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Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis
Abstract
Zymomonas mobilis ZM4/Ac^R (pZB5), a mutant recombinant strain with increased acetate resistance, has been isolated following electroporation of Z. mobilis ZM4/Ac^R. This mutant strain showed enhanced kinetic characteristics in the presence of 12 g sodium acetate l^−1 at pH 5 in batch culture on 40 g glucose, 40 g xylose l^−1 medium when compared to ZM4 (pZB5). In continuous culture, there was evidence of increased maintenance energy requirements/uncoupling of metabolism for ZM4/Ac^R (pZB5) in the presence of sodium acetate; a result confirmed by analysis of the effect of acetate on other strains of Z. mobilis.Nomenclature mCell maintenance energy coefficient (g g^−1 h^−1) Maximum overall specific growth rate (1 h^−1) Maximum specific ethanol production rate (g g^−1 h^−1) Maximum specific total sugar utilization rate (g g^−1 h^−1) Biomass yield per mole of ATP (g mole^−1Ethanol yield on total sugars (g g^−1) Biomass yield on total sugars (g g^−1) True biomass yield on total sugars (g g^−1)
... In Eq. (2), z max and z min are the maximum and minimum inhibition terms. For xylose consumption, substrate [ ] is the sum of the xylose and glucose concentrations, i.e. it is assumed that the presence of glucose reduces the AA inhibition of the xylose consumption rate [36,37]. For glucose, substrate [ ] is the glucose concentration only. ...
... For the reaction rate term describing biomass growth, z min was set equal to z max , i.e. it is assumed that there is a significant inhibition of this reaction rate [36]. The values of z min , z max and S [ ] min were obtained via kinetic fitting using data available in the literature from two co-fermentations that used glucose and xylose by a mutant strain with increased acetate resistance, Zymomonas mobilis ZM4/AcR (pZB5) [37]. All other model parameters used were those reported by [31]. ...
... Data points are experimental data: glucose (stars), xylose (diamonds), ethanol (triangle) and ethanol (diamond). Jeon et al. reported experimental data[37]. Continuous lines in a) and b) are the predictions obtained using the model without AA inhibition terms proposed by Grisales Díaz and Willis, 2019[31] and in c) and d) are the predictions using the model proposed in this work. ...
This paper considers the economic assessment and optimisation of a bioethanol production process using corn stover (CS) as the feedstock. This includes a comparison between the use of batch and fed-batch reactors with and without deacetylation. As a basis of the study, a kinetic model describing the co-fermentation of substrates producing ethanol using Zymomonas mobilis is proposed. The model extends work available in the literature to include acetate inhibition. The reported optimisation studies include realistic variations in feedstock quality, deacetylation, a mechanical pre-treatment stage and a green recovery system: extractive distillation with vapour compression. Results indicate that the use of fed-batch reactors using a deacetylation stage achieves an ethanol yield of between 267 and 334 L/ton dry basis of CS and economic potential of between 0.4 and 5.5 MM USD/ year higher than the use of batch reactors. This also has the lowest energy requirements in the product recovery stage (3.2-3.4 MJ-fuel/kg-ethanol or 1.6-1.8 MJ/kg-ethanol). Omitting de-acetylation prior to hydrolysis/co-fermentation increases the minimum ethanol selling price and energy requirements by ~3-14% and ~8-30%, respectively.
... Unfortunately, because Z. mobilis strains are inherently resistant (conferred by several indigenous plasmids) to a variety of antibiotics (5,6), genetic manipulation of Z. mobilis strains remains limited. Among the diverse transformation protocols proposed for Z. mobilis, electroporation is the most effective method (7)(8)(9)(10)(11)(12)(13); however, transformation efficiencies vary greatly depending on the plasmids and strains used. ...
... Similar to other bacteria, genetically modified Z. mobilis strains are increasingly being used to define the genomic structure and function of various biotechnological applications (4,(10)(11)(12)(13). The construction of these strains frequently requires gene targeting, including chromosomal gene deletions/mutations and chromosomal insertions of heterologous DNA segments that require antibiotic resistance markers, which are a common option for selectable markers in genetic manipulation of Z. mobilis strains (12,13). ...
... The electroporation data reported for Z. mobilis ZM4 are limited (10,11) and the efficiency was shown to be very low in our preliminary experiments. In order to improve the transformation and integration efficiency in ZM4 by electroporation, we first performed extensive evaluation and optimization of various parameters by using the broad host range vector pBBR1MCS-2 and three shuttle plasmids that could replicate in ZM4 (unpublished data). ...
Flippase expression was carried out in Zymomonas mobilis strain ZM4. The FRT-flank-ed selection marker gene was first integrated into the ZM4 chromosome by homologous recombination. The Saccharomyces cerevisiae flp gene was then introduced under the control of the ZM4 gap gene promoter (Pgap, encoding glyceraldehyde-3-phosphate dehydrogena-se) or the l bacteriophage cI 857 -p R contained in the broad-host-range cloning vector pBBR1-MCS-2. This study demonstrated that flp was expressed and that the deletion frequency of the FRT-flanked marker gene was very high (approx. 100 %). In addition, the flp gene ex-pression vector could be conveniently removed from the resulting unmarked Z. mobilis mutants by serially transferring the cells three times into antibiotic-free medium, thereby establishing an efficient method for constructing unmarked Z. mobilis mutants.
... The present experiments have been designed as part of a systematic study on rate limitation in xylose-utilizing strains of recombinant Z. mobilis and have focused initially on the effects of XK over-expression. The acetate-resistant recombinant strain of Z. mobilis ZM4/ Ac R (pZB5), based on ZM4 (pZB5) from Zhang et al. [1] and further developed by Jeon et al. [12], was selected for this investigation and the broad host range vector pBBR1MCS-2 [13] has been used to introduce additional copies of the xylB gene (expressing XK) into this strain. Detailed evaluations on xylose-and glucosebased media have been used to determine the effect of XK over-expression on its fermentation kinetics. ...
... All bacterial strains and plasmids used in this study are listed in Table 1. The Z. mobilis strain used in the present investigation ZM4/Ac R (pZB5) was an acetateresistant mutant of the strain ZM4 (pZB5) [12]. E. coli DH5a cultures were incubated overnight in Luria Broth [14] at 37°C with antibiotic concentrations specified below. ...
... The conditions for transformation of Z. mobilis ZM4/ Ac R (pZB5) via electroporation were described previously [12]. During electroporation, to prevent plasmid restriction from host controlled restriction and modification system, the phage protein ''ocr'' [23], a Type-Onee restriction inhibitor purchased from Epicentre Ò (USA), was used according to the ManufacturerÕs Instructions. ...
The broad host range vector pBBR1MCS-2 has been evaluated as an expression vector for Zymomonas mobilis. The transformation efficiency of this vector was 2 x 10(3) CFU per mug of DNA in a recombinant strain of Z. mobilis ZM4/AcR containing the plasmid pZB5. Stable replication for this expression vector was demonstrated for 50 generations. This vector was used to study xylose metabolism in acetate resistant Z. mobilis ZM4/AcR (pZB5) by over-expression of xylulokinase (XK), as previous studies had suggested that XK could be the rate-limiting enzyme for such strains. Based on the above vector, a recombinant plasmid pJX1 harboring xylB (expressing XK) under control of a native Z. mobilis promotor Ppdc was constructed. When this plasmid was introduced into ZM4/AcR (pZB5) a 3-fold higher XK expression was found compared to the control strain. However, fermentation studies with ZM4/AcR (pZB5, pJX1) on xylose medium did not result in any increase in rate of growth or xylose metabolism, suggesting that XK expression was not rate-limiting for ZM4/AcR (pZB5) and related strains.
... Lawford et al. (1998) evolved a method for extension of more glucose in ethanoic acid that consists of media culture for the sake of advancement of fermentation performance of recombinant Zymomonas (Table 6.3). Recombinant plasmid pZB5 also transformed into an ethanolic-acid-tolerant strain (Joachimsthal et al. 1998) and a mutant (recombinant Z. mobilis) (pZB5) strain modified to acetate tolerance (Jeon et al. 2002). Overexposure of enzyme xylulokinase in a xylose-metabolizing recombinant strain was also established as a result of another recombinant strain (pZB5, pjX1) (Jeon et al. 2002). ...
... Recombinant plasmid pZB5 also transformed into an ethanolic-acid-tolerant strain (Joachimsthal et al. 1998) and a mutant (recombinant Z. mobilis) (pZB5) strain modified to acetate tolerance (Jeon et al. 2002). Overexposure of enzyme xylulokinase in a xylose-metabolizing recombinant strain was also established as a result of another recombinant strain (pZB5, pjX1) (Jeon et al. 2002). The adaptable laboratory evolution approach has been used for the improvement of ethanolic-acid ability (Wang et al. 2016). ...
Zymomonas mobilis strains are examined as the model organism in the industries because of having many potential advantages. Different research showed that different strains of Z. mobilis produced high amount of ethanol and the sugar because it can easily utilize xylose and arabinose in addition to glucose. We can improve the strains of Z. mobilis such as by adaptive laboratory evolution (ALE) by many methods. It is a very important method for the improvement of different attributes of common industrial strains. These strains have used as advanced model organism for genetic method and inverse metabolic engineering. The ED pathway of Z. mobilis gives an alternative way for the production of bio refineries and valuable byproducts like sorbitol, succinic acid, levan, and isobutanol. The metabolic engineering using Z. mobilis gives advanced biofuel production. The techniques adopted for strain improvement and future guidelines for this were discussed.
... Two types of sugar (i.e., xylose and glucose) are simultaneously fermented into ethanol by recombinant Zymomonas mobilis strain ZM4 (pZB5) (Z. mobilis ZM4 (pZB5)) [28]. The fermentation temperature is kept around 32°C and the substrates stay at the reactor for 36 h until the fermentation process finishes. ...
... where c Z is the concentration of recombinant Z. mobilis ZM4 (pZB5), which uses glucose and xylose as carbon sources and converts them into ethanol simultaneously [28]. The expression for the microbial growth rate is given by: ...
Biofuel is a clean and renewable energy source and is considered a promising alternative to traditional fossil fuels. The economic viability is crucial in promoting large-scale adoption and long-term sustainability of biofuel. Most of the current literature on biofuel economics assumes the individual biofuel manufacturing processes are independent of each other. Consequently, the interrelationships between parameters within and across processes regarding manufacturing cost and biofuel yield are not well investigated. In this paper, a system-level cost model for cellulosic biofuel manufacturing is established across multiple production processes to investigate the relationships between the individual process characteristics and the system performance to reduce the overall cost under the constraint of biofuel yield. Two numerical case studies are conducted to illustrate the effectiveness of the proposed model. Compared with the baseline case, the cost-effective case shows that 12.8% of the total cost is reduced without ethanol yield loss.
... This result could be explained by two scenarios. One is affected by by-product inhibition such as acetic acid, which is one of the distinctive by-products produced under aerobic condition by Z. mobilis (Jeon et al. 2002;Yang et al. 2009); the other is caused by oxidative stress. However, the aerobic fermentation results indicated that similar amounts of acetic acid were produced from both the deficient strain (1.5 g l −1 ) and the parental strain ZM4 (1.2 g l −1 acetic acid) after 60 h under the same operation time. ...
... However, the aerobic fermentation results indicated that similar amounts of acetic acid were produced from both the deficient strain (1.5 g l −1 ) and the parental strain ZM4 (1.2 g l −1 acetic acid) after 60 h under the same operation time. Previous studies (Joachimsthal et al. 1998;Jeon et al. 2002) reported that approximately 8 g l −1 acetic acid at pH 5.0 can inhibit the cell growth of ZM4. Therefore, the results indicated that the inhibited cell growth of ZM4 gnlΔ was unlikely caused by such small amount of acetic acid produced. ...
The physiological characteristics and the potential gluconolactone production of the gluconolactonase-deficient strain, Zymomonas mobilis ZM4 gnlΔ, were investigated via growth inhibitory assay and biotransformation of glucose and fructose into gluconolactone and sorbitol, respectively. The results of ethanol fermentation studies performed in the presence of high concentration of glucose (>200 g l⁻¹) under fermentative or aerobic conditions indicated that a significant reduction of volumetric ethanol productivity from the strain of ZM4 gnlΔ was noticeable due to the reduced rates of specific growth, sugar uptake, and biomass yield as compared with those of the parental strain ZM4. The biotransformation prepared at pH 6.0 using the permeabilized cell indicated that gluconic acid from ZM4 gnlΔ was still produced as a major product (67 g l⁻¹) together with sorbitol (65 g l⁻¹) rather than gluconolactone after 24 h. Only small amount of gluconolactone was transiently overproduced up to 9 g l⁻¹, but at the end of biotransformation, all gluconolactone were oxidized into gluconic acid. This indicated that autolysis of gluconolactone at the pH led to such results despite under gluconolactonase inactivation conditions. The physiological characteristics of ZM4 gnlΔ was further investigated under various stress conditions, including suboptimal pH (3.5~6.0), temperature (25~40 °C), and presence of growth inhibitory molecules including hydrogen peroxide, ethanol, acetic acid, furfural, and so forth. The results indicated that ZM4 gnlΔ was more susceptible at high glucose concentration, low pH of 3.5, and high temperature of 40 °C and in the presence of 4 mM H2O2 comparing with ZM4. Therefore, the results were evident that gluconolactonase in Z. mobilis contributed to industrial robustness and anti-stress regulation.
... Although these results revealed that furfural had effects on multiple aspects of cellular metabolism at the transcriptional level and membrane might play important roles in response to furfural, developing a strain tolerant to furfural and acetic acid inhibitors is also difficult by using rational engineering strategies due to our poor understanding of their underlying molecular mechanisms. On the other hand, different sodium acetate-tolerant Z. mobilis strains such as ZM4/AcR (Joachimsthal et al. 1998), Z. mobilis ZM4/Ac R (pZB5) (Jeon et al. 2002), and ZM4/Ac R (pZB5, pJX1) (Jeon et al. 2005) have been obtained from previous studies. Although genome changes associated with Z. mobilis sodium acetate-tolerant mutant ZM4/Ac R were characterized by using microarray comparative genome sequencing (CGS) and 454pyrosequencing (Yang et al. 2010a), developing an acetic acid-tolerant strain will also be a difficult task for Z. mobilis. ...
... Actually, Joachimsthal et al. first screened an acetatetolerant Z. mobilis strain (ZM4/Ac R ) by chemical mutagenesis and selection in the presence of acetate (Joachimsthal et al. 1998), and also used as a host for constructing of engineered tolerant Z. mobilis strain, i.e., ZM4/Ac R (pZB5) and ZM4/Ac R (pZB5, pJX1) (Jeon et al. 2002(Jeon et al. , 2005Mohagheghi et al. 2004;Yamada et al. 2002). However, all studies are focusing on sodium acetate inhibition, not acetic acid. ...
Furfural and acetic acid from lignocellulosic hydrolysates are the prevalent inhibitors to Zymomonas mobilis during cellulosic ethanol production. Developing a strain tolerant to furfural or acetic acid inhibitors is difficul by using rational engineering strategies due to poor understanding of their underlying molecular mechanisms. In this study, strategy of adaptive laboratory evolution (ALE) was used for development of a furfural and acetic acid-tolerant strain. After three round evolution, four evolved mutants (ZMA7-2, ZMA7-3, ZMF3-2, and ZMF3-3) that showed higher growth capacity were successfully obtained via ALE method. Based on the results of profiling of cell growth, glucose utilization, ethanol yield, and activity of key enzymes, two desired strains, ZMA7-2 and ZMF3-3, were achieved, which showed higher tolerance under 7 g/l acetic acid and 3 g/l furfural stress condition. Especially, it is the first report of Z. mobilis strain that could tolerate higher furfural. The best strain, Z. mobilis ZMF3-3, has showed 94.84 % theoretical ethanol yield under 3-g/l furfural stress condition, and the theoretical ethanol yield of ZM4 is only 9.89 %. Our study also demonstrated that ALE method might also be used as a powerful metabolic engineering tool for metabolic engineering in Z. mobilis. Furthermore, the two best strains could be used as novel host for further metabolic engineering in cellulosic ethanol or future biorefinery. Importantly, the two strains may also be used as novel-tolerant model organisms for the genetic mechanism on the "omics" level, which will provide some useful information for inverse metabolic engineering.
... Zymomonas mobilis is of interest as a potential biocatalyst for large-scale ethanol production from lignocellulosic materials due to its high ethanol fermentation yields (2)(3)(4)(5). Recombinant strains of this gram-negative bacterium are capable of efficiently converting both glucose and xylose to ethanol, and under some conditions can also tolerate acetic acid and other inhibitory components typically present in biomass hydrolyzates. ...
... This averaged Y X/ATP value is consistent with the range of 4.62-9.90 g/mol recently reported for a pair of recombinant Z. mobilis strains fermenting glucose and xylose mixtures (4). The rate of ATP consumption for maintenance was calculated as the difference between the specific rate of ATP production and the estimated specific rate of ATP consumption for growth-mediated processes by rearranging Equation 1. ...
The growth (glucose-and xylose-associated processes) and non-growth-mediated (maintenance energy requirements) characteristics of a metabolically engineered strain of Zymomonas mobilis capable of fermenting both glucose and xylose to ethanol have been investigated in mixed sugar fermentations. Ideally such a microorganism will also tolerate acetic acid and other inhibitory components typically present in biomass hydrolyzates. We have measured substrates and products concentrations as well as intracellular adenosine-5'-triphosphate (ATP) concentrations across a 2-factor, 3-level factorial design using the methods of analysis, techniques, and calculations described previously (1). Batch fermentations were conducted using a 10% (w/v) total sugar concentration (5% glucose/5% xylose mixture) at a temperature of 30 °C at pH 5.0, 5.3, or 6.0 in the presence of varying initial amounts of acetic acid (0, 4, and 8 g/L). Results show large differences in Z. mobilis fermentation kinetics but only modest changes in intracellular ATP levels across the experimental design space. Ethanol process yields varied between 56.6% and 92.3% of theoretical depending upon the test conditions, and illustrate how pH strongly influences the inhibitory effect of acetic acid on fermentation kinetics. For example, maximum specific growth rates varied between a low of 0.06 h -1 observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.20 h -1 obtained at pH 6 without any acetic acid present. Reflecting this trend, calculated rates of ATP consumption for growth-mediated-processes ranged from a low of 6.2 g ATP/g DCM-h at pH 5 and 8 g/L acetic acid to a high of 20.9 g ATP/g DCM-h at pH 6 without acetic acid. ATP requirements for maintenance showed an opposite pattern, with the highest maintenance requirement, 12.9 g ATP/g DCM-h, obtained at pH 5 and 8 g/L acetic acid, and the lowest, 0.2 g ATP/g DCM-h, at pH 6 without acetic acid present. Despite the more than sixty-fold difference in maintenance requirements observed across the design space, maximum levels of free intracellular ATP only varied within a narrow range of 1.5 to 3.8 mg ATP/g DCM. These findings provide quantitative information about how pH and acetic acid concentration affect fermentation kinetics. A mathematical model is being developed to describe how the rate of ATP consumption for maintenance varies as a function of the concentrations of undissociated acetic acid and dissociated acetate ion, which are modulated by pH (hydronium ion concentration), as well as the concentration of ethanol. Preliminary results suggest that in this system the concentration of undissociated acetic acid is the strongest determinant of maintenance requirements. INTRODUCTION Zymomonas mobilis is of interest as a potential biocatalyst for large-scale ethanol production from lignocellulosic materials due to its high ethanol fermentation yields (2-5). Recombinant strains of this gram-negative bacterium are capable of efficiently converting both glucose and xylose to ethanol, and under some conditions can also tolerate acetic acid and other inhibitory components typically present in biomass hydrolyzates. However, the fermentation performance characteristics of Z. mobilis, particularly its operable pH range and ability to grow in the presence of ethanol, are influenced by a variety of factors, including sugar type(s) and concentration, acetic acid concentration, and temperature. A distinctive and significant characteristic of xylose-and glucose-fermenting Z. mobilis strains is the uncoupling of ethanol production from cell growth that occurs towards the end of batch mixed sugar fermentations. This uncoupling behavior is often observed after glucose is depleted, whereupon cell growth rate decreases, eventually falling to zero, while the remaining xylose is fermented to ethanol (Figure 1). This phenomenon, in combination with Z. mobilis using the low ATP yield Entner-Doudoroff glycolytic pathway (which generates only 1 net mol of intracellular ATP per mol of glucose or xylose consumed), results in relatively more sugars being available for ethanol production compared with other ethanologens. We have speculated that lower ATP accumulation levels may underlie the uncoupling phenomenon, as well as the greater sensitivity to inhibition by acetic acid or ethanol or low pH observed when fermenting xylose. Presumably, the amount of ATP available for growth processes falls late in fermentation as increasing amounts of ATP become required for cell maintenance as sugar concentrations decrease and inhibitory products like ethanol accumulate.
... Among these mutants, environmental stress-tolerant mutant, and antibiotic-sensitive strains have showed some potential in industrial applications. For example, the acetate-tolerant Z. mobilis mutant (AcR) was generated by chemical mutagenesis and selection in the presence of acetate [47], and used as a host for constructing of engineered tolerant Z. mobilis strain for bio-ethanol production, that is ZM4/Ac R (pZB5) [48][49][50]. ...
... For example, Lawford and Rousseau et al. developed a process via addition of extra glucose in acetic acid-containing media for improving fermentation performance of recombinant Zymomonas [82]. Recombinant plasmid pZB5 was also transferred into an acetic acid-tolerant strain (ZM4/Ac R ) [47], and a mutant recombinant Z. mobilis ZM4/Ac R (pZB5) strain with increased acetate resistance was obtained [48]. Overexpression of xylulokinase in a xylose-metabolising recombinant strain was also performed, and resulted in another recombinant ZM4/Ac R (pZB5, pJX1) [83]. ...
Biosynthesis of liquid fuels and biomass-based building block chemicals from microorganisms have been regarded as a competitive alternative route to traditional. Zymomonas mobilis possesses a number of desirable characteristics for its special Entner-Doudoroff pathway, which makes it an ideal platform for both metabolic engineering and commercial-scale production of desirable bio-products as the same as Escherichia coli and Saccharomyces cerevisiae based on consideration of future biomass biorefinery. Z. mobilis has been studied extensively on both fundamental and applied level, which will provide a basis for industrial biotechnology in the future. Furthermore, metabolic engineering of Z. mobilis for enhancing bio-ethanol production from biomass resources has been significantly promoted by different methods (i.e. mutagenesis, adaptive laboratory evolution, specific gene knock-out, and metabolic engineering). In addition, the feasibility of representative metabolites, i.e. sorbitol, bionic acid, levan, succinic acid, isobutanol, and isobutanol produced by Z. mobilis and the strategies for strain improvements are also discussed or highlighted in this paper. Moreover, this review will present some guidelines for future developments in the bio-based chemical production using Z. mobilis as a novel industrial platform for future biofineries.
... Concentrations of potential inhibitors have been determined also for the various raw materials used in this study. Such information provides possible reasons for reduced fermentation productivities, as well as potential targets for further strain enhancement (Joachimsthal et al. 1998;Kim et al. 2000a;Jeon et al. 2002;Pienkos and Zhang 2009;Yang et al. 2010a). ...
... Sorghum straw, SCT and A. donax (Adx) hydrolysates were similar in their characteristics, while fermentation of the hydrolysates of the woody raw materials (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities, the latter possibly because of higher concentrations of inhibitory compounds (e.g. higher furfural concentrations in oil mallee). As previously reported (Joachimsthal and Rogers 2000;Jeon et al. 2002;Yang et al. 2010b), improved strains of Z. mobilis can be developed to partially overcome some of the inhibitory effects that result from inhibitory components such as acetate. ...
To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis.
Dilute acid pretreatment (2% H(2) SO(4) ) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60-75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment.
Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates.
The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food-based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.
... Zymomonas mobilis is another ethanologenic organism that have a huge potential in industrial bioethanol production due to its desirable characteristics such as high specific rate of sugar uptake, increased ethanol yield and non-requirements for aerobic conditions [282]. Researchers are also using novel metabolic strategies to make this bacterium a suitable candidate for large-scale production of bioethanol [283][284][285]. Some breakthroughs have already been achieved using this organism. ...
Biofuels are fast advancing as alternative sources of renewable energy due to their non-polluting features and cost-competitiveness in comparison to fossil fuels. However, in order to fast-track their development, focus is shifting towards the use of technologies that will maximize their yields. Nanoparticles are gaining increasing interest amongst researchers due to their exquisite properties, which enable them to be applied in diverse fields such as agriculture, electronics, pharmaceuticals and food industry. They are also being explored in biofuels in order to improve the performance of these bioprocesses. This review critically examines the various studies in literature that have explored nanoparticles in biofuel processes such as biohydrogen, biogas, biodiesel and bioethanol production, towards enhancing their process yields. Furthermore, it elucidates the different types of nanomaterials (metallic, nanofibers and nanotubes) that have been used in these bioprocesses. It also evaluates the effects of immobilized nanoparticles on biofuels such as biodiesel, and the ability of nanoparticles to effectively suppress inhibitory compounds under certain conditions. A short section is included to discuss the factors that influence the performance of nanoparticles on biofuels production processes. Finally, the review concludes with suggestions on improvements and possible further research aspects of these bioprocesses using nanoparticles.
... Zymomonas mobilis is another ethanologenic organism that have a huge potential in industrial bioethanol production due to its desirable characteristics such as high specific rate of sugar uptake, increased ethanol yield and non-requirements for aerobic conditions [282]. Researchers are also using novel metabolic strategies to make this bacterium a suitable candidate for large-scale production of bioethanol [283][284][285]. Some breakthroughs have already been achieved using this organism. ...
Biofuels are fast advancing as alternative sources of renewable energy due to their non-polluting features and cost-competitiveness in comparison to fossil fuels. However, in order to fast-track their development, focus is shifting towards the use of technologies that will maximize their yields. Nanoparticles are gaining increasing interest amongst researchers due to their exquisite properties, which enable them to be applied in diverse fields such as agriculture, electronics, pharmaceuticals and food industry. They are also being explored in biofuels in order to improve the performance of these bioprocesses. This review critically examines the various studies in literature that have explored nanoparticles in biofuel processes such as biohydrogen, biogas, biodiesel and bioethanol production, towards enhancing their process yields. Furthermore, it elucidates the different types of nanomaterials (metallic, nanofibers and nanotubes) that have been used in these bioprocesses. It also evaluates the effects of immobilized nanoparticles on biofuels such as biodiesel, and the ability of nanoparticles to effectively suppress inhibitory compounds under certain conditions. A short section is included to discuss the factors that influence the performance of nanoparticles on biofuels production processes. Finally, the review concludes with suggestions on improvements and possible further research aspects of these bioprocesses using nanoparticles.
... Jeon et al. also found similar characteristics in a saccharification of acid-treated woody biomass such as pine, oil mallee, and eucalyptus [45]. One possible suggestion to improve fermentability of saccharified biomass pre-treated with acid is using the ethanol producing strains that are tolerant to lignin derivatives and sugar degradation compounds such as acetic acid and furfural [46]. Also, those inhibitory compounds can be removed by means of charcoal or ion exchange resins prior to fermentation [47,48]. ...
... Cara untuk menanggulangi masalah tersebut meliputi perbaikan proses perlakuan awal, seperti penggunaan kondisi perlakuan awal yang sedang (mild), dan kombinasi metode kimiawi dan enzimatik pada tahap detoksifikasi, juga penggunaan strain yang tahan terhadap inhibitor melalui adaptasi, mutasi atau rekayasa metabolik. Contoh penggunaan strain penghasil etanol (etanologenik) yang toleran terhadap inhibitor adalah pembuatan strain mutan Z. mobilis yang tahan terhadap asam asetat (Joachimsthal et al. 1998;Jeon et al. 2002), isolasi Z. mobilis yang dapat tumbuh pada hasil hidrolisis (hidrolisat) bahan berkayu keras (Lawford dan Rousseau 2000), adaptasi S. cerevisiae terhadap inhibitor yang dihasilkan bahan berlignoselulosa (Keating et al. 2006), introduksi gen-gen untuk ekspresi asam fenilakrilik dekarboksilase (yang dapat memecah senyawa aromatik asam ferulik dan sinamik) (Zaldivar et al. 2001) dan laccase (yang dapat memecah senyawa aromatik koniferil aldehida) pada S. cerevisiae (Zaldivar et al. 2001;Kiiskinen dan Saloheimo 2004). ...
Biomass as raw material of bioethanol Bioethanol is the world's most produced alternative biofuel as considered environmentally friendly compared to biodiesel. World's production on bioethanol has been increasing due to the fluctuation of fossil fuel price. There is increasing interest in fermentation for ethanol production from various raw materials including lower cost lignocellulosic materials such as agricultural/forestry residues and high yield biomass energy crops. Reports and reviews have been focusing on lowering cost production such as using lignocellulosic biomass feedstock, genetic manipulation of microorganism for higher ethanol production using lignocellulosic hydrolisat, and efficient fermentation technology. Indonesia has potential of using cellulosic biomass for ethanol production for alternative energy. The use of lignocellulosic biomass will reduce the impact of using foodstuff for biofuel production. Abundant lignocellulosic energy is potentially converted into simple sugars and then further converted into biofuel energy. Other potential factor is the use of indigenous Indonesian microbes for cellulosic enzymes production for lignocellulose hydrolysis and then also potential indigenous microbes for fermentation of these sugars into biofuels. Several challenges shoud be considered both technology difficulty and non-technique constraints, including improvement for hydrolysis process for producing simple sugars and microbe improvement for utilizing all sugars C5 and C6 produced by lignocellulosic hydrolisate. Agriculture waste management and identification, and government support is others non-techniques challenge to be considered.
... Acetate-resistant strains have been reported to be capable of producing ethanol at high acetate concentration. 8 This research aims to study the elimination of inhibition effect of furfural on fermentation by removing it from the aqueous solution. ...
Ethanol can be produced from lignocellulosic biomass through fermentation; however, some byproducts from lignocellulosics, such as furfural compounds, are highly inhibitory to the fermentation and can substantially reduce the efficiency of ethanol production. In this study, commercial and polymer-derived activated carbons were utilized to selectively remove the model fermentation inhibitor, furfural, from water solution during bioethanol production. The oxygen functional groups on the carbon surface were found to influence the selectivity of sorbents between inhibitors and sugars during the separation. After inhibitors were selectively removed from the broth, the cell growth and ethanol production efficiency was recovered noticeably in the fermentation. A sorption/desorption cycle was designed, and the sorbents were regenerated in a fixed-bed column system using ethanol-containing standard solution. Dynamic mass balance was obtained after running four or five cycles, and regeneration results were stable even after twenty cycles.
... Ethanol concentrations above 60 g/l were achieved with input sugar concentrations of 65 g/l xylose and 65 g/l glucose (Joachimsthal et al., 1999). In subsequent studies an acetate resistant recombinant strain has been characterized (Jeon et al., 2002) as acetate presents an important inhibition problem in lignocellulosic hydrolystates from acid pretreatment. . Further genetic manipulation should be simpler than for yeasts as Z. mobilis is a procaryotic microorganism with a relatively small genome (approximately 2 Â 10 6 base pairs). ...
T he increasing focus on sustainable industrial processes for the chemical industry has resulted recently in a number of new bio-based initiatives. Environmental pressures and a shift towards the use of agricultural-based raw materials, as well as rapid devel-opments in the science supporting biotechnology, have stimulated this interest. The recent sequencing of the human genome and the associated sequencing of industrial bacterial and yeast genomes have also played their part. In addition, the fields of metabolic engineering, bioinformatics and computer-based modelling and process optimization are opening up opportunities for new products and cost reductions. In the present review, a number of these new industrial bio-based processes are identified. A case study on fuel ethanol, using agricultural residues and based on a genetically-engineered micro-organism (rec Zymomonas mobilis), is presented as such a process provides an opportunity for reducing fuel ethanol production costs as well as facilitating the infrastructure for other higher value bio-based products.
... The major inhibition mechanisms were possibly the intracellular de-energization and acidification (Kim et al. 2000). A recombinant strain was generated by transforming plasmid pZB5 into the AcR background, which can utilize both xylose and glucose with increased acetate resistance and improved fermentation characteristics in the presence of 12 g/L NaAc (Jeon et al. 2002). However, strain AcR was generated while many systems biology tools were being developed or had yet to be conceived, and the molecular mechanism of AcR sodium acetate tolerance was elusive until recently (Yang et al. 2010a). ...
The development and use of robust ethanologenic microorganisms resistant to industrially relevant pretreatment inhibitors
will be a critical component in the successful generation of biofuel on the industrial scale. Recent progress to understand
the genetic basis of pretreatment inhibitor tolerance using genomics and systems biology tools for metabolic engineering for
the model ethanologenic bacterium Zymomonas mobilis is reviewed in this chapter. The importance of accurate genome annotations and the integration of systems biology data for
annotation improvement are highlighted, and case studies that describe the identification and characterization of the Z. mobilis nhaA, hfq, and himA inhibitor tolerance related gene targets are presented.
... Usually studies concerning maintenance correlate an elevated value to an environmental stress. For example, in Zymomonas mobilis fermentations, increased acetate concentration (Jeon et al., 2002), reduced pH (Lawford and Ruggiero, 1990), and elevated temperature or ethanol concentration (Fieschko and Humphrey, 1983), have each resulted in a greater maintenance. Bhattacharya and Dubey (1995) showed that the maintenance of E. coli fermentations increased with increased expression of a plasmid-borne recombinant protein, and similarly attributed this result to an increased metabolic burden for cells. ...
We studied the physiological response of Escherichia coli central metabolism to the expression of heterologous pyruvate carboxylase (PYC) in the presence and absence of pyruvate oxidase (POX). These studies were complemented with expression analysis of central and intermediary metabolic genes and conventional in vitro enzyme assays to evaluate glucose metabolism at steady-state growth conditions (chemostats). The absence of POX activity reduced nongrowth-related energy metabolism (maintenance coefficient) and increased the maximum specific rate of oxygen consumption. The presence of PYC activity (i.e., with POX activity) increased the biomass yield coefficient and reduced the maximum specific oxygen consumption rate compared to the wildtype. The presence of PYC in a poxB mutant resulted in a 42% lower maintenance coefficient and a 42% greater biomass yield compared to the wildtype. Providing E. coli with PYC or removing POX increased the threshold specific growth rate at which acetate accumulation began, with an 80% reduction in acetate accumulation observed at a specific growth rate of 0.4 h-1 in the poxB-pyc+ strain. Gene expression analysis suggests utilization of energetically less favorable glucose metabolism via glucokinase and the Entner-Doudoroff pathway in the absence of functional POX, while the upregulation of the phosphotransferase glucose uptake system and several amino acid biosynthetic pathways occurs in the presence of PYC. The physiological and expression changes resulting from these genetic perturbations demonstrate the importance of the pyruvate node in respiration and its impact on acetate overflow during aerobic growth.
Zymomonas mobilis is a gram-negative facultative anaerobic spore, which is generally recognized as a safe. As a promising ethanologenic organism for large-scale bio-ethanol production, Z. mobilis has also shown a good application prospect in food processing and food additive synthesis for its unique physiological characteristics and excellent industrial characteristics. It not only has obvious advantages in food processing and becomes the biorefinery chassis cell for food additives, but also has a certain healthcare effect on human health. Until to now, most of the research is still in theory and laboratory scale, and further research is also needed to achieve industrial production. This review summarized the physiological characteristics and advantages of Z. mobilis in food industry for the first time and further expounds its research status in food industry from three aspects of food additive synthesis, fermentation applications, and prebiotic efficacy, it will provide a theoretical basis for its development and applications in food industry. This review also discussed the shortcomings of its practical applications in the current food industry, and explored other ways to broaden the applications of Z. mobilis in the food industry, to promote its applications in food processing.
This chapter utilizes the cellulosic biofuel manufacturing system consisting of several processes as an example to demonstrate the formulation of a system‐level energy model. It introduces the knowledge related to cellulosic biofuel manufacturing followed by a detailed description of the major processes involved in cellulosic biofuel manufacturing. The chapter illustrates the formulation of the energy consumption model for cellulosic biofuel manufacturing, which involves discussions on four energy‐related factors in cellulosic biofuel manufacturing, including heating energy, energy loss, reaction energy, and energy recovery. It explores particle swarm optimization to solve the proposed energy consumption minimization problem. The chapter provides case study based on the energy model and energy consumption optimization problem. It also illustrates comparisons between the baseline case and an optimal case of the cellulosic biofuel manufacturing system. Therefore, the solution with the lowest energy consumption is selected as the near‐optimal solution.
In recent days, varieties of applications were derived from novel nano materials. One among them is the addition of nanomaterial in biofuel. "Biofuel" is a renewable and bio degradable liquid fuel mainly produced from domestic vegetable oil, plant oils and recycled cooking oils. It is an alternative fuel used as a blend in CI engines, which reduces green house gas emissions also. Recent research proved that the blend of biodiesel perform well in cold temperatures. Recently, nanosized metal/metal oxide particles were suspended in a bio fuel (base fluid), by which the properties of the base fluid gets enhanced significantly. By changing its thermo physical properties it is possible to produce energy efficient bio fuel. Based on the number of experimental investigations from literature, it is noted that with a wide variety of nano additives the fuel properties and the engine performance could be significantly improved. In this chapter, an overview on the Production of Bio fuel by the addition of metal and metal oxide nanoparticle was discussed. This chapter initially explains about the studies on thermal conductivity, viscosity, specific heat capacity, heat transfer and rheological properties performed with nano particle addition in different base fluids. Then provides the detail on improvement made in bio fuel production by the metal/metal oxide nanoparticle addition. Finally, the chapter concludes with possible further research aspects of bio fuel production using nanoparticle.
运动发酵单胞菌（Zymomonas mobilis）是目前已知唯一能够在厌氧条件下利用Entner-Doudoroff（ED）途径代谢葡萄糖、果糖和蔗糖产乙醇的革兰氏阴性细菌，具有乙醇发酵速率高和对糖表观收率高、乙醇耐受性好及生物安全（generally regarded as safe，GRAS）等特点。基于合成生物学方法和代谢工程改造，可以作为纤维素乙醇及其他生物基产品生物炼制的细胞工厂。本文综述了运动发酵单胞菌独特的生理特点及其作为细胞工厂在不同领域的应用，重点介绍了构建运动发酵单胞菌作为底盘细胞，实现工业产品规模化经济生产涉及的系统生物学、合成生物学及代谢工程改造相关方法、技术与工具等方面的进展及瓶颈。同时探讨了持续开发、完善、应用高效精准的基因编辑技术、代谢途径精准时空调控方法及高通量自动筛选检测手段，在运动发酵单胞菌基因组精简优化以及生物固碳与固氮等方面取得的突破，推动合成生物学理论研究和实践应用的发展。
Zymomonas mobilis, the only microorganism known to use the Entner-Doudoroff (ED) pathway anaerobically, can produce ethanol naturally from glucose, fructose and sucrose with many desirable traits such as ethanol production at high rate and yield and merit with biosafety (generally regarded as safe, GRAS), which has attracted more attention to be engineered as cell factories to produce biofuels and other bio-based products from lignocellulosic biomass. With the rapid development of novel technologies such as next-generation sequencing (NGS) and CRISPR-Cas genome editing as well as the accumulation of knowledge from studies on its physiology and modifications through metabolic engineering and systems biology, it is necessary to summarize accomplishments achieved recently to further explore the advantages of Z. mobilis, expediting the development and deployment of the robust synthetic microbial chass for the goals of "build to understand" and "build to apply" in the systems and synthetic biology era. In this review, we critically comment on the unique physiological characteristics of Z. mobilis and its potentials as a synthetic chassis to be engineered as microbial cell factories for producing diverse biochemicals economically, with a focus on the advances and challenges of developing efficient and effective tools and techniques for engineering this bacterium, taking advantages of methodologies developed with system biology and synthetic biology as well as metabolic engineering. We also prospect on future research for developing Z. mobilis as an attractive microbial chassis to be able to fix CO2 and N2 for biochemical production through genome optimization and metabolic engineering to advance the principles of synthetic biology and explore its potentials on biotechnological applications, which need unceasing effort to improve, develop and deploy efficient and effective genome editing tools, strategies for fine-tuning metabolic and regulatory pathways timely and spatially, as well as automatic high-throughput screening and quantification approaches.
As a promising alternative to fossil fuels, cellulosic biofuel has obtained considerable interest due to its potential for mitigating global climate change and enhancing energy security. However, the widespread adoption of cellulosic biofuel is taking place in a slower pace than expected. One major challenge is that the cellulosic biofuel production is still highly energy-intensive. In fact, the energy contained in cellulosic biofuel is less than the energy required for its production. To address this issue, in this paper, an analytical system-level energy model is proposed to characterize the fundamental relationships between total energy consumption and biofuel production parameters in cellulosic biofuel production systems. Furthermore, an optimization strategy based on Particle Swarm Optimization (PSO) is adopted to minimize the energy consumption of cellulosic biofuel production while maintaining the desired biofuel yield. A baseline case is implemented for analyzing energy consumption, and the results show that pretreatment consumes most energy among all processes and the water/biomass ratio is the most significant energy driver. In addition, the optimal solution results in a 21.09% reduction in the total energy consumption compared to the baseline case.
Zymomonas mobilis is a promising organism for the production of biofuels from lignocellulosic biomass as it natively produces ethanol at high yields and at rates far greater than other microorganisms, including Saccharomyces cerevisiae. This makes Z. mobilis attractive not only for ethanol production but potentially other products as well. One limitation of Z. mobilis is that it cannot natively ferment the pentose sugars, xylose and arabinose, present in lignocellulosic hydrolysates. Over the past few decades, a number of strains have been engineered that produce ethanol from lignocellulosic sugars. While many advances have been made, many challenges still remain. This chapter reviews the basic physiology of Z. mobilis and the numerous efforts devoted to engineering strains capable of producing ethanol and other chemicals from lignocellulosic sugars.
Four different plasmids were electro transformed into Zymomonas mobilis ZM4 and CP4, two important ethanol-producing strains. The results showed that the best source strain for preparing plasmids was the transformed host strain itself, and Escherichia coli JM110 as the source strain could yield significantly higher transformation efficiencies than Top10. The optimal recovery time of transformed ZM4 or CP4 cells to obtain maximum number of transformants and highest transformation efficiency was 11 h for pZB21-mini, pZB21 and pZA22, but 24 or 20 h for pBBR1MCS-2. The optimal electric field strength for pZB21-mini was 13.25 kV /cm in ZM4 and 14.0 kV /cm in CP4.But for pZA22 and pBBR1MCS-2, it was 11.75 kV /cm in ZM4 and 12.5 kV /cm in CP4; for pZB21, also 12.5 kV /cm in CP4.These plasmids were shown to be more stable in ZM4 than in CP4 by serial transfer to antibioticfree medium and the 3 plasmids were more stable than pBBR1MCS-2. The results will help to support the genetic and biotechnological research of Z. mobilis by providing information about some of the most important factors that influence the transformation of ZM4 and CP4, and also providing insights into the similarities and differences in their restriction-modification (R-M) systems.
Ethanol production with different concentrations of cellulose Avicel as the substrate was evaluated in a batch system using a mono-culture of a cellulolytic ethanol producing strain (Clostridium thermocellum strain LQRI) and specific co-cultures of LQRI in combination with one of the the non-cellulolytic ethanol producing strains (Thermoanaerobacter ethanolicus strain X514 or Thermoanaerobacter ethanolicus 39E) which was added to the LQRI mono-culture after six days. Results showed that lactate concentration, which was significantly higher in the specific co-culture of LQRI+39E than of LQRI+X514 (**p<0.01), dramatically increased after the non-cellulolytic ethanol producing strain was added to the LQRI mono-culture, while no significant differences were observed in the rate of decrease of acetate concentration between the specific co-cultures and the controls. Reducing sugar concentration increased in the specific co-culture of LQRI+39E and the control, while it decreased in the specific co-culture of LQRI+X514.The maximum concentrations of reducing sugar in the specific co-culture of LQRI+39E and the control were 2837.5 μg·mL-1 and 2254.1 μg·mL-1 respectively, while the minimum concentration in the specific co-culture of LQRI+X514 was 466.7 μg·mL-1. Most importantly, ethanol concentration in the specific co-cultures was significantly higher than that in the control. The ethanol concentration in the specific co-culture of LQRI+X514 increased dramatically after X514 was added to the mono-culture after the 6th day, and the rate of ethanol increase in LQRI+X514 was higher than that in LQRI+39E; the maximum ethanol concentrations were 64.1 mmol·L-1 and 20.0 mmol·L-1 respectively. Additionally, the same results were found for different concentrations of cellulose, with 1% and 2% Avicel as the substrate, which suggested further that the ethanol production abilities were promoted in LQRI mono-culture where X514 or 39E was added after six days. X514 can not only utilize hexose, but also ferment pentose to produce ethanol in the co-culture. However, the ethanol production abilities and end product distributions were closely related to the features of the added strain in the co-culture system.
Producing sugars from lignocellulose is the most challenging but holds the greatest promise for the potential scale of the resource availability. Different simplified processing pathways for the sugar platform are illustrated in this chapter. The separation, purification, and detoxification of biomass hydrolyzates are of particular interest in this chapter. Several methods have been suggested for detoxification of pretreated or acid-hydrolyzed biomass, including: overliming, ammonium hydroxide, use of adsorbents such as activated carbon or polymeric adsorbents, solvent extraction, ion exchange, flocculation, treatment with microbes, treatment with enzymes. The sections describe some of the findings reported on these methods. Many methods of reducing the inhibitory nature of biomass hydrolysates have been developed, though all add cost and complexity to the operations of a biorefinery. The optimal solution will likely be specific to the biomass feedstock, downstream processing needs, and local opportunities for synergy with other biomass processing facilities.
Over the past decade, ethanol has emerged as the single most-important alternative fuel to gasoline. In many countries, a blend of gasoline and alcohol (gasohol) is being supplied as a liquid fuel for transportation. Bioethanol produced from agricultural feed stocks is renewable in nature and reduces the problem of ghg emissions associated with petroleum-derived gasoline. While most current processes for production of bioethanol are dependent on microbial fermentation of food feedstocks (e.g., corn and sugarcane), second-generation technology based on the fermentation of nonfood feedstocks (e.g., corn stover and switch grass) is under development. Successful commercialization of bioethanol production requires an efficient microbe, rapid hydrolysis of feedstock into fermentable sugars and an optimized fermentation process. This article consolidates the current state of the art in upstream processing of cellulose for bioethanol production with bacteria. Recent advances in microbial cocultures involving one or more bacteria for efficient production of bioethanol are also discussed. The importance of engineering bacterial processes for efficient cellulosic bioethanol production is emphasized.
The Zymomonas mobilis ZM4 strain with excellent ethanol-producing capabilities was the first strain of Z. mobilis, which was sequenced. This strain is resistant to transformation, and no previous study has shown a detailed protocol for electrotransfer of ZM4 with foreign DNA. In this work, many electrical and biological parameters were selected and evaluated in order to optimize the electrotransformation of ZM4. First, improved transformation efficiencies of 11 896, 99, 96 and 5989 transformants/μg DNA were separately achieved with shuttle plasmid pZB21-mini (3082 bp), pZB21 (5930 bp), pZA22 (6994 bp) and broad-host-range vector pBBR1MCS-2 (5144 bp) all prepared from Escherichia coli JM110. The crucial factors affecting the transformation efficiency included the source of the plasmid (the best strain was ZM4), origin and size of the plasmids, growth phase of the cells (the most ideal phase was early log phase with OD600 of 0.3–0.4), the electric field strength (generally 11.75 kV/cm–13.25 kV/cm) and the recovery time (3–24 h). Further, based upon the optimal transformation protocol mentioned above for replicative plasmids in ZM4, (i) the electrotransformation by recombinant plasmid pBBR1MCS-2-Pgap-FLP (6880 bp) was an immediate success with the transformation efficiency 102 transformants/μg DNA; (ii) the site-specific integration efficiencies (expressed in terms of “per μg of DNA”) of 3–6 integrating transformants was obtained using the integrating plasmid pBR328-ldhR-cml-ldhL (7447 bp). This study will assist genetic and biotechnological research of ZM4 and other Z. mobilis strains by providing information about suitable vectors and a more universal and reliable procedure for introducing DNA into this strain.
A current challenge of the cellulosic ethanol industry is to improve the resistance of inhibitors present in biomass hydrolysates. RNA-binding protein gene lsm6 was cloned from industrial Saccharomyces cerevisiae ZU-E8, which is able to conferment glucose and xylose, and transformed into ZU-E8 via expression vector pRS426. The positive transformant ZU-910 with over-expressing lsm6 was identified on the culture plates using high concentration of acetate and re-screened by fermentation test. Fermentation by the recombinants was performed in a medium containing 80g/L xylose and 2g/L acetic acid or 20g/L NH(4)Ac/NaAc. After 96h shaking-flask fermentation, ZU-910 utilized 90.2% xylose with an ethanol yield of 26.9g/L, which was 8.5- and 10-fold higher than ZU-E8. Further, in the corn stover hemicellulosic hydrolysate fermentation, both the xylose conversion and ethanol production by ZU-910 was larger by 50% and 40% than ZU-E8. ZU-910 has also enhanced tolerance against furfural and SO(4)(2-).
The impact of the two adaptation-induced mutations in an improved xylose-fermenting Zymomonas mobilis strain was investigated. The chromosomal mutation at the xylose reductase gene was critical to xylose metabolism by reducing xylitol formation. Together with the plasmid-borne mutation impacting xylose isomerase activity, these two mutations accounted for 80 % of the improvement achieved by adaptation. To generate a strain fermenting xylose in the presence of high acetic acid concentrations, we transferred the two mutations to an acetic acid-tolerant strain. The resulting strain fermented glucose + xylose (each at 5 % w/v) with 1 % (w/v) acetic acid at pH 5.8 to completion with an ethanol yield of 93.4 %, outperforming other reported strains. This work demonstrated the power of applying molecular understanding in strain improvement.
To better understand the DNA restriction-modification (R-M) systems for more amenable strain development of the alternative industrial ethanologen, Zymomonas mobilis, three gene knockout mutants were constructed. The gene knockout mutants were tested for their DNA restriction activities by the determination of transformation efficiency using methylated and unmethylated foreign plasmid DNAs. Inactivation of a putative mrr gene encoded by ZMO0028 (zmrr) resulted in a 60-fold increase in the transformation efficiency when unmethylated plasmid DNA was used. This indicated that the putative mrr gene may serve as a type IV restriction-modification system in Z. mobilis ZM4. To assign the function of a putative type I DNA methyltransferase encoded by ZMO1933 (putative S subunit) and ZMO1934 (putative M subunit), the putative S subunit was inactivated. The gene inactivation of ZMO1933 resulted in a 30-fold increase in the transformation efficiency when methylated plasmid DNA was introduced, indicating that the putative S subunit possibly serves as a part of functional type I R-M system(s). Growth studies performed on the mutant strains indicate inactivation of the type I S subunit resulted in a lower maximum specific glucose consumption rate and biomass yield, while inactivation of the type IV Zmrr had the opposite effect, with an increase in the maximum specific growth rate and biomass yield.
During ethanol fermentation, bacterial strains may encounter various stresses, such as ethanol and acid shock, which adversely affect cell viability and the production of ethanol. Therefore, ethanologenic strains that tolerate abiotic stresses are highly desirable. Bacteria of the genus Deinococcus are extremely resistant to ionizing radiation, ultraviolet light, and desiccation, and therefore constitute an important pool of extreme resistance genes. The irrE gene encodes a general switch responsible for the extreme radioresistance of D. radiodurans. Here, we present evidence that IrrE acting as a global regulator confers high stress tolerance to a Zymomonas mobilis strain. Expression of the gene protected Z. mobilis cells against ethanol, acid, osmotic, and thermal shock. It also markedly improved cell viability, the expression levels and enzyme activities of pyruvate decarboxylase and alcohol dehydrogenase, and the production of ethanol under both ethanol and acid stress. These data suggest that irrE is a potentially promising gene for improving the abiotic stress tolerance of ethanologenic bacterial strains.
The application of systems biology tools holds promise for rational industrial microbial strain development. Here, we characterize a Zymomonas mobilis mutant (AcR) demonstrating sodium acetate tolerance that has potential importance in biofuel development. The genome changes associated with AcR are determined using microarray comparative genome sequencing (CGS) and 454-pyrosequencing. Sanger sequencing analysis is employed to validate genomic differences and to investigate CGS and 454-pyrosequencing limitations. Transcriptomics, genetic data and growth studies indicate that over-expression of the sodium-proton antiporter gene nhaA confers the elevated AcR sodium acetate tolerance phenotype. nhaA over-expression mostly confers enhanced sodium (Na(+)) tolerance and not acetate (Ac(-)) tolerance, unless both ions are present in sufficient quantities. NaAc is more inhibitory than potassium and ammonium acetate for Z. mobilis and the combination of elevated Na(+) and Ac(-) ions exerts a synergistic inhibitory effect for strain ZM4. A structural model for the NhaA sodium-proton antiporter is constructed to provide mechanistic insights. We demonstrate that Saccharomyces cerevisiae sodium-proton antiporter genes also contribute to sodium acetate, potassium acetate, and ammonium acetate tolerances. The present combination of classical and systems biology tools is a paradigm for accelerated industrial strain improvement and combines benefits of few a priori assumptions with detailed, rapid, mechanistic studies.
Zymomonas mobilis is one of the most promising microorganisms for bioethanol production. However, its practical use on industrial scale is impeded by its high sensitivity to acetate, which is present in high concentration in pretreated biomass. This research develops an adaptive mutation method for generating acetate-tolerant strains for bioethanol production. The goal is to obtain Zymomonas mobilis strain capable of growing and producing ethanol in the presence of acetate at a concentration typical of a pretreated biomass (2-3%). The interplay between the ability of fermentative production of ethanol and acetate tolerance will be investigated through careful fermentation studies. The potential cross-tolerance to other inhibitors, commonly present in pretreated biomass will be evaluated. A preliminary study on the mechanism of acetate tolerance at the cell membrane level will be conducted. The strain developed through this research will be useful in bioethanol production from biomass. The insights into tolerance mechanisms gained through this study will allow a more rational approach to further engineer a better producing strain. M.S. Committee Chair: Dr. Rachel Chen; Committee Member: Dr. Athanassios Sambanis; Committee Member: Dr. Sankar Nair
Efficient conversion of lignocellulosic biomass requires biocatalysts able to tolerate inhibitors produced by many pretreatment processes. Recombinant Zymomonas mobilis 8b, a recently developed integrant of Zymomonas mobilis 31821(pZB5), tolerated acetic acid up to 16 g l(-1) and achieved 82%-87% (w/w) ethanol yields from pure glucose/xylose solutions at pH 6 and temperatures of 30 degrees C and 37 degrees C. An ethanol yield of 85% (w/w) was achieved on glucose/xylose from hydrolysate produced by dilute sulfuric acid pretreatment of corn stover after an overliming' process was used to improve hydrolysate fermentability.
Intracellular adenosine-5'-triphosphate (ATP) levels were measured in a metabolically engineered Zymomonas mobilis over the course of batch fermentations of glucose and xylose mixtures. Fermentations were conducted over a range of pH (5-6) in the presence of varying initial amounts of acetic acid (0-8 g/L) using a 10% (w/v) total sugar concentration (glucose only, xylose only, or 5% glucose/5% xylose mixture). Over the design space investigated, ethanol process yields varied between 56.6% and 92.3% +/- 1.3% of theoretical, depending upon the test conditions. The large variation in process yields reflects the strong effect pH plays in modulating the inhibitory effect of acetic acid on fermentation performance. A corresponding effect was observed on maximum cellular specific growth rates, with the rates varying between a low of 0.15 h(-1) observed at pH 5 in the presence of 8 g/L acetic acid to a high of 0.32 +/- 0.02 h(-1) obtained at pH 5 or 6 when no acetic acid was initially present. While substantial differences were observed in intracellular specific ATP concentration profiles depending upon fermentation conditions, maximum intracellular ATP accumulation levels varied within a relatively narrow range (1.5-3.8 mg ATP/g dry cell mass). Xylose fermentations produced and accumulated ATP at much slower rates than mixed sugar fermentations (5% glucose, 5% xylose), and the ATP production and accumulation rates in the mixed sugar fermentations were slightly slower than in glucose fermentations. Results demonstrate that higher levels of acetic acid delay the onset and influence the extent of intracellular ATP accumulation. ATP production and accumulation rates were most sensitive to acetic acid at lower values of pH.
High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals,
and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and
faster specific rates when compared to yeasts, no commercial scale fermentations currently exist which use
Z. mobilis for the manufacture of fuel ethanol. This may change
with the recent announcement of a Dupont/Broin partnership to develop a process for conversion
of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and
their fermentation characteristics, are described in the present review. The review also addresses opportunities
offered by Z. mobilis for higher value products through its metabolic
engineering and use of specific high activity enzymes.
Lactic acid bacteria have a profoundly negative influence on the fermentation performance of Zymomonas mobilis. Lactic acid bacteria are an important part of the steeping process in starch wet-milling. Their prevalence and acid pH optimum for growth make these organisms opportunistic contaminants of continuous ethanol fermentations, where the feedstock is enzymatic starch hydrolysate. At concentrations of lactic acid in the range of 3–10 g/L, cell density, product yield, and productivity are reduced. However, under similar operating conditions, a chemostat culture fed a synthetic glucose medium with addeddl-lactic acid (range 0–13 g/L) did not exhibit a decrease in either yield or productivity. In pH-stat (5.0) batch cultures, with about 11 g/L lactic acid added to the medium, growth and conversion efficiency were only very slightly inhibited. At pH 4.5, this amount of lactic acid caused a 23% decrease in growth yield, but only a 3% reduction in ethanol yield. Acetic acid is a minor metabolic byproduct of lactic cultures, and at pH 5, the growth rate and growth yield of Z. mobilis were inhibited 17 and 23% respectively, by the addition of 3.37 g/L lactic acid. Ethanol yield was decreased only 3.5% by this amount of acetic acid.
It is concluded that lactic acid per se is not the causative inhibitory agent. Also, although a possible role for acetic acid and unidentified natural antibiotic substances is implied, the mechanism whereby lactic acid bacteria negatively affect Zymomonas remains unresolved.
This study examined the continuous cofermentation performance characteristics of a dilute-acid “prehydrolysate-adapted” recombinant Zymomonas 39676:pZB4L and builds on the pH-stat batch fermentations with this recombinant that we reported on last year. Substitution of yeast extract by 1% (w/v) corn steep liquor (CSL) (50% solids) and Mg (2 mM) did not alter the coferm entation performance. Using declared assumptions, the cost of using CSL and Mg was estimated to be 12.5c/gal of ethanol with a possibility of 50% cost reduction using fourfold less CSL with 0.1% diammonium phosphate. Because of competition for a common sugar transporter that exhibits a higher affinity for glucose, utilization of glucose was complete whereas xylose was always present in the chemostat effluent. The ethanol yield, based on sugar used, was 94% of theoretical maximum. Altering the sugar ratio of the synthetic dilute acid hardwood prehydrolysate did not appear to significantly change the pattern of xylose utilization. Using a criterion of 80% sugar utilization for determining the maximum dilution rate (D
max), changing the composition of the feed from 4% xylose to 3%, and simultaneously increasing the glucose from 0.8 to 1.8% shifted D
max from 0.07 to 0.08/h. With equal amounts of both sugars (2.5%), D
max was 0.07/h. By comparison to a similar investigation with rec Zm CP4:pZB5 with a 4% equal mixture of xylose and glucose, we observed that at pH 5.0, the D
max was 0.064/h and shifted to 0.084/h at pH 5.75. At a level of 0.4% (w/v) acetic acid in the CSL-based medium with 3% xylose and 1.8% glucose at pH 5.75, the D
max for the adapted recombinant shifted from 0.08 to 0.048/h, and the corresponding maximum volumetric ethanol productivity decreased 45%, from 1.52 to 0.84 g/(L·h). Under these conditions of continuous culture, linear regression of a Pirt plot of the specific rate of sugar utilization vs D showed that 4 g/L of acetic acid did not affect the maximum growth yield (0.030 g dry cell mass/g sugar), but did increase the maintenance coefficient twofold, from 0.46 to 1.0 g of sugar/(g of cell·h).
Recombinant Zymomonas mobilis CP4:pZB5 was grown with pH control in batch and continuous modes with either glucose or xylose as the sole carbon and energy source. In batch cultures in which the ratio of the final cell mass concentration to the amount of sugar in the medium was constant (i.e., under conditions that promote “coupled growth”), maximum specific rates of glucose and xylose consumption were 8.5 and 2.1 g/(g of cell…h), respectively; maximum specific rates of ethanol production for glucose and xylose were 4.1 and 1.0 g/(g of cell…h), respectively; and average growth yields from glucose and xylose were 0.055 and 0.034 g of dry cell mass (DCM)/g of sugar respectively. The corresponding value of YATP for glucose and xylose was 9.9 and 5.1 g of DCM/mol of ATP, respectively. YATP for the wild-type culture CP4 with glucose was 10.4g of DCM/mol of ATP. For single substratechem ostat cultures in which the growth rate was varied as the dilution rate (D), the maximum or “true” growth yield (max Ya/s) was calculated from Pirt plots as the inverse of the slope of the best-fit linear regression for the specific sugar utilization rate as a function of D, and the “maintenance coefficient” (m) was determined as the y-axis intercept. For xylose, values of max Y
s/s and m were 0.0417g of DCM/g of xylose (YATP=6.25) and 0.04g of, xylose/(g of cell…h), respectively. However, with glucose there was an observed deviation from linearity, and the data in the Pirt plot was best fit with a second-order polynomial in D. At D>0.1/h, YATP=8.71 and m=2.05g of glu/(g of cell…h) whereas at D<0.1/h, YATP=4.9g of DCM/mol of ATP and m=0.04g of glu/(g of cell…h). This observation provides evidence to question the validity of the unstructured growth model and the assumption that Pirt's maintenance coefficient is a constant that is in dependent of the growth rate. Collectively, these observations with individual sugars and the values assign ed to various growth and fermentation parameters will be useful in the development of models to predict the behavior of rec Zm in mixed substrate fermentations of the type associated with biomass-to-ethanol processes.
Acetic acid acts to promote uncoupling withZymomonas. At pH 5, 36% of acetic acid is in the uncharged and undissociated form (HAc), which is able to permeate the plasma membrane.
The transmembrane ΔpH drives the accumulation of acetic acid, which results in the acidification of the cytoplasm. The consequential
increase in maintenance metabolism represents a diversion of energy that would otherwise be available for growth. At pH 5,
the growth ofZ. mobilis (ATCC 29191) was 50% inhibited with 8.3 g/L acetic acid (50 mM HAc) and completely inhibited by 11 g/L. Addition of 6 g/L acetic acid caused the glucose-to-ethanol conversion efficiency
to decrease from 98 to 90% of theoretical maximum. The growth yield coefficient for glucose was 50% decreased by 2.3 g/L acetic
acid (13.5 mM HAc) from 0.036 to 0.018 g cell/g glucose. However, the specific (ethanol) productivity of batch cultures was enhanced by
<5 g/L acetic acid (<30 mM HAc). For continuous cultures, the acetic acid sensitivity depends on the growth rate (dilution rate), but an increase in
specific productivity can be achieved at proportionately lower concentrations of acetic acid. At a growth rate of 0.112/h,
the addition of 1.7 g/L acetic acid to the 5% glucose feed resulted in an increase in specific productivity from 2.68 to 5.87
g ethanol/g cell/h. The uncoupling effect of acetic acid could be beneficial in terms of improving the productivity in closed,
continuous fermentations, such as cell recycle or immobilized cell reactors.
Zymomonas mobilis CP4(pZB5) is a recombinant bacterium that can produce ethanol from both xylose and glucose. The ethanol-producing efficiency
of this organism is substantially impeded by toxic substances present in pretreated hydrolyzates or solid biomass substrates.
Acetic acid and furfural (a pentose degradation product) are highly toxic to this organism at levels envisioned for a pretreated-hardwood
liquid hydrolyzate. In addition, lignin degradation products and 5-hydroxymethylfurfural (a hexose degradation product) have
a moderately toxic effect on the organism. Of the compounds studied, organic acids and aldehydes were found to be more inhibitory
than lignin acids or the one alkaloid studied. Acetone:water and methanol extracts of solid biomass samples from red oak,
white oak, and yellow poplar are toxic toZymomonas cell growth and ethanol production, with the extracts from white oak being the most toxic.
pH-dependent energetic uncoupling ofZymomonas by acetic acid occurs by virtue of the permeability of the plasma membrane to the undissociated form of acetic acid (HAc)
and the acidification of the cytoplasm resulting from the uptake of HAc and the consequential diversion of energy away from
biosynthetic processes (growth) in order to maintain constant intracellular pH. Energetic uncoupling is manifested by an increase
in specific productivity. The degree of uncoupling caused by HAc depends on a rather complex interaction between several different
variables including membrane permeability, the transmembrane δpH and the concentraiton of undissociated form of acetic acid
in the medium. Within the pH range of 5.0–5.5, maximal energic uncoupling is produced by 30–38 mM HAc. For practical purposes,
in terms of the concentration of acetic acid, this corresponds to about 5 and 15 g/L at pH 5.0 and 5.5, respectively. Assuming
any upper limit concentration of acetic acid in hydrolysate fermentation media of about 12 g/L, inhibition of Z.mobilis in terms of both ethanol yield and productivity is avoided by controlling the pH in the range of 5.5–6.0.
The substrate fermentation range of the ethanologenic bacterium Zymomonas mobilis was expanded to include the pentose sugar, L-arabinose, which is commonly found in agricultural residues and other lignocellulosic biomass. Five genes, encoding L-arabinose isomerase (araA), L-ribulokinase (araB), L-ribulose-5-phosphate-4-epimerase (araD), transaldolase (talB), and transketolase (tktA), were isolated from Escherichia coli and introduced into Z. mobilis under the control of constitutive promoters that permitted their expression even in the presence of glucose. The engineered strain grew on and produced ethanol from L-arabinose as a sole C source at 98% of the maximum theoretical ethanol yield, based on the amount of consumed sugar. This indicates that arabinose was metabolized almost exclusively to ethanol as the sole fermentation product, with little by-product formation. Although no diauxic growth pattern was evident, the microorganism preferentially utilized glucose before arabinose, apparently reflecting the specificity of the indigenous facilitated diffusion transport system. This microorganism may be useful, along with the previously developed xylose-fermenting Z. mobilis (M. Zhang, C. Eddy, K. Deanda, M. Finkelstein, and S. Picataggio, Science 267:240-243, 1995), in a mixed culture for efficient fermentation of the predominant hexose and pentose sugars in agricultural residues and other lignocellulosic feedstocks to ethanol.
The ethanol-producing bacterium Zymomonas mobilis was metabolically engineered to broaden its range of fermentable substrates to include the pentose sugar xylose. Two operons
encoding xylose assimilation and pentose phosphate pathway enzymes were constructed and transformed into Z. mobilis in order to generate a strain that grew on xylose and efficiently fermented it to ethanol. Thus, anaerobic fermentation of
a pentose sugar to ethanol was achieved through a combination of the pentose phosphate and Entner-Doudoroff pathways. Furthermore,
this strain efficiently fermented both glucose and xylose, which is essential for economical conversion of lignocellulosic
biomass to ethanol.
Nuclear Magnetic Resonance (NMR) spectroscopy is proving to be a very valuable technique for characterizing the metabolic status of a range of microbial fermentations. This non-invasive method allows us not only to determine the presence of particular metabolites, but also to monitor reaction rates, enzyme activities and transport mechanisms in vivo. Despite the low levels of the carbon-13 isotope (1.1%), natural-abundance 13C-NMR studies have proven useful in monitoring the progress of various fermentation processes. Furthermore, 31P-NMR can provide noninvasive information relating to cellular metabolism, and on the energy status of the cells. This results from the facility with NMR to identify various nucleotide phosphates and other energy-rich compounds in the cell, as well as to characterize changes in the intracellular pH from the chemical shifts of internal phosphate and other phosphorylated intermediates. In this review, we will summarize the use of NMR as an analytical tool in biotechnology and also discuss examples that illustrate how NMR can be used to obtain significant information on the characteristics of ethanol fermentations in both yeasts and bacteria.
Maintenance energy plays an important role both in basic kinetic studies as well as in process development. However the determination of the maintenance energy coefficient is based so far on indirect time consuming measurements assuming linearity between growth rate and substrate uptake rate. This paper presents a simple and fast method using cell recycling techniques for the direct determination of maintenance energy coefficient.
Growth of Zymomonas mobilis ZM4 in media containing sodium acetate was inhibited above 12 g sodium acetate/l at pH 5.0. Following mutagenesis of ZM4, an acetate-tolerant strain was isolated in continuous culture that grew in the presence of 20 g sodium acetate/l at pH 5.0. In continuous culture with complete cell recycle at 30 deg C and pH 5.4 using media containing 110 g glucose/l, the maintenance energy coefficient (m) for the mutant was found to increase from 1.9 g glucose/g cell dry wt.h at 12 g sodium acetate/l up to 3.9 g/g.h at 20 g/l.
Zymomonas mobilis is being considered for the industrial production of ethyl alcohol. Expansion of its substrate range of glucose, fructose and sucrose could be advantageous, but genetic manipulation of Z. mobilis is restricted as it is resistant to transformation. We present a protocol using electroporation for high efficiency transformation of this bacterium. Optimal parameters included cooled cells (0–4 C), use of 10% glycerol as an osmotic support medium, a pulse in the 12 kV/cm range for 10 ms and outgrowth on GYx medium prior to selection. The routine efficiency achieved was greater than 1.0 107/g DNA, a major increase over other transformation methods in which yields ranged from 100–2000/g DNA.
The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch
and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and
65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose
utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with
ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable
of glucose and xylose utilization.
The inhibitory effects of six lignocellulose degradation products on glucose fermentation by Saccharomyces cerevisiae and Zymomonas mobilis on xylose fermentation by Pichia stipitis and Candida shehatae were studied in batch cultures. Toxic compounds were added in varying concentrations and subsequent inhibitions on growth and ethanol production were quantified. Vanillin was shown to be a strong inhibitor of both growth and ethanol production by xylose fermenting yeasts and S. cerevisiae when it was added to the culture media at a concentration of 1 g l−1. Fermentative activities of Z. mobilis were greatly sensitive to the presence of hydroxybenzaldehyde (0.5 g l−1). Analysis of culture media extracts showed that some of the inhibitors, particularly vanillin and furaldehyde, could be assimilated by the tested microbial strains which resulted in the partial recovery in both growth and ethanol production processes on prolonged incubation.
The fermentation characteristics and effects of lignocellulosic toxic compounds on recombinant Zymomonas mobilis ZM4(pZB5), which is capable of converting both glucose and xylose to ethanol, and its parental strain, ZM4, were characterized using 13C and 31P nuclear magnetic resonance (NMR) in vivo. From the 31P NMR data, the levels of nucleoside triphosphates (NTP) of ZM(pZB5) using xylose were lower than those of glucose. This can be related to the intrinsically slower assimilation and/or metabolism of xylose compared to glucose and is evidence of a less energized state of ZM4(pZB5) cells during xylose fermentation. Acetic acid was shown to be strongly inhibitory to ZM4(pZB5) on xylose medium, with xylose utilization being completely inhibited at pH 5.0 or lower in the presence of 10.9 g/L of sodium acetate. From the 31P NMR results, the addition of sodium acetate caused decreased NTP and sugar phosphates, together with acidification of the cytoplasm. Intracellular deenergization and acidification appear to be the major mechanisms by which acetic acid exerts its toxic effects on this recombinant strain.
The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture o 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L·h), and a yield (Y
p/s) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40–60 h); a slow bleed of concentrated cells may be required to overcome this problem.
In vivo nuclear magnetic resonance studies of ethanol fermentation characteristics and acetic acid inhibition of a recombinant Zymomonas mobilis ZM4 (pZB5)
- Kim Is Barrow
- Rogers Kd
- Pl
Kim IS, Barrow KD, Rogers PL (2000b) In vivo nuclear magnetic resonance studies of ethanol fermentation characteristics and acetic acid inhibition of a recombinant Zymomonas mobilis ZM4 (pZB5). Appl. Biochem. Biotechnol. 84–86: 357–370.