High productivity ethanol fermentation on a mineral medium using a flocculent strain ofZymomonas mobilis

Summary A flocculent strain ofZymomonas mobilis (ZM4F JM1) was isolated in continuous culture. The parent strain, ZM4F, had lost its flocculating properties. The isolation was done in a conical fermentor at high dilution rate. Ethanol production by the new strain was then compared on a rich and mineral medium. The mineral medium showed high performance and could be used for industrial production of ethanol since it reduced one hundred fold the vitamin cost of the fermentation.     

Ethanol production by Zymomonas mobilis CP4 from sugar cane chips

Summary The fermentation of large sugar cane chips (1.0–1.5 in) to ethanol by Zymomonas mobilis CP4 (Z. mobilis) was studied in two glass fermentors operating with culture circulation for agitation (the EX-FERM type): a. A laboratory scale(2.5 liter) cylindrical vessel; b. A bench scale (8 liter) wide vessel. Z. mobilis cultures consumed 89–96% of the cane sucrose, converting it to ethanol by 90–97% of the theoretical yield in the laboratory scale fermentor and by 83–90% in the bench scale fermentor culture. Comparative Saccharomyces spp. cultures in laboratory fermentor consumed 96–98% of the cane sucrose, with ethanol conversion of only 75–79% of the theoretical yield.These preliminary results indicated that sucrose in agricultural size sugar cane chips was ethanol fermentable as compared to small size sugar cane chips or to sugar cane juice. Z. mobilis CP4 cultures converted sucrose more efficiently to ethanol than Saccharomyces spp. as shown in the laboratory scale fermentor studies.The ethanol yields in a wide bench scale fermentor cultures were slightly lower than in a laboratory fermentor.     

Continuous ethanol production from sugar beet molasses using an osmotolerant mutant strain of Zymomonas mobilis

A laboratory process was established for ethanol production by fermentation of sugar beet molasses with the bacterium Zymomonas mobilis. Sucrose in the molasses was hydrolyzed enzymatically to prevent levan formation. A continuous system was adopted to reduce sorbitol formation and a two-stage fermentor was used to enhance sugar conversion and the final ethanol concentration. This two-stage fermentor operated stably for as long as 18 d. An ethanol concentration of 59.9 g/l was obtained at 97% sugar conversion and at high ethanol yield (0.48 g/g, 94% of theoretical). The volumetric ethanol productivity (3.0 g/l·h) was superior to that of batch fermentation but inferior to that of a single-stage continuous system with the same medium. However, the thanol concentration was increased to a level acceptable for economical recovery. The process proposed in this paper is the first report of successful fermentation of sugar beet molasses in the continuous mode using the bacterium Z. mobilis.     

Draft Genome Sequence of the Flocculating Zymomonas mobilis Strain ZM401 (ATCC 31822)

Zymomonas mobilis ZM401 is a flocculating strain which can be self-immobilized within fermentors for a high-cell-density culture to improve ethanol productivity, as well as high-gravity fermentation to increase ethanol titer, due to its improved ethanol tolerance associated with the morphological change. Here, we report its draft genome sequence.     

Influence of the flocculating agent in sedimentation and performance of a non flocculent strain of Zymomonas mobilis in the ethanol production process

The influence of the flocculating agent was studied in the performance (measured by microbial growth and ethanol production) of a non flocculent strain of Zymomonas mobilis, as well as the potentiality of the sedimentation process in the separation of the biomass from the fermentation broth. Among the flocculating agents studied, it was verified that both tannin and the polyelectrolyte yielded good results with regard to cellular performance. However, with regard to sedimentation tannin is more adequate to be used in processes involving Zymomonas mobilis.     

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.     

Continuous high rate production of ethanol by Zymomonas mobilis in an attached film expanded bed fermentor

Summary Experiments were conducted with Zymomonas mobilis in an attached film expanded bed (AFEB) fermentor at different dilution rates, using a feed glucose concentration of 100 gm/l. Vermiculite was used as the bed material for attachment of the bacterial film. Ethanol volumetric productivities were maximum at a dilution rate of 3.6hr^-1. The productivities were 105 gm/l-hr and 210 gm/l-hr based on total fermentor volume and bed volume, respectively.     

A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis.     

Effect of ventilation on the production of acetaldehyde by Zymomonas mobilis

The production of acetaldehyde, a flavoring agent in food, by Zymomonas mobilis was carried out in batch culture. The volatilization rate constant (k_v) of acetaldehyde and the initial volumetric oxygen transfer coefficient (k_La⁰) in an Erlenmeyer flask with a cotton-plug (cotton-flask) and an aerated-flask with a forced-air system (aerated-flask) were measured. The culture environment in the aerated-flask was found to be very different from that in the cotton-flask. Cell growth in a cotton-flask was strongly inhibited, making practical acetaldehyde production in cotton-flask very difficult. On the other hand, acetaldehyde production in the aerated-flask increased while the fermentation time decreased with increases in the air flow rate. The k_v value of acetaldehyde in a jar fermentor was affected mainly by air flow rate. By considering both the oxygen transfer rate and the ventilation effect on the culture, it was possible to scale-up from the aerated-flask to a jar fermentor. In the jar fermentor, production of acetaldehyde and growth inhibition by acetaldehyde were affected mainly by the k_La⁰ and k_v, values, respectively. The overall production of acetaldehyde in the jar fermentor under the optimum k_La⁰ and k_v conditions was 6.64 g/l (Y_p/s: 0.27), which was about 1.5 times higher than the maximum concentration obtained in the aerated-flask.     

R-Plasmid Transfer in Zymomonas mobilis

Conjugal transfer of three IncP1 plasmids and one IncFII plasmid into strains of the ethanol-producing bacterium Zymomonas mobilis was obtained. These plasmids were transferred at high frequencies from Escherichia coli and Pseudomonas aeruginosa into Z. mobilis and also between different Z. mobilis strains, using the membrane filter mating technique. Most of the plasmids were stably maintained in Z. mobilis, although there was some evidence of delayed marker expression. A low level of chromosomal gene transfer, mediated by plasmid R68.45, was detected between Z. mobilis strains. Genetic evidence suggesting that Z. mobilis may be more closely related to E. coli than to Pseudomonas or Rhizobium is discussed.     

The flocculation of bacteria using cationic synthetic flocculants and chitosan

Summary Bacteria are flocculated with high molecular weight cationic synthetic flocculants and chitosan. High charge density polymers are the most effective of the synthetic flocculants. Only chitosan is effective in flocculating the E. coli and B. subtilis cultures in complex broth. The difference in effectiveness between the synthetic flocculants and chitosan for flocculating E. coli, B. subtilis and Z. mobilis may be attributed to hydrogen bonding between the polysaccharide flocculant and cell surface polymers in addition to electrostatic interactions, and, in complex media, complexation of synthetic polymers with anionic polyelectrolytes.     

Expression of a Lactose Transposon (Tn951) in Zymomonas mobilis

The potential utility of Zymomonas mobilis as an organism for the commercial production of ethanol would be greatly enhanced by the addition of foreign genes which expand its range of fermentable substrates. We tested various plasmids and mobilizing factors for their ability to act as vectors and introduce foreign genes into Z. mobilis CP4. Plasmid pGC91.14, a derivative of RP1, was found to be transferred from Escherichia coli to Z. mobilis at a higher frequency than previously reported for any other plasmids. Both tetracycline resistance and the lactose operon from this plasmid were expressed in Z. mobilis CP4. Plasmid pGC91.14 was stably maintained in Z. mobilis at 30°C but rapidly lost at 37°C.     

Vacuum fermentation for ethanol production using strains of Zymomonas mobilis

Summary Using strains of Z.mobilis, a vacuum fermentation system has been evaluated. The system was designed with the fermentor at atmospheric pressure and an external vacuum vessel (50 mm Hg). Sequential operation of the vacuum vessel was under microprocessor control. The use of Z.mobilis together with the two-stage design of the vacuum system has been found to overcome the problems of oxygen addition and the possibility of contamination reported previously for vacuum fermentations with yeasts. The productivity of 85 g/1/h found in the continuous cell recycle experiments was similar to that reported previously for a strain of S.cerevisiae.     

Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis

Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis.     

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Summary Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of -galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed -galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.     

Ethanol production from wheat flour by Zymomonas mobilis

Starch from wheat flour was enzymatically hydrolyzed and used for ethanol production by Zymmonas mobilis. The addition of a nitrogen source like ammonium sulfate was sufficient to obtain a complete fermentation of the hdyrolyzed strach. In batch culture a glucose concentration as high as 223 g/l could be fermented (conversion 99.5%) to 105 g/l of ethanol in 70 h with an ethanol yield of 0.47 g/g (92% of theoretical). In continuous culture the use of a flocculent strain and a fermentor with an internal settler resulted (D=1,4 h⁻¹) in a high ethanol productivity of 70.7 g/l·h with: ethanol concentration 49.5 g/l, ethanol yield 0.50 g/g (98% of theoretical and substrate conversion 99%.     

Complete Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Centrotype ATCC 29191

Zymomonas mobilis is an ethanologenic bacterium that has been studied for use in biofuel production. Of the sequenced Zymomonas strains, ATCC 29191 has been described as the phenotypic centrotype of Zymomonas mobilis subsp. mobilis, the taxon that harbors the highest ethanol-producing Z. mobilis strains. ATCC 29191 was isolated in Kinshasa, Congo, from palm wine fermentations. This strain is reported to be a robust levan producer, while in recent years it has been employed in studies addressing Z. mobilis respiration. Here we announce the finishing and annotation of the ATCC 29191 genome, which comprises one chromosome and three plasmids.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.     

Continuous high-ethanol fermentation from cane molasses by a flocculating yeast

Repeated-batch fermentation by a flocculating fusant, Saccharomyces cerevisiae HA 2, was done in a molasses medium that contained 20% (w/v) total sugar, at 30°C in an automatically controlled fermentor, and the effects of ethanol concentration on the specific growth rate and the specific production rate of ethanol were studied. Both the specific growth rate and the specific production rate of ethanol fell with increase of ethanol concentration, and there was a linear correlation between each rate and the concentration of thanol. The maximum specific growth rate (μ_max) and the maximum specific production rate of ethanol (q_max) were 0.12 h⁻¹ and 0.1 g ethanol/10⁹ cells·h, respectively. The specific growth rate and the specific production rate of ethanol fell to zero at ethanol concentration of 89 g/l and 95 g/l, respectively. The number of viable cells, calculated from the linear inhibition equation, was 1.3 × 10⁹ cells/ml for production of 85 g/l ethanol at a dilution rate (D₁) of 0.2 h⁻¹. Based on this estimation, a laboratory-scale continuous fermentation, using two fermentors in series, was done. In the second fermentor, 85 g/l ethanol was produced at a dilution rate (D₁) of 0.2 h⁻¹ by the active feedig of the fermented mash from the first fermentor into the second fermentor by pumping (hereafter called active feeding). To maintain the number of viable cells above 10⁹ cells/ml in the second fermentor, a active feeding ratio of more than 23% was required. Under these conditions, 81 g/l ethanol was produced in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹, and the high ethanol productivity of 20.3 g/l·h could be achieved. A bench-scale continuous fermentation, using two fermentors in series, with a active feeding ratio of 25% was done. An ethanol concentration of 84 g/l in the second fermentor at a dilution rate (D_t) of 0.25 h⁻¹ was achieved, just as it was in the laboratory-scale fermentation test.     

Expression of a xylose-specific transporter improves ethanol production by metabolically engineered Zymomonas mobilis

Zymomonas mobilis is a promising organism for biofuel production as it can produce ethanol from glucose at high rates. However, Z. mobilis does not natively ferment C5 sugars such as xylose. While it has been engineered to do so, the engineered strains do not metabolize these sugars at high rates. Previous research has identified some of the bottlenecks associated with xylose metabolism in Z. mobilis. In this work, we investigated transport as a possible bottleneck. In particular, we hypothesized that the slow uptake of xylose through the promiscuous Glf transporter may limit the efficiency of xylose metabolism in Z. mobilis. To test this hypothesis, we expressed XylE, the low-affinity xylose transporter from Escherichia coli, in a xylose-utilizing strain of Z. mobilis. Our results show that the expression of this pentose-specific transporter improves the rate of xylose utilization in Z. mobilis; however, this enhancement is seen only at high xylose concentrations. In addition, we also found that overexpression of the promiscuous Z. mobilis transporter Glf yielded similar results, suggesting that the transport bottleneck is not due to the specificity, but rather the capacity for sugar uptake.