Underlying Mechanism of Uncoupled Cell Growth and Ethanol Fermentation of Zymomonas mobilis using Different Nitrogen Sources

Background: Microbial growth needs C, N, P, S as well as metal ions such as magnesium, which is a major cofactor for enzymes involved in various metabolic activities. Yeast extract is widely used as nitrogen supply as well as vitamins and growth factors to sustain microbial growth in the culture medium. Zymomonas mobilis is a model ethanologenic bacterium for ethanol production, and has been developing as a chassis for diverse biochemical production. Although yeast extract is routinely used to prepare rich medium (RM) for Z. mobilis, the glucose consumption and ethanol production of Z. mobilis in RM were not coupled with cell growth in some studies. Results: In this study, the effects of different nitrogen sources as well as the supplementation of additional nitrogen source into RM and minimum medium (MM) on cell growth and ethanol fermentation of Z. mobilis were investigated to understand the uncoupled cell growth and ethanol fermentation for ecient carbon utilization and optimal ethanol productivity of Z. mobilis. Our results indicated that nitrogen sources such as yeast extract from different companies affected cell growth, glucose utilization, and the corresponding ethanol production. We also quantied the concentrations of major ion elements in different organic nitrogen sources using the quantitative analytic approach of Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), and demonstrated that metal ions such as magnesium in the media affected glucose consumption, cell growth, and ethanol fermentation. The effect of magnesium on gene expression was further investigated using RNA-Seq transcriptomics, and our result indicated that the lack of Mg 2+ triggered stress responses while decreasing energy-consuming metabolism. Mg

Results: In this study, the effects of different nitrogen sources as well as the supplementation of additional nitrogen source into RM and minimum medium (MM) on cell growth and ethanol fermentation of Z. mobilis were investigated to understand the uncoupled cell growth and ethanol fermentation for e cient carbon utilization and optimal ethanol productivity of Z. mobilis. Our results indicated that nitrogen sources such as yeast extract from different companies affected cell growth, glucose utilization, and the corresponding ethanol production. We also quanti ed the concentrations of major ion elements in different organic nitrogen sources using the quantitative analytic approach of Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES), and demonstrated that metal ions such as magnesium in the media affected glucose consumption, cell growth, and ethanol fermentation. The effect of magnesium on gene expression was further investigated using RNA-Seq transcriptomics, and our result indicated that the lack of Mg 2+ triggered stress responses while decreasing energy-consuming metabolism.
Conclusions: Our work demonstrated that concentrations of metal ions such as magnesium and molybdenum in nitrogen sources are essential for vigorous cell growth, and the difference of Mg 2+ concentration in different yeast extract was one of the major factors affecting the coupling of cell growth and ethanol fermentation in Z. mobilis. We also revealed that genes responsive for Mg 2+ de ciency in the medium were majorly related to stress responses and energy conservation. The importance of metal ions on cell growth and ethanol fermentation suggested that metal ions should become one of the parameters for monitoring the quality of commercial nitrogen sources and optimizing microbial culture medium for economic biochemical production.
Rich medium (RM) containing carbon source such as glucose, nitrogen source such as yeast extract, and KH 2 PO 4 is commonly used to culture Z. mobilis. Nitrogen source in the medium was reported to affect the growth of Z. mobilis. For example, the morphology of Z. mobilis CP3 changed when cultured in medium with low nitrogen source [11]. Although nitrogen sources like peptone, corn steep liquid, and even N 2 can be used to sustain the cell growth of Z. mobilis [10][11][12], yeast extract preserving the B vitamins that naturally occur in yeast is the preferred nitrogen sources.
Yeast extract is the hydrolysate of yeast cells through autolysis or enzymatic hydrolysis, which can provide nitrogenous compounds, carbon, sulfur, trace nutrients, vitamin B complex and other important growth factors for various microorganisms. Despite that the total nitrogen content and free amino acid nitrogen in different yeast extracts are monitored, yeast extracts produced by different companies have different trace components such as growth factors and metal ions due to the differences in their production processes, which will affect the microbial cell growth since the vitamins and metal ions (e.g. Mg 2+ , MoO 4 2− , Cu 2+ , Zn 2+ , and Fe 2+ ) are cofactors of enzymes involved in various metabolic activities.
Our previous results and literature reports showed that cell growth of ZM4 and its ethanol production were uncoupled in a few studies. Glucose consumption was not coupled with cell growth, and glucose was not completely consumed for ethanol production when cells reached stationary phase [13][14][15][16][17][18][19]. This uncoupling phenomenon between growth and fermentation performance has also been reported in other microorganisms including yeast affecting the economic bioproduct production [20,21]. For example, ethanol production of yeast cells was reported to be related to the length of uncoupling phase during the batch fermentation process [22].
The uncoupling of growth and fermentation was majorly due to the stressful physical and chemical growth condition such as extreme temperature, pH and toxic compounds in the media, limited nutrient sources of carbon, nitrogen and metal ions used for fermentation were also reported to affect cell growth and fermentation performance [19][20][21]23]. Although the effect of diverse nitrogen sources on cell growth and fermentation performance has been reported in various microorganisms such as the ethanologen yeast and lactate-producer Sporolactobacillus inulinus [24][25][26], metal ions within nitrogen sources and their impact on cell growth and fermentation has not been investigated extensively with limited information available.
To understand the phenomenon of growth and fermentation uncoupling for both e cient carbon utilization and maximum ethanol productivity, we investigated the effects of different nitrogen sources on the growth and ethanol production of Z. mobilis in this study to help achieve the goal of optimal titer, rate, and yield (TRY) for economic bioethanol production using Z. mobilis. Our results demonstrated that glucose consumption and ethanol production can be coupled with cell growth by changing the nitrogen sources in the rich medium, and the concentration of metal ions such as Mg 2+ and MoO 4 2− in the nitrogen source could be one of the major factors affecting cell growth and ethanol production.

Results And Discussions
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 KH 2 PO 4, and yeast extract as the source of nitrogen, minerals and vitamins, 10 mM NH 4 Cl was added into RM using the yeast extract from OXOID (RM OXOID ) to increase inorganic nitrogen content. However, extra NH 4 Cl supplemented into RM OXOID 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 RM OXOID , although the nal biomass in terms of OD 600 value did increased slightly corresponding to the increase of peptone added (Fig. 1).
Subsequently, different concentrations of tryptone were added in RM OXOID . The results showed that the addition of extra tryptone could signi cantly increase both cell biomass and glucose consumption of ZM4 (Fig. 2). The addition of more than 5 g/L tryptone in RM OXOID can not only enhance both cell growth and glucose utilization of ZM4, but also couple the growth and ethanol fermentation of ZM4. The nal highest OD 600 value of ZM4 in RM OXOID 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 RM OXOID was further evaluated by comparing cell growth as well as glucose utilization and ethanol production of ZM4 in both RM OXOID and RM OXOID +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 speci c growth rate, ethanol yield, and productivity (Table 1). Effect of exchanging yeast extract from OXOID with different brand ones in RM As brie y 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 RM OXOID as discussed above ( Fig. 1, 2, 3), we also tested the effect of yeast extract from different companies including those from Becton Dickinson (YE BD ) and Sangon Biotech Co., Ltd (YE SG , 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.
Page 6/21 YE OXOID in RM OXOID was replaced with different nitrogen sources including peptone (P), tryptone (T), CSL, YE BD , and YE SG . 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 RM OXOID , while the biomass of ZM4 in RM(P) was the lowest (Fig. 4B). It seemed that tryptone was better than YE OXOID and peptone, which was consistent with above experiments of supplementation of nitrogen source into RM OXOID (Fig. 1, 2).
Except that the speci c 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 speci c growth rates of ZM4 in other RM were almost the same (Fig. 4A). Compared with YE OXOID as the sole nitrogen source, YE BD , YE SG and 5%CSL all increased the nal 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 RM BD was three times more than that in RM OXOID , the time for complete glucose utilization in RM BD was about two-thirds shorter than that in RM OXOID (Fig. 4B, C). YE SG was almost as good as YE BD 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 (NH 4 ) 2 SO 4 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 (NH 4 ) 2 SO 4 . The representative organic nitrogen sources used to replace (NH 4 ) 2 SO 4 in MM included YE BD , YE OXOID , and 5%CSL.
Our results demonstrated that the replacement of inorganic nitrogen source (NH 4 ) 2 SO 4 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 YE BD or YE OXOID as the sole nitrogen source (Fig. 5), which was different from those when YE BD or YE OXOID was used as the sole nitrogen source in RM (Fig. 4). ZM4 grew better in RM with YE BD than that with YE OXOID as the nitrogen source (Fig. 4). Therefore, some ingredients must be existing in MM that RM lacks when YE OXOID was used as the nitrogen source, which leaves to the compositions of NaCl and metal ions of Na 2 MoO 4 and MgSO 4 that are different in the ingredients of these two media ( of Mg 2+ and MoO 4 2− in MM BD had no effect on the growth of ZM4, but ZM4 had a delay period in MM OXOID lacking MoO 4 2− , and the biomass decreased in MM OXOID lacking Mg 2+ (Fig. 6) played important role on cell growth of Z. mobilis, and Mg 2+ 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 Mg 2+ in the yeast extract from OXOID was 8.23 mg/L less than that from Becton Dickinson. Mg 2+ 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 RM OXOID was not as good as in RM BD (Table 2).
Since 0.5 g/L MgSO 4 ·7H 2 O containing 49 mg/L Mg 2+ was provided in MM which was su cient for cell growth (Table 2, 4), the growth difference of Z. mobilis in MM OXOID and MM BD was therefore not as obvious as that in RM OXOID and RM BD (Fig. 4, 5). The concentration of Mg 2+ 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 con rmed the importance of su cient Mg 2+ concentration in the media for optimal cell growth and ethanol fermentation. To verify the impact of Mg 2+ on cell growth, different concentrations of Mg 2+ were then added into the RM OXOID and MM OXOID without MgSO 4 (MM OXOID △MgSO 4 ), and the growth of ZM4 in these media was measured by the Bioscreen C (Fig. 7). Our results indicated that in both RM OXOID and MM OXOID △MgSO 4 , even a small amount of Mg 2+ at 4 mg/L could obviously boost cell growth.
The cell growth, glucose utilization, and ethanol production were further investigated using the shake ask experiment with OD 600 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 Mg 2+ in RM OXOID or MM OXOID △MgSO 4 allowed ZM4 to grow as well as in RM BD or MM BD △MgSO 4 . At the same time, cell growth, glucose consumption, and ethanol production of ZM4 in RM OXOID with 8 mg/L Mg 2+ added were coupled as in RM BD (Fig. 8), which suggested that Mg 2+ 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 con rmation of the impact of Mg 2+ 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 Mg 2+ 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 RM OXOID and RM BD (RM OXOID /RM BD , Fig. 9A, Table S1) as well as cultured in RM and RM OXOID +8 Mg (RM OXOID /RM OXOID +8 Mg, Fig. 9B, Table S2), respectively.
Although genes up-regulated in RM OXOID 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 RM OXOID and RM BD (RM OXOID /RM BD , Fig. 9A, Table S1) as well as cultured in RM OXOID and RM OXOID +8 Mg (RM OXOID /RM OXOID +8 Mg, Fig. 9B, Table S2), respectively. Two and eight of these down-regulated and upregulated ones are common between these two comparisons ( Fig. 9, Table S1, S2).
These results indicated that Mg 2+ , a key element of cofactor, is essential for vigorous cell growth, and the lack of Mg 2+ 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 Mg 2+ into RM OXOID (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 RM BD compared with RM + 8 Mg (Fig. 9C), the transcriptomics study also further con rmed our hypothesis that the difference of Mg 2+ concentrations in different nitrogen sources is one of the determinants affecting the coupling of cell growth and ethanol fermentation in Z. mobilis.

Conclusion
The uncoupling of cell growth and fermentation will increase the cost of microbial biochemical production. The effects of nitrogen sources on cell growth and ethanol fermentation performance of Z. mobilis ZM4 were investigated to understand the uncoupling of cell growth and ethanol fermentation of ZM4 in this study. Through the supplementation and replacement of inorganic or organic nitrogen sources in both RM and MM, we found that YE BD , YE SG , or 5%CSL were better than YE OXOID . We also quanti ed the ion elements in different nitrogen sources using ICP-OES, and demonstrated that the difference of metal ions of MgSO 4 and Na 2 MoO 4 in yeast extract could be one of the major factors affecting cell growth and ethanol fermentation. We further veri ed the impact of Mg 2+ on cell growth of ZM4 by supplementing various concentrations of Mg 2+ into the medium, and identi ed genes involved in stress response and energy conservation responsive for the uncoupling of cell growth and ethanol fermentation when the medium was lack of Mg 2+ using RNA-Seq transcriptomics approach. These ndings can be used as a reference for the selection and/or modi cation of nitrogen sources for economic ethanol production using Z. mobilis ZM4. The concentrations of ion elements in nitrogen sources affecting cell growth and fermentation performance can also be used as a parameter for optimizing and monitoring the components of nitrogen sources for e cient cell growth and fermentation using other microorganisms.
The recipes of different rich medium (RM) and minimum medium (MM) with different nitrogen sources used in this work were listed in Tables 3 and 4, respectively. Table 3 Recipes  Table 4 Recipes

Growth test by Bioscreen C
The seed culture of Z. mobilis was centrifuged to remove RM medium. Cells were resuspended with test medium. Bioscreen C assays were carried out as described previously [16, [27][28][29][30][31] except that cells were inoculated into Bioscreen C wells containing a total volume of 200 µL test medium at an initial OD 600 value of 0.05 and incubated without shaking at 30 °C. Triplicate were used for each condition, and turbidity measurements (OD 600 ) were taken every 15 min till cells grew into stationary phase.

Flask fermentation and analytical analysis
The seed culture of Z. mobilis was used to inoculate shake ask containing 80% of test medium at an initial OD 600 of 0.1, and cultured at 30 ℃, 100 rpm. At least two replicates were used for each condition.
The OD 600 values of the bacterial culture was measured by UV-visible spectrophotometer UV-1800 (AoYi Instrument Co., Ltd, Shanghai, China) every 3 h. At the same time, 1-mL culture was centrifuged at 12,000 rpm for 1 min to obtain the supernatant for measuring the glucose and ethanol concentrations in the culture. Biosensor analyzer M-100 (Sieman Technology Co., Ltd., Shenzhen, China) was used for quick assessment of the concentrations of glucose and ethanol. The supernatant was also ltered through a 0.45-µm lter before applying on a Shimadzu LC-2030 high pressure liquid chromatography (HPLC) with refractive index detector (RID). Bio-Rad Aminex HPX-87H (300 × 7.8 mm) column was used to separate the fermentation products, and 0.005 M H 2 SO 4 was used as the mobile phase at a ow rate of 0.5 mL/min. Temperatures of detector and column were 40 and 60 ℃, respectively.
One percent (w/v) of different organic nitrogen sources were prepared in ddH 2 O and then lter-sterilized.
The mobilis was used as the reference for RPKM calculation [33,36]. The RPKM value of each gene was then imported into JMP Genomics (Ver. 9.0), and data normalization and statistical analysis were conducted to identify differentially expressed genes when three different media were used. Duplicate samples were used for each condition.      Cell growth (A), nal OD600 value (B), as well as glucose (Glu) consumption and ethanol (Eth) production (C) of ZM4 in RM with different nitrogen sources. At least two independent experiments were carried out with similar results. Values are the mean of one representative experiment with two or more technical replicates. Error bars represent standard deviations.

Figure 5
Cell growth, glucose (Glu) consumption and ethanol (Eth) production of ZM4 in MM with different nitrogen sources. At least two independent experiments were carried out with similar results. Values are the mean of one representative experiment with two or more technical replicates. Error bars represent standard deviations.

Figure 6
Page 20/21 Cell growth, glucose (Glu) consumption and ethanol (Eth) production of ZM4 in MM with original inorganic nitrogen sources replaced by YEBD and YEOXOID, respectively(A), as well as in above two media with Mg2+ removed (B), or MoO42-removed (C). At least two independent experiments were carried out with similar results. Values are the mean of one representative experiment with two or more technical replicates. Error bars represent standard deviations. were included as control. At least two independent experiments were carried out with similar results.
Values are the mean of one representative experiment with two or more technical replicates. Error bars represent standard deviations.

Figure 8
Cell growth, glucose (Glu) consumption and ethanol (Eth) production of ZM4 in RMOXOID, RMOXOID+8Mg (RMOXOID with 8 mg/L Mg2+ added), RMBD, and RMSG. At least two independent experiments were carried out with similar results. Values are the mean of one representative experiment with two or more technical replicates. Error bars represent standard deviations.

Figure 9
Volcano plots of signi cantly differentially expressed genes of Z. mobilis cultured in RMOXOID and RMBD (RMOXOID/RMBD, A), RMOXOID and RMOXOID+8Mg (RMOXOID/RMOXOID+8Mg, B), as well as RMBD and RMOXOID+8Mg (RMBD/RMOXOID+8Mg, C). RMOXOID+8Mg is RMOXOID with 8 mg/L Mg2+ added. X-axis is the log2-based ratios between two conditions examined, and Y-axis is the -log10(P-value) of the difference. The dots above the horizontal red dash line indicate genes signi cantly differentially expressed, and the vertical red dash line indicate genes signi cantly differentially expressed with ratio greater than 2 (log2-based ratio greater than 1). Gene name with red and blue color font indicates upregulated and down-regulated ones, respectively. Gene name with bold font indicates common ones between different comparisons of RMOXOID/RMBD, RMOXOID/RMOXOID+8Mg, and