Identification of PTSFru as the major fructose uptake system of Clostridium acetobutylicum

As a member of the saccharolytic clostridia, a variety of different carbohydrates like glucose, fructose, or mannose can be used as carbon and energy source by Clostridium acetobutylicum ATCC 824. Thirteen phosphoenolpyruvate-dependent phosphotransferase systems (PTS) have been identified in C. acetobutylicum, which are likely to be responsible for the uptake of hexoses, hexitols, or disaccharides. Here, we focus on three PTS which are expected to be involved in the uptake of fructose, PTS^Fru, PTS^ManI, and PTS^ManII. To analyze their individual functions, each PTS was inactivated via homologous recombination or insertional mutagenesis. Standardized comparative batch fermentations in a synthetic medium with glucose, fructose, or mannose as sole carbon source identified PTS^Fru as primary uptake system for fructose, whereas growth with fructose was not impaired in PTS^ManI and slightly altered in PTS^ManII-deficient strains of C. acetobutylicum. The inactivation of PTS^ManI resulted in slower growth on mannose whereas the loss of PTS^ManII revealed no phenotype during growth on mannose. This is the first time that it has been shown that PTS^Fru and PTS^ManI of C. acetobutylicum are directly involved in fructose and mannose uptake, respectively. Moreover, comprehensive comparison of the fermentation products revealed that the loss of PTS^Fru prevents the solvent shift as no butanol and only basic levels of acetone and ethanol could be determined.     

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.     

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.     

By-products in the fermentation of sucrose by different Zymomonas-strains

Summary Eight Zymomonas strains were compared with respect to their sucrose hydrolysing activity and subsequent ethanol, levan and sorbitol formation. The ethanol yields obtained were within narrow limits, 0.40–0.43 g·g^-1 of sucrose. The distribution of by-products differed significantly between the strains tested. A low sucrose hydrolysis rate seemed to be associated with the formation of levan and a high sucrose hydrolysis rate with the formation of sorbitol through accumulation of monomeric sugars. Fructo-oligomers consisting of two fructose and one glucose unit represented the greatest loss of sucrose in the fermentation conditions used.     

Physiological and genetic analysis of the glucose-fructose permeation system in two Synechocystis species

Fructose is toxic for Synechocystis PCC 6714 and 6803, strains which grow chemoheterotrophically on glucose. This toxicity, as well as fructose uptake, were inhibited by glucose or by its non-metabolized analogue 3-O-methyl-glucose. The results suggested that both sugars were transported by the same permeation system, the affinity for fructose, estimated from the corresponding K _m and K _i, being very low. The unicity of the permeation system was further established by the isolation of spontaneous mutants showing the expected pleiotropic phenotype, Glu^–, Fru^r, transport^–, and by the simultaneous re-acquisition of the relevant wild type characteristics in mutant cells transformed by wild type DNA. The genetic nature of this mutation is discussed in view of the impossibility to isolate spontaneously reversed wild type clones from the transport deficient mutants.Abbreviations OMG 3-O-methyl-glucose - DCMU 3-(3,4 dichlorophenyl)-1,1-dimethylurea     

Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

A continuous thermophilic cellulose fermentation in an upflow reactor by aClostridium thermocellum-containing mixed culture

Summary A continuous thermophilic cellulose fermentation by aCl. thermocellum-containing mixed culture was carried out in an upflow reactor for a period of 100 days. The cellulose conversion rate was finally 0.35 g.1^–1.h^–1. Evidence that the fermentation process was influenced by both pH and dilution rate was given by the changes of concentration of the main fermentation products, acetic acid and ethanol. The role of cellodextrins and glucose as reactive intermediates in the process of cellulose breakdown was established.     

Effect of temperature on sucrose to ethanol conversion by Zymomonas mobilis strains

Summary Two strains of Zymomonas mobilis were tested for their ability to ferment sucrose to ethanol at elevated temperatures (30–42.5°C). The optimal temperature for efficient sucrose to ethanol conversion was 35°C with 22–27 h fermentation time and 75% conversion efficiency. Increases in magnesium concentration improved one of the strains at 40°C from 38 to 76% ethanol yield efficiency.     

Modeling of ethanol fermentation using Zymomonas mobilis ATCC 10988 grown on the media containing glucose and fructose

A model of ethanol fermentation by Zymomonas mobilis ATCC 10988 on the medium containing glucose and fructose is proposed. This model was developed on the basis of metabolic analysis and many experimental findings. When glucose was used as the substrate, the dependence of the carbon fraction (α) assimilating to biomass on the specific growth rate (μ) could be well correlated to α = 0.25μ + 0.012. This correlation resulted in a novel equation for specific glucose uptake rate, which could describe the Z. mobilis fermentation in both batch and continuous modes. When fructose and glucose were both presented in the liquid medium, the model could predict the uptake of glucose and fructose as well as the formation of biomass, ethanol and sorbitol by Z. mobilis. All parameters used in the model were independently evaluated on the basis of various experimental findings. Good agreement was found between the model predictions and data of Z. mobilis fermentation on media containing both glucose and fructose. The proposed model could also describe the behavior of ethanol fermentation on sucrose medium supplemented with immobilized invertase.     

Fructose production from sucrose and high fructose syrup: A mycelial fungal system

Summary The use of Mucor sp. M105 and Fusarium sp. F5 in the production of fructose from sugarcane sucrose and high fructose syrup (HFS) was investigated. Although Mucor sp. could not utilize sucrose as the sole carbon and energy source for cell growth, Mucor sp. preferentially utilized glucose in a glucose:fructose (1:1) mixture during fermentation to ethanol. In contrast, Fusarium sp. utilized sucrose as sole carbon source by secretion of extracellular hydrolytic enzymes that degraded the disaccharide. In Fusarium sp., glucose formation in the medium was faster than fructose. Due to the low consumption rate of fructose, this substrate remained in the fermentation broth. The application of these biological systems for the production of fructose from either sucrose or HFS is discussed.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Ethanol production from cane juice by Zymomonas mobilis

Summary Zymomonas mobilis Z 7 fermented 100 to 200 g.l^{- 1} sucrose in cane juice to ethanol without addition of cofactors or mineral salts in 1 ltr laboratory and 100 ltr pilot plant fermenters. Ethanol yields (E_yield) were from 60 to 88% with fermentation times of 20 to 29 h at 35 °C.Nomenclature V_s max g.1^-1 .h^-1 maximum sucrose hydrolysis rate - V_g max g.1^-1 .h^-1 maximum glucose uptake rate - V_fmax g.1^-1 .h^-1 maximum fructose uptake rate - V_e max g.1^-1 .h^-1 maximum ethanol production rate - S_h g.1^-1 sucrose hydrolyzed at t_ferm - G_u g.1^-1 glucose utilized at t_ferm - F_u g.1^-1 fructose utilized at t_ferm - E_max g.1^-1 ethanol produced at t_ferm - G_i g.1^-1 initial free glucose (before sucrose hydrolysis) - E_yield g.g^-1 ethanol produced divided by the theoretical ethanol yield from sucrose hydrolyzed - t_ferm h fermentation time to ethanol max     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Effect of calcium and sodium salts on ethanol production in high sugar fermentation by free cells ofZymomonas mobilis

Summary Addition of calcium carbonate enhanced ethanol production byZymomonas mobilis ZM4 and a mutant (ZMI₂), especially at higher concentrations (200–400 g/L) of glucose and sucrose, as well as at higher temperature (42°C) by the mutant. Calcium and sodium carbonates neutralized the acid produced in the medium and enhanced the ethanol production. The Na salts were less effective in the parent strain and were not favourable for the mutant. Ca²⁺ ions played a direct role in augmenting ethanol production as evidenced by the effect of calcium chloride at controlled pH (5.5).     

Continuous fermentation of apple juice by immobilized yeast cells

Summary The sugar content of an apple juice was continuously converted into ethanol bySaccharomyces cerevisiae entrapped in Ca-alginate gel. The average values characterizing the process were: fermentation efficiency, 84.7±4.2%, ethanol concentration in the mash, 38.9–1.9 g·l^–1 and volumetric productivity, 6.3±0.5 g·l^–1·h^–1.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia     

Lactic acid fermentation by Lactobacillus casei in free cell form and immobilised on gluten pellets

A comparative study of the fermentation of a range of carbohydrate substrates, at various temperatures, was carried out using a commercial Lactobacillus casei strain in a free cell form and immobilised on gluten pellets. This strain required yeast extract, l-cysteine HCl and Mn²⁺ at 5, 0.5 and 0.1 g l^–1, respectively, for maximum growth and lactic acid production. Sugar fermentation using free cells showed preference in the order glucose, sucrose, fructose while lactose was poorly utilised. Optimum temperature for growth and lactic acid production over (18–30 h) was 43 °C. L. casei was successfully immobilised on gluten pellets and fermented glucose and sucrose in a shorter time (18 h) with increased lactic acid production (42 and 41 g l^–1 on glucose and sucrose, respectively).     

Ethanol production from sucrose by immobilizedZymomonas mobilis cells in polyurethane foam

Summary The possibility of using polyurethane foam as a support for the immobilization ofZymomonas mobilis cells to carry out sucrose conversion to ethanol was investigated. Sucrose hydrolysis efficiencies of 90% and higher, volumetric reactor productivity of 20 gL^–1h^–1 and final ethanol concentration of 6.3% (v/v) at a dilution rate of 0.4 h^–1 show the good performance of polyurethane foams for whole cell immobilization.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.