common between these two comparisons (Fig. 9, Table S1, S2).
Genes related to stress responses were up-regulated while genes related to protein secretion were down-regulated in RMOXOID medium compared with in RMOXOID+8 Mg or RMBD medium (Fig. 9, Table S1, S2). For example, genes encoding catalase (ZMO0918), general stress protein CsbD (ZMO0740), NADH dehydrogenase (ZMO1113), and succinate-semialdehyde dehydrogenase SSADH (ZMO1754) were up-regulated when RMOXOID was used compared with those in RMOXOID+8 Mg or RMBD (Fig. 9, Table S1, S2). These results indicated that Mg2+, a key element of cofactor, is essential for vigorous cell growth, and the lack of Mg2+ will trigger energy-consuming stress responses while slowing down energy-consuming metabolism with genes encoding levansucrase (ZMO0374) and protein secretion-related (ZMO0934) down-regulated (Fig. 9, Table S1, S2).
Considering the facts that the supplementation of 8 mg/L Mg2+ into RMOXOID (RM + 8 Mg) could restore the coupling of cell growth and ethanol fermentation of Z. mobilis (Fig. 8), and there were only one gene up-regulated (ZMO2034) and one down-regulated (ZMO1522) when Z. mobilis grew in RMBD compared with RM + 8 Mg (Fig. 9C), the transcriptomics study also further confirmed our hypothesis that the difference of Mg2+ concentrations in different nitrogen sources is one of the determinants affecting the coupling of cell growth and ethanol fermentation in Z. mobilis.