Flocculating Zymomonas mobilis is a promising host to be engineered for fuel ethanol production from lignocellulosic biomass

Whereas Saccharomyces cerevisiae uses the Embden‐Meyerhof‐Parnas pathway to metabolize glucose, Zymomonas mobilis uses the Entner‐Doudoroff (ED) pathway. Employing the ED pathway, 50% less ATP is produced, which could lead to less biomass being accumulated during fermentation and an improved yield of ethanol. Moreover, Z. mobilis cells, which have a high specific surface area, consume glucose faster than S. cerevisiae, which could improve ethanol productivity. We performed ethanol fermentations using these two species under comparable conditions to validate these speculations. Increases of 3.5 and 3.3% in ethanol yield, and 58.1 and 77.8% in ethanol productivity, were observed in ethanol fermentations using Z. mobilis ZM4 in media containing ～100 and 200 g/L glucose, respectively. Furthermore, ethanol fermentation bythe flocculating Z. mobilis ZM401 was explored. Although no significant difference was observed in ethanol yield and productivity, the flocculation of the bacterial species enabled biomass recovery by cost‐effective sedimentation, instead of centrifugation with intensive capital investment and energy consumption. In addition, tolerance to inhibitory byproducts released during biomass pretreatment, particularly acetic acid and vanillin, was improved. These experimental results indicate that Z. mobilis, particularly its flocculating strain, is superior to S. cerevisiae as a host to be engineered for fuel ethanol production from lignocellulosic biomass.     

Ethanol production from paper sludge by simultaneous saccharification and co‐fermentation using recombinant xylose‐fermenting microorganisms

Simultaneous saccharification and co‐fermentation (SSCF) of waste paper sludge to ethanol was investigated using two recombinant xylose‐fermenting microbes: Zymomonas mobilis 8b and Saccharomyces cerevisiae RWB222. S. cerevisiae RWB222 produced over 40 g/L ethanol with a yield of 0.39 g ethanol/g carbohydrate on paper sludge at 37°C, while similar titers and yields were achieved by Z. mobilis 8b at 30°C. Both S. cerevisiae RWB222 and Z. mobilis 8b exhibited decreasing cell viability at 37°C when producing over 40 g/L ethanol. A high ethanol concentration can account for S. cerevisiae RWB222 viability loss, but ethanol concentration was not the only factor influencing Z. mobilis 8b viability loss at 37°C. Over 3 g/L residual glucose was observed at the end of paper sludge SSCF by Z. mobilis 8b, and a statistical analysis revealed that a high calcium concentration originating from paper sludge, a high ethanol concentration, and a high temperature were the key interactive factors resulting in glucose accumulation. The highest ethanol yields were achieved by SSCF of paper sludge with S. cerevisiae RWB222 at 37°C and Z. mobilis 8b at 30°C. With good sugar consumption at 37°C, S. cerevisiae RWB222 was able to gain an improvement in the polysaccharide to sugar yield compared to that at 30°C, whereas Z. mobilis 8b at 30°C had a lower polysaccharide to sugar yield, but a higher sugar to ethanol yield than S. cerevisiae. Both organisms under optimal conditions achieved a 19% higher overall conversion of paper sludge to ethanol than the non‐xylose utilizing S. cerevisiae D5A at its optimal process temperature of 37°C. Biotechnol. Bioeng. 2010;107: 235–244. © 2010 Wiley Periodicals, Inc.     

Growth ofZymomonas on lactose: Gene cloning in combination with mutagenesis

Summary Wild-type strains ofZymomonas mobilis have a limited substrate range of glucose, fructose and sucrose. In order to expand this substrate range, transconjugants ofZ. mobilis containing Lac⁺ plasmids have been constructed. Although -galactosidase is expressed in such strains, they lack the ability to grow on lactose. We now report the development ofZ. mobilis strains capable of growth on lactose. This was achieved in two stages. First, a broad host range plasmid was constructed (pRUT102) which contained the lactose operon under the control of aZ. mobilis promoter plus genes for galactose utilization.Z. mobilis CP4.45 containing pRUT102 was then subjected to mutagenesis combined with continued selection pressure for growth on lactose. One strain,Z. mobilis SB6, produced a turbid culture that yielded 0.25% ethanol from 5% lactose (plus 2% yeast extract) in 15 days.     

Cloning and expression of amyE gene from Bacillus subtilis in Zymomonas mobilis and direct production of ethanol from soluble starch

Two proven secretion signal zmo130 and zmo331 native to Zymomonas mobilis were fused to the N terminal of ??-amylase from Bacillus subtilis and transformed into 5 different strains of Z. mobilis separately. It was found that the signal zmo130 could direct the extracellular secretion of the expressed ??-amylase with high activity, but zmo331 could not. Fermentation experiments demonstrated that the recombinant Z. mobilis CICC 10225(p130A) exhibited the highest level of ethanol production, which is nearly 50% of the theoretical yield of ethanol from soluble starch, but another recombinant Z. mobilis ATCC 31821(p130A) took the shortest fermentation time of approximately 3 days, with the second high level of ethanol yield. The recombined strains in our study could be an important target for the following genetic engineering of next amylase in order to hydrolyze starch completely.     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.     

Detrimental effect of increasing sugar concentrations on ethanol production from maize or decorticated sorghum mashes fermented with <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> or <Emphasis Type="Italic">Zymomonas mobilis</Emphasis>

The efficiency of ethanol fermentation, as affected by grain source (maize and decorticated red sorghum), total sugar concentration (13 or 20° Plato) and type of microorganism (Saccharomyces cerevisiae or Zymomonas mobilis) was studied. Maize mashes yielded 0.32 l ethanol kg⁻¹ ground grain whereas mashes prepared with decorticated red sorghum produced 0.28 l ethanol kg⁻¹. Both microorganisms yielded similar amounts of ethanol. However, high-gravity mashes (20° Plato) yielded lower amounts of ethanol compared to counterparts adjusted to 13° Plato (0.28 vs. 0.22 l ethanol kg⁻¹ ground grains). In decorticated sorghum mashes adjusted to 20° P, Z. mobilis produced 40 ml kg⁻¹ more ethanol compared to S. cerevisiae. In addition, Z. mobilis had a lower dependency on nitrogenous compounds.     

Alcohol production from pineapple waste

Saccharomyces cerevisiae andZymomonas mobilis were grown on pineapple waste and their alcohol production characteristics compared. The pineapple waste consisted of 19% cellulose, 22% hemi-cellulose, 5% lignin and 53% cell soluble matters but concentration of soluble sugars, which included 5.2% sucrose, 3.1% glucose and 3.4% fructose, was relatively low and pretreatment of the substrate was needed. Pretreatment of pineapple waste with cellulase and hemi-cellulase and then fermantation withS. cerevisiae orZ. mobilis produced about 8% ethanol from pineapple waste in 48 h.

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Résumé On a fait croîtreSaccharomyces cerevisiae etZymomonas mobilis sur des déchets d'ananas, et on a comparé les caractéristiques de leur production d'alcool. Le déchet d'ananas consistait en 19% de cellulose, 22% d'hémicellulose, 5% de lignine et 53% de matières cellulaires solubles. Mais la concentration en sucres solubles qui comprenait 5.2% de sucrose, 3.1% de glucose et 3.4% de fructose, était relativement faible. Le prétraitement du substral s'avérait donc nécessaire. Le p_r étraitement ces déchets d'ananas avec la cellulase et l'hemicellulase, suivi de la fermentation parS. cerevisiae ouZ. mobilis ont produit environ 8% d'ethanol à partir de résidus d'ananas en 48 h.

     

Fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae and Zymomonas mobilis ZM4 with penicillin G

Traditional fermentation of paddy malt mash (containing 18.1% w/v dextrose equivalent) to paddy arrack using paddy husk as source of inoculum yielded very low level of ethanol (4.25% v/v). Use of yeast isolates obtained from paddy husk as well as a potent ethanol producer like Zymomonas mobilis ZM4 and their combinations in the fermentation revealed that a combination of an yeast isolate PH 03 (Saccharomyces cerevisiae) and Z. mobilis ZM4 produced synergistically and statistically more ethanol (10.1% v/v) than the individual and other combination of cultures. In this process, addition of penicillin G at a concentration of 20 U/ml rather than heat sterilization, helped retention of the limited amylase activity in the mash for simultaneous saccharification and fermentation over 7 d at 30°C. About 98.5% of the carbohydrate was accountable in the fermentation which yielded 86.7% of the theoretical yield of ethanol, apart from biomass and acids.     

The Composition of Jerusalem Artichoke (Helianthus tuberosus L.) Spirits Obtained from Fermentation with Bacteria and Yeasts

The composition of spirits distilled from fermentation of Jerusalem artichoke (Helianthus tuberosus L.) tubers was compared by means of gas chromatography. The microorganisms used in the fermentation processes were the bacterium Zymomonas mobilis, strains 3881 and 3883, the distillery yeast Saccharomyces cerevisiae, strains Bc16a and D₂ and the Kluyveromyces fragilis yeast with an active inulinase. The fermentation of mashed tubers was conducted using a single culture of the distillery yeast Saccharomyces cerevisiae and the bacterium Zymomonas mobilis (after acid or enzymatic hydrolysis) as well as Kluyveromyces fragilis (sterilized mashed tubers). The tubers were simultaneously fermented by mixed cultures of the bacterium or the distillery yeast with K. fragilis. The highest ethanol yield was achieved when Z. mobilis 3881 with a yeast demonstrating inulinase activity was applied. The yield reached 94 % of the theoretical value. It was found that the distillates resulting from the fermentation of mixed cultures were characterized by a relatively lower amount of by‐products compared to the distillates resulting from the single species process. Ester production of 0.30–2.93 g/L, responsible for the aromatic quality of the spirits, was noticed when K. fragilis was applied for ethanol fermentation both in a single culture process and also in the mixed fermentation with the bacterium. Yeast applied in this study caused the formation of higher alcohols to concentrations of 7.04 g/L much greater than those obtained with the bacterium. The concentrations of compounds other than ethanol obtained from Jerusalem artichoke mashed tubers, which were fermented by Z. mobilis, were lower than those achieved for yeasts.     

EVALUATION OF BIOETHANOL PRODUCTION FROM CAROB PODS BY Zymomonas mobilis AND Saccharomyces cerevisiae IN SOLID SUBMERGED FERMENTATION

Bioethanol production from carob pods has attracted many researchers due to its high sugar content. Both Zymomonas mobilis and Saccharomyces cerevisiae have been used previously for this purpose in submerged and solid-state fermentation. Since extraction of sugars from the carob pod particles is a costly process, solid-state and solid submerged fermentations, which do not require the sugar extraction step, may be economical processes for bioethanol production. The aim of this study is to evaluate the bioethanol production in solid submerged fermentation from carob pods. The maximum ethanol production of 0.42 g g^?1 initial sugar was obtained for Z. mobilis at 30°C, initial pH 5.3, and inoculum size of 5% v/v, 9 g carob powder per 50 mL of culture media, agitation rate 0 rpm, and fermentation time of 40 hr. The maximum ethanol production for S. cerevisiae was 0.40 g g^?1 initial sugar under the same condition. The results obtained in this research are comparable to those of Z. mobilis and S. cerevisiae performance in other culture mediums from various agricultural sources. Accordingly, solid submerged fermentation has a potential to be an economical process for bioethanol production from carob pods.     

Construction and Analysis of High-Ethanol-Producing Fusants with Co-Fermentation Ability through Protoplast Fusion and Double Labeling Technology

Double labeling of resistance markers and report genes can be used to breed engineered Saccharomyces cerevisiae strains that can assimilate xylose and glucose as a mixed carbon source for ethanol fermentation and increased ethanol production. In this study Saccharomyces cerevisiae W5 and Candida shehatae 20335 were used as parent strains to conduct protoplast fusion and the resulting fusants were screened by double labeling. High performance liquid chromatography (HPLC) was used to assess the ethanol yield following the fermentation of xylose and glucose, as both single and mixed carbon sources, by the fusants. Interestingly, one fusant (ZLYRHZ7) was demonstrated to have an excellent fermentation performance, with an ethanol yield using the mixed carbon source of 0.424 g g⁻¹, which compares with 0.240 g g⁻¹ (W5) and 0.353 g g⁻¹ (20335) for the parent strains. This indicates an improvement in the ethanol yield of 43.4% and 16.7%, respectively.     

Development of new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by recombinant strain

The development of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial strains has been studied intensively [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445; G. Birol, Z.Ï. Önsan, B. Kirdar, S.G. Oliver, Ethanol production and fermentation characteristics of recombinant Saccharomyces cerevisiae strains grown on starch, Enzyme Microb. Technol. 22 (1998) 672–677; F. Kobayashi, Y. Nakamura, Effect of repressor gene on stability of bioprocess with continuous conversion of starch into ethanol using recombinant yeast, Biochem. Eng. J. 18 (2004) 133–141; F. Kobayashi, Y. Nakamura, Mathematical model of direct ethanol production from starch in immobilized recombinant yeast culture, Biochem. Eng. J. 21 (2004) 93–101; M.M. Altintas, K.Ö. Ülgen, B. Kirdar, Z.Ï. Önsan, S.G. Oliver, Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors, Enzyme Microb. Technol. 31 (2002) 640–647; K.Ö. Ülgen, B. Saygili, Z.Ï. Önsan, B. Kirdar, Bioconversion of starch into ethanol by a recombinant Saccharomyces cerevisiae strain YPG-AB, Process Biochem. 37 (2002) 1157–1168]. Saccharomyces cerevisiae YPB-G strain secretes a bifunctional fusion protein containing enzymatic activity of the B. subtilis alpha-amylase and of the Aspergillus awamori glucoamylase [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445], and therefore is distinguished in relation to SSFSE step. In this work we have used the experimental data, presented in the paper [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445] to develop two-hierarchic-level unstructured mathematical model describing kinetics of direct bioconversion of starch to ethanol. The first level has modeled enzymatic hydrolysis of starch to glucose by bifunctional protein and the second level includes utilization and bioconversion of glucose to ethanol by yeasts. The second level has unified the enzymatic degradation of starch, and glucose metabolization to ethanol by microorganisms. The response surface analysis was used to develop the rates models. A hybrid genetic algorithm and a decomposition approach were used in the nonlinear parameters identification procedure. The proposed model demonstrated excellent flexibility for different operational conditions of SSFSE process, and can be used successfully to describe microbial physiology of genetically modified strains.     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.     

A high-throughput transient gene expression system for switchgrass (Panicum virgatum L.) seedlings

Background

Fermentations using Escherichia coli KO11, Saccharomyces cerevisiae 424A(LNH-ST), and Zymomonas mobilis AX101 are compared side-by-side on corn steep liquor (CSL) media and the water extract and enzymatic hydrolysate from ammonia fiber expansion (AFEX)-pretreated corn stover.

Results

The three ethanologens are able produce ethanol from a CSL-supplemented co-fermentation at a metabolic yield, final concentration and rate greater than 0.42 g/g consumed sugars, 40 g/L and 0.7 g/L/h (0-48 h), respectively. Xylose-only fermentation of the tested ethanologenic bacteria are five to eight times faster than 424A(LNH-ST) in the CSL fermentation. All tested strains grow and co-ferment sugars at 15% w/v solids loading equivalent of ammonia fiber explosion (AFEX)-pretreated corn stover water extract. However, both KO11 and 424A(LNH-ST) exhibit higher growth robustness than AX101. In 18% w/w solids loading lignocellulosic hydrolysate from AFEX pretreatment, complete glucose fermentations can be achieved at a rate greater than 0.77 g/L/h. In contrast to results from fermentation in CSL, S. cerevisiae 424A(LNH-ST) consumed xylose at the greatest extent and rate in the hydrolysate compared to the bacteria tested.

Conclusions

Our results confirm that glucose fermentations among the tested strains are effective even at high solids loading (18% by weight). However, xylose consumption in the lignocellulosic hydrolysate is the major bottleneck affecting overall yield, titer or rate of the process. In comparison, Saccharomyces cerevisiae 424A(LNH-ST) is the most relevant strains for industrial production for its ability to ferment both glucose and xylose from undetoxified and unsupplemented hydrolysate from AFEX-pretreated corn stover at high yield.     

Recent Developments in the Zymomonas Process for Ethanol Production

Abstract

The bacterium Zymomonas mobilis, which is used in the tropics to make pulque and alcoholic palm wines, appears to have considerable potential for industrial alcohol fermentations. Some of the advantages of the Zymomonas process reported in studies from our laboratory^1-24 are

1. There are significantly higher specific rates of sugar uptake and ethanol production compared to those found for yeasts.

2. Considerably higher volumetric ethanol productivities found in continuous cell recycle systems (up to 120 to 200 g/hr).

3. There are higher ethanol yields and lower biomass production than for yeasts. The lower biomass concentrations would seem to be a consequence of the lower metabolic energy available for growth. Zymomonas metabolize glucose via the Entner-Doudoroff pathway while yeasts convert glucose to ethanol via glycolysis.

4. Zymomonas cultures grow anaerobically and, unlike yeasts, do not require the controlled addition of oxygen to maintain viability at high cell concentrations.

5. The ethanol tolerance of some selected strains of Zymomonas is comparable if not higher than strains of Saccharomyces cerevisiae. Ethanol concentrations of 85 g/(up to 11% v/v) have been achieved in continuous culture and up to 130 g/(16% v/v) in batch culture.     

Characterization of the two alcohol dehydrogenases of Zymomonas mobilis

The two alcohol dehydrogenases of Zymomonas mobilis, a fermentative bacterium, have been characterized kinetically and physiologically, and a preliminary biochemical characterization has been made. The two isozymes are separable electrophoretically and have been purified separately by affinity chromatography. One, a tetramer, has a subunit molecular weight of 34,700, very close to that of yeast alcohol dehydrogenase. It is the more cathodally migrating of the two isozymes on electrophoresis at pH 8.6, and has been designated ZADH-I. The second, a dimer, has a slightly smaller subunit size (31,100), and has been designated ZADH-II. Both contain approximately one zinc atom per subunit, but have very different amino acid compositions. The two isozymes have clearly different apparent Michaelis constants and show different kinetics. The kinetics of ZADH-I are similar to those of ADH-II of yeast. ZADH-I is more stable during purification than ZADH-II, can oxidize a wider range of alcohols, and has a pH optimum of 9.5, more similar to that of yeast alcohol dehydrogenase. Strains of Z. mobilis cultured in low-sucrose media lose the ZADH-II irreversibly, suggesting that the gene may be on a plasmid. The fermentative ability of Z. mobilis, based as it is on the Entner-Doudoroff pathway, is clearly different from that of yeast. Under all conditions tested, Z. mobilis produced more acetaldehyde than did laboratory strains of Saccharomyces cerevisiae. At high sugar concentrations it grew poorly and produced little ethanol while yeast grew and fermented readily. As with yeast, the ratio of NAD to NADH in the cell cannot be perturbed by changes in medium composition when both isozymes are present. Allyl alcohol resistant mutants of this bacterium show reduced alcohol dehydrogenase activity and alcohol production and increased acetaldehyde excretion.     

Evolutionary Engineering of Saccharomyces cerevisiae for Anaerobic Growth on Xylose

Xylose utilization is of commercial interest for efficient conversion of abundant plant material to ethanol. Perhaps the most important ethanol-producing organism, Saccharomyces cerevisiae, however, is incapable of xylose utilization. While S. cerevisiae strains have been metabolically engineered to utilize xylose, none of the recombinant strains or any other naturally occurring yeast has been able to grow anaerobically on xylose. Starting with the recombinant S. cerevisiae strain TMB3001 that overexpresses the xylose utilization pathway from Pichia stipitis, in this study we developed a selection procedure for the evolution of strains that are capable of anaerobic growth on xylose alone. Selection was successful only when organisms were first selected for efficient aerobic growth on xylose alone and then slowly adapted to microaerobic conditions and finally anaerobic conditions, which indicated that multiple mutations were necessary. After a total of 460 generations or 266 days of selection, the culture reproduced stably under anaerobic conditions on xylose and consisted primarily of two subpopulations with distinct phenotypes. Clones in the larger subpopulation grew anaerobically on xylose and utilized both xylose and glucose simultaneously in batch culture, but they exhibited impaired growth on glucose. Surprisingly, clones in the smaller subpopulation were incapable of anaerobic growth on xylose. However, as a consequence of their improved xylose catabolism, these clones produced up to 19% more ethanol than the parental TMB3001 strain produced under process-like conditions from a mixture of glucose and xylose.     

Fermentation of lactose by Zymomonas mobilis carrying a Lac+ recombinant plasmid

Lac⁺ recombinant plasmids encoding a β-galactosidase fused protein and lactose permease of Escherichia coli were introduced Zymomonas mobilis. The fused protein was expressed with 450 to 5,860 Miller units of β-galactosidase activity, and functioned as lactase. Raffinose uptake by Z. mobilis CP4 was enhanced in the plasmid-carrying strain over the plasmid-free strain, suggesting that the lactose permease was functioning in the organism. Z. mobilis carrying the plasmid could produce ethanol from lactose and whey, but could not grow on lactose as the sole carbon source. It was found that the growth of the organism was inhibited by either galactose of the galactose liberated from lactose.     

Growth requirements and the establishment of a chemically defined minimal medium inZymomonas mobilis

Summary A chemically defined minimal medium which fulfils the growth requirements of differentZymomonas mobilis strains has been established. The kinetics of ethanol production of the strains ATCC 10988, CU1, CP4 and 11163 grown on the minimal medium at different glucose concentrations were measured. All strains produced ethanol at rates similar to those on complete medium. The minimal medium described is suitable to study spontaneous metabolic deficiciencies and regulation of enzyme activities inZ.mobilis.