Simultaneous saccharification and ethanol fermentation of sago starch using immobilized Zymomonas mobilis

Simultaneous saccharification and ethanol fermentation (SSF) of sago starch using amyloglucosidase (AMG) and immobilized Zymomonas mobilis ZM4 on sodium alginate was studied. The immobilized Zymomonas cells were more thermo-stable than free Zymomonas cells in this system. The optimum temperature in the SSF system was 40°C, and 0.5% (v/w) AMG concentration was adopted for the economical operation of the system. The final ethanol concentration obtained was 68.3 g/l and the ethanol yield, Y_p/s, was 0.49 g/g (96% of the theoretical yield). After 6 cycles of reuse at 40°C with 15% sago starch hydrolysate, the immobilized Z. mobilis retained about 50% of its ethanol fermenting ability.     

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.     

Conversion of sugar-beet particles to ethanol by the bacteriumZymomonas mobilis in solid-state fermentation

Summary The possibility of usingZymomonas mobilis as the microorganism, in solid-state fermentation of sugar-beet particles was investigated. The major factors affecting the process were investigated and related to ethanol yield and productivity. Ethanol yield of 0.48 g/g sugar, volumetric productivity of 12 g/L h, and final ethanol concentration of 130 g/L show the good performance ofZ.mobilis in a solid-state fermentation.     

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.     

Continuous ethanol production from sago starch using immobilized amyloglucosidase and Zymomonas mobilis

To produce ethanol more economically than in a conventional process, it is necessary to attain high productivity and low production cost. To this end, a continuous ethanol production from sago starch using immobilized amylogucosidase (AMG) and Zymomonas mobilis cells was studied. Chitin was used for immobilization of AMG and Z. mobilis cells were immobilized in the form of sodium alginate beads. Ethanol was produced continuously in an simultaneous saccharification and ethanol fermentation (SSF) mode in a pacekd bed reactor. The maximum ethanol productivity based on the void volume, V_v, was 37 g/l/h with ethanol yield, Y_p/s, 0.43 g/g (84% of the theoretical ethanol yield) in this system. The steady-state concentration of ethanol (46 g/l could be maintained in a stable manner over two weeks at the dilution rate of 0.46 h⁻.     

Formation of higher alcohols and phenol by strains of zymomonas sp.

Strains of the bacteria Zymomonas sp. were studied for their ability to form higher alcohols. In a complex growth medium, six strains were shown to produce significant amounts of 1-propanol, 1-butanol, 2-methyl-1-butanol, 3-methyl-1-butanol, 2-methyl-2-butanol, pentanols, secondary hexyl-alcohols, and trace amounts of n-hexanol. When resting cells of these organisms were placed into a fermentation medium containing glucose and Tris-buffer, Z. mobilis 8938 produced increased levels of 1-butanol, and secondary hexyl-alcohols at concentrations of 13.5 mg/liter and 5.8 mg/liter, respectively. Another strain, Z. mobilis subsp. mobilis B 806, stimulated the formation of 1-propanol and 1-butanol at concentrations of 14.9 mg/liter and 23.52 mg/liter, respectively. Amino acids or amino acid precursors were then added to the fermentation medium. The presence of threonine and α-ketobutyric acid stimulated Z. mobilis 8938 to produce 82.6 mg/liter secondary hexyl-alcohols and 8.0 mg/liter n-hexanol, respectively. Isoleucine and valine increased the production of 2-methyl-1-butanol (394.0 mg/liter) and 3-methyl-1-butanol (113.4 mg/liter), respectively, by Z. mobilis subsp. mobilis B 806. Glutamine enhanced the formation of 2-methyl-2-butanol production to concentrations 38.8 mg/liter in Zymomonas strain B 806. Additional experiments suggested that higher alcohol production could also be accomplished in the absence of glucose when cells were allowed to metabolize the precursors only. The effect of aromatic amino acids on phenol production was determined using resting cells of Zymomonas sp. The maximum yield of phenol (111.6 mg/liter) was found by Zymomonas strain 8938 in the presence of tyrosine. The addition of phenylalanine also stimulated this strain to form 71.4 mg/liter of phenol.     

Characteristics of Zymomonas mobilis immobilized by photo-crosslinkable resin in ethanol fermentation

Zymomonas mobilis, an ethanol-producing bacterium, was immobilized in hydrophilic photo-crosslinked resin gels to form a biocatalyst. The molecular structure of the photo-crosslinkable resin could be modulated so as to minimize a disadvantage of this bacterium—poor-tolerance to salts in molasses. Characteristics of Z. mobilis immobilized by photo-crosslinkable resin gel, such as fermentability, cell growth in gel, the potential of gel materials, diffusion of materials, and salt distribution are discussed. ENTG-3800 photo-crosslinkable resin was selected as the most suitable entrapping material for Z. mobilis, especially in using molasses.     

Isolation of Noninhibitory Strains of Zymomonas mobilis

Wild-type Zymomonas mobilis strains inhibit the growth of Escherichia coli. We report the first isolation of noninhibitory strains, called Zymomonas inhibition negative (Zin⁻), after treatment with N-methyl-N′-nitro-N-nitrosoguanidine. A standardized soft-agar overlay procedure for detecting E. coli growth inhibition was also developed.     

Fermentative lactic acid production with a metabolically engineered yeast immobilized in photo-crosslinkable resins

In this study, the immobilization technique involving photo-crosslinkable resin gels was used for lactic acid production. Saccharomyces cerevisiae OC-2T T165R, a metabolically engineered yeast that produces optically pure l(+)-lactic acid, was immobilized in hydrophilic photo-crosslinked resin gels as a biocatalyst. Three resin gels, TEP 1, TEP 2 and TEP 3, were examined and all of them showed high performance as to lactic acid production. Resin gel TEP 1, which exhibited the highest productivity among the resin gels was used for 15 consecutive batch fermentations without decreases in productivity and mechanical deformation, indicating that it was a suitable carrier for long-term lactic acid fermentation. Moreover, the use of the immobilization technique can improve the productivity of the metabolically engineered yeast in the fermentation with or without extraction, showing promise for using the immobilized engineered yeast for lactic acid production.     

Flow rate and bead size as critical parameters for immobilized-yeast reactors

The productivity of immobilized yeast cell reactors varies with a number of parameters, including flow, amount and growth rate of yeast, bead size and type of medium. Variation of these parameters has a pronounced effect on reaction rate. This paper presents typical ranges for these productivities and demonstrates the patterns of changes that take place when bead size, flow and reaction medium are varied. Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for the production of ethanol. The productivity of immobilized yeast in a batch reactor (0.2 g ethanol/g yeast · h) was only two-thirds that of free cells suspended at an equivalent cell density (0.3 g ethanol/g yeast · h). Different flow rates and bead sizes were used to ‘optimize’ the productivity. The productivity of 3.34 mm beads at a flow rate of 8.8 litre h^?1(superficial velocity: 0.12 cm s^?1) was 95% higher than that at 1.0 l h^?1. Maximum productivities of 0.34, 0.27, 0.22 g/g yeast· h were obtained (at a flow rate of 8.8 l h^?1) for 9.2% yeast-immobilized beads of 3.34, 4.45 and 5.65 mm in diameter, respectively.     

Cellulosic ethanol production by Zymomonas mobilis harboring an endoglucanase gene from Enterobacter cloacae

Enterobactercloacae was isolated from the gut of the wood feeding termite, Heterotermesindicola, and a 2.25-kb fragment conferring cellulase activity was cloned in Escherichiacoli. The cloned fragment contained a 1083-bp ORF which could encode a protein belonging to glycosyl hydrolase family 8. The cellulase gene was introduced into Zymomonasmobilis strain Microbial Type Culture Collection centre (MTCC) on a plasmid and 0.134 filter paper activity unit (FPU)/ml units of cellulase activity was observed with the recombinant bacterium. Using carboxymethyl cellulose and 4% NaOH pretreated bagasse as substrates, the recombinant strain produced 5.5% and 4% (V/V) ethanol respectively, which was threefold higher than the amount obtained with the original E.cloacae isolate. The recombinant Z. mobilis strain could be improved further by simultaneous expression of cellulase cocktails before utilizing it for industrial level ethanol production.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

Summary The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.     

Effects of ethanol concentration on immobilized Zymomonas mobilis for continuous production of ethanol

The effects of ethanol concentration on the ethanol productivity and activity of immobilized Zymomonas mobilis cells during continuous fermentation of glucose has been studied at various ethanol concentrations. On changing the inlet ethanol concentration, Po, from 0.0 kg/m³ to any other level, 8 h were required to fully experience the effects of a change in Po, whereas 8 h to 2 days, depending on Po, were required to reach the steady state on switching back to the ethanol free medium. The volumetric ethanol productivity decreased from 92.5 to 0.0 kg/m³·h as the ethanol concentration in the bioreactor was changed from 46.3 to 126 kg/m³. The activity of the immobilized cells recovered up to 63% in 2 days even after exposing the cells to 126 kg/m³ of ethanol.     

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.     

Ethanol production by immobilized whole cells ofZymomonas mobilis in a continuous flow expanded bed bioreactor and a continuous flow stirred tank bioreactor

Continuous ethanol fermentation by immobilized whole cells ofZymomonas mobilis was investigated in an expanded bed bioreactor and in a continuous stirred tank reactor at glucose concentrations of 100, 150 and 200 g L^–1. The effect of different dilution rates on ethanol production by immobilized whole cells ofZymomonas mobilis was studied in both reactors. The maximum ethanol productivity attained was 21 g L^–1 h^–1 at a dilution rate of 0.36 h^–1 with 150 g glucose L^–1 in the continuous expanded bed bioreactor. The conversion of glucose to ethanol was independent of the glucose concentration in both reactors.     

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.     

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.     

Growth ofZymomonas mobilis CP4 on mannitol

Summary The ethanologenZymomonas mobilis has a restricted substrate range, namely glucose, fructose and sucrose. It would be useful to expand its substrate range to include other carbohydrates.Z. mobilis was screened for growth on 30 different carbohydrates and organic acids. A single spontaneous mutant,Z mobilis CP4.60, was isolated which illustrated feeble growth on mannitol as the sole carbohydrate source after three months of incubation. Growth ofZ. mobilis CP4.60 for several months in continuous culture with excess mannitol, and including a round of NTG (N-methyl-N'-nitro-N-nitrosoguanidine) mutagenesis in the chemostat, led to the isolation a sequential series of mutants (CP4.62, CP4.64 and CP4.66), each with improved growth rates on mannitol. Metabolism of mannitol byZ. mobilis is oxygen-dependent, resulting in limited production of ethanol and incresed production of lactic acid. This is an initial example of extension of the substrate range ofZymomonas. The conversion of mannitol to fructose could be via an altered alcohol dehydrogenase.     

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.