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.     

The kinetics of ethanol production by Zymomonas mobilis on fructose and sucrose media

Summary Z.mobilis is strain ZM4 was grown on 250 g/l fructose and sucrose media in batch culture and on 100 and 150 g/l sucrose media in continuous culture. With fructose, a significant reduction in the growth rate and the cell yield was apparent although the other kinetic parameters were similar to those previously reported for fermentation of glucose. With sucrose the major differences were a reduction in ethanol yield, (due to levan formation) and a lower final ethanol concentration. Ethanol inhibition of sucrose metabolism occurred at relatively low ethanol concentrations compared to those inhibiting glucose metabolism.     

Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Summary Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units withZ. mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.     

Stability and expression of a cloned α-glucosidase gene inZymomonas mobilis grown in batch and continuous culture

Summary Strains ofZymomonas mobilis containing an -glucosidase gene cloned fromBacillus brevis strain 27-7 (NRRL B-4389) on the plasmid pNSW358 showed varying degrees of stability in batch culture under non-selective conditions. After 45 generations of growth in continuous culture, pNSW358 was stable inZ.mobilis strain ZM6100 and the specific activity of -glucosidase in these cells was 2.7 nmol/min/mg protein. Lysed cell extracts confirmed the activity of the -glucosidase enzyme in ZM6100(pNSW358) with 21 g/1 ethanol in 50 (82% theoretical conversion of maltose to ethanol). ZM6100(pNSW358) whole cells showed a very slow conversion rate on maltose as a sole carbon source with only 5.3 g/1 ethanol after 30 days on 100 g/l maltose medium.     

Kinetic studies on a flocculent strain of Zymomonas mobilis

Summary A flocculent mutant of Zymomonas mobilis has been isolated and kinetic studies carried out in batch and continuous culture. By comparison with the parent strain the specific rates of glucose uptake and ethanol production were decreased by 20%. Cell recycle and semibatch cultures with the flocculent strain resulted in relatively high productivities (viz. 50 g/l/h). However semibatch culture had the additional advantages of an increased ethanol concentration (viz. 82 g/l) and a more stable and controlled environment for cell separation.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Ethanol production from cassava and sago starch using Zymomonas mobilis

Summary Cassava and sago starch were evaluated for their feasibilities as substrates for ethanol production using Zymomonas mobilis ZM4 strain. Before fermentation, the starch materials were pretreated employing two commercial enzymes, Termamyl (thermostable -amylase) and AMG (amyloglucosidase). Using 2 l/g of Termamyl and 4 l/g of AMG, effective conversion of both cassava and sago starch into glucose was found with substrate concentration up to 30%(w/v) dry substances. Fermentation study performed using these starch hydrolysates as substrates resulted in ethanol yield at an average of 0.48g/g by Z. Mobilis ZM4.     

Characterization of a superior thermotolerant mutant ofZymomonas mobilis ZM4 for ethanol production in glucose medium

Summary Nitrosoguanidine-induced, stable theromotolerant mutant (ZMI₂) ofZymomonas mobilis ZM4 was found to possess almost normal cell morphology, and a better ethanol tolerance at 42°C than the parent strain (ZM4). Its kinetic parameters, in converting different concentrations of glucose to ethanol, were comparable to ZM4 at 30°C, and significantly superior at 42°C. In a 200 g/L glucose medium in a pH-stat (5.0) at 42°C, the mutant yielded more ethanol (71.0 g/L) (improved to 73.7 g/L at pH 5.5) and alcohol dehydrogenase (ADH) than the parent strain. The ADH levels in both the strains were repressed, depending upon the increased level of sugar and degree of temperature.     

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.     

Effect of acid or enzymatic hydrolysis on ethanol production by Zymomonas mobilis growing on Jerusalem artichoke juice

Summary Ethanol production from the inulin of Jerusalem artichoke byZ. mobilis was studied in batch and continuous fermentations. Both acid or enzymatic hydrolysis were used. In continuous cultures enzymatic hydrolysis showed better results. Ethanol productivities of 17.7 and 29.0 g/l.h were obtained at output concentrationsca 35 g/l (% of conversion 99 and 83; ethanol yield 0.45 g/g). The hydrolysed juice could be used without any nutrient addition.     

Kinetics of alcohol production by zymomonas mobilis at high sugar concentrations

Summary Studies on the growth ofZ.mobilis revealed that high concentrations of glucose (10-25%) can be efficiently and rapidly converted to ethanol in batch culture. By comparison withS. carlsbergensis,Z.mobilis had specific glucose uptake rates and specific ethanol productivies several times greater than the yeast.Z.mobilis also had ethanol yields of up to 97% of a theoretical value.     

Kinetic studies on a highly productive strain of Zymomonas mobilis

Summary Previous studies have demonstrated that Zymomonas mobilis is a very promising organism for ethanol production. In the present study comparative kinetic data from batch and continuous cultures on glucose media are presented which show that a new strain of Z. mobilis has higher specific rates of growth and ethanol production as well as a higher tolerance to ethanol.     

Ethanol production byZymomonas mobilis in continuous culture at high glucose concentrations

Summary Ethanol production byZ.mobilis has been studied in continuous culture with 10, 15 and 20% glucose media. At 10% glucose, steady state conditions were achieved under glucose-limited conditions. At 15 and 20% glucose, the glucose was not fully metabolized even at low dilution rates and oscillatory behavior was evident. It is proposed that ethanol inhibition of growth is responsible for these phenomena. Comparison of kinetic parameters with those from previously published batch data revealed similar values. The maintenance energy coefficient (m) forZ.mobilis was relatively high and was calculated as 1.6 g/g/h for 10% glucose and 3.1 g/g/h for 15% glucose.     

Ethanol production with artificially flocculatedZymomonas cells

Zymomonas mobilis ZM3 was flocculated with a synthetic polyelectrolyte and titanium hydrous oxide. Continuous culture on a glucose medium in a gaslift tower reactor with internal recycle resulted in a stable production of 70 g/l ethanol, with an 8 fold increase in productivity compared to a similar free cell culture.     

Role of antimicrobial agents in simultaneous saccharification and fermentation of paddy malt mash to ethanol by mixed cultures of Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4

Summary Among various antimicrobial plant extracts, chemicals and antibiotics used for simultaneous saccharification and fermentation, penicillin G prevented contamination and did not inhibit amylase activity and growth of the synergistic co-cultures Saccharomyces cerevisiae PH03 and Zymomonas mobilis ZM4 during a 7-day fermentation of paddy malt (25.0%) mash (18.0% dextrose equivalent) to ethanol at 30°C and pH 5.5. The treatment yielded 10.1% (v/v) ethanol from the mash which was significantly more than that of the boiled and fermented mash (9.3% v/v) and equal to that of the mash boiled and fermented (10 2% v/v) after added amylases treatment. Most of the other compounds (kanamycin, streptomycin, polymyxin, tetracycline) had growth inhibitory effect especially on Z.mobilis.     

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.     

Kinetic analysis of ethanol production by an acetate-resistant strain of recombinant Zymomonas mobilis

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 m Cell 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^–1 Ethanol yield on total sugars (g g^–1)Biomass yield on total sugars (g g^–1)True biomass yield on total sugars (g g^–1)     

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.     

Simultaneous ethanol production from lactose byKluyveromyces fragilis andZymomonas mobilis

Ethanol production from lactose byKluyveromyces fragilis NRRL 665 in monoculture and coculture with strains ofZymomonas mobilis was studied. One of the strains,Z. mobilis NRRL 1960, when cocultured withK. fragilis, produed 55.2 g/l of ethanol, whereasK. fragilis in monoculture procuded only 36 g/l ethanol from 200 g/l lactose medium. Increased Qp (g ethanol produced/g biomass/h) and Qs (g substrate consumed/g biomass/h) were observed in coculture than in monoculture. However, the residual sugar concentration increased in coculture; this increase might be due to the slow utilization rate of galactose.     

High productivity ethanol fermentations with Zymomonas mobilis using continuous cell recycle

Summary Cell recycle studies have been carried out with a strain of Zymomonas mobilis selected for its improved ethanol tolerance and faster rates of glucose uptake and ethanol production. As part of the investigation a capilliary cross-flow microfiltration unit with polyamide membranes has been evaluated in view of its potential advantages (low cost and ability to withstand repeated cleaning with caustic soda). The results demonstrate that ethanol concentrations of 60–65g/l can be sustained at productivities ranging from 120–200g/l/h.