Horst W. Doelle

Zymomonas Mobilis—Science and Industrial Application

Zymomonas mobilis is undoubtedly one of the most unique bacterium within the microbial world. Known since 1912 under the names Termobacterium mobilis, Pseudomonas linderi, and Zymomonas mobilis, reviews on its uniqueness have been published in 1977 and 1988. The bacterium Zymomonas mobilis not only exhibits an extraordinarily uniqueness in its biochemistry, but also in its growth behavior, energy production, and response to culture conditions, as well as cultivation techniques used. This uniqueness caused great interest in the scientific, biotechnological, and industrial worlds. Its ability to couple and uncouple energy production in favor of product formation, to respond to physical and chemical environment manipulation, as well as its restricted product formation, makes it an ideal microorganism for microbial process development. This review explores the advances made since 1987, together with new developments in the pure scientific and applied commercial areas.

Purification and kinetic characteristics of pyruvate decarboxylase and ethanol dehydrogenase from Zymomonas mobilis in relation to ethanol production

SummaryAlcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis were partially purified and characterized. Alcohol dehydrogenase exhibits a pH optimum at 6.5 and pyruvate decarboxylase a major peak at pH 6.0 and a minor one at pH 4.3. The molecular weights were estimated to be 147,300±14,700 and 219,700±20,400 daltons, respectively. Both enzymes exhibit hyperbolic saturation curves with their respective substrates. Whereas alcohol dehydrogenase was inhibited by ethanol (Ki=6.86×10−4 M) and NAD+ (Ki=1.44×10−4 M), no inhibition was observed with pyruvate decarboxylase at similar concentrations of ethanol. Neither of the enzymes responded to sulphydryl-binding reagents, but differed in their response to a number of divalent metals. The results are compared with those of the respective enzymes from yeast and discussed with a view towards ethanol production limitations by Zymomonas mobilis.

Screening and characterization of bioflocculant produced by isolated Klebsiella sp.

Abstract Sixteen strains of polymer-producing bacteria were isolated from the activated sludge samples taken from two seafood processing plants in Southern Thailand. Their culture broths possessed the ability to flocculate kaolin suspension in the presence of 1% CaCl2. Based on the flocculating activity, the strain S11 was selected and identified to be a Klebsiella sp. using the partial 16S rRNA sequencing method. The growth of the isolated Klebsiella sp. was maximal (1.026 g l−1 dry cell mass) after 1 day cultivation while the highest polymer yield (0.973 g l−1) was achieved after 5 days cultivation. The flocculating activity of the culture broth, however, was highest after 2 days cultivation. The polymer was identified to be an acidic polysaccharide containing neutral sugar and uronic acid as its major and minor components, respectively. Results on the properties of the partially purified polysaccharide from Klebsiella sp. S11 revealed that it consisted of galactose, glucose and mannose in an approximate ratio of 5:2:1. It was soluble in acidic or basic solutions but not in organic solvents. Its molecular mass was greater than 2 × 106 Da. Infrared spectra showed the presence of hydroxyl, carboxyl and methoxyl groups in its molecules. Differential scanning calorimetry of the polysaccharide indicated the crystalline melting point (Tm) at 314 °C. The optimum dosage of polysaccharide to give the highest flocculating activity was 15 mg l−1 in the presence of 1% CaCl2.

Levansucrase from Zymomonas mobilis

SummaryLevansucrase (EC 2.4.1.10) from Zymomonasmobilis was purified from protamine sulfate treated cell free extracts by DEAE-trisacryl ionexchange chromatography to produce a 330-fold partial purification with an 11 % yield. Levansucrase was inhibited completely by glucose (30mM) and ethanol (1.6 M). These concentrations of glucose and ethanol were exceeded by cells growing in cultures containing an initial sucrose concentration of 200 gl−1.

Regulation of glucose metabolism in bacterial systems

In the past 10 years there have been rapid developments in the elucidation of the mechanisms of the Pasteur (or oxygen) and the Crabtree (or glucose repression of the respiratory chain) Effects in bacterial systems, which convincingly exhibit the difference between the regulatory mechanisms in yeast and bacteria. The presented review will demonstrate that the enzyme phosphofructokinase plays no role in the mechanism of the Pasteur effect and that there exists no glucose repression on biomass formation in bacteria under aerobic conditions. Endproduct formation is caused aerobically and anaerobically by an oversupply of NADHP2, whereas biomass correlates to energy supply. This development indicates very strongly that the mechanism of the Pasteur effect may be reflected solely in the change of glucose uptake rates and must therefore be sought at the cell membrane. In regard to the Crabtree Effect, the question arises whether there exists such a mechanism in bacteria. The variability of the bacterial electron transport systems, the lack of cytochrome a as terminal oxidase together with bioenergetic investigations indicate that the Crabtree effect may give cause for an alteration but not for a cessation of respiratory activity.

Gas chromatographic separation and determination of microquantities of the esters of the tricarboxylic acid cycle acids and related compounds

A column was developed which separated and determined microquantities of the methyl esters of seven tricarboxylic acid cycle acids, viz. fumaric, succinic, malic, α-ketoglutaric, cis-aconitic, citric, and isocitric acid. The 2 ft. × 1 4 in. O.D. 10% Reoplex 400 on acid-washed Chromosorb W column also separated the key intermediate of the dicarboxylic acid cycle, glyoxylate, from the tricarboxylic acid cycle acids. In addition, the column allowed determination of microquantities of the esters of pyruvic, lactic, glycollic, oxalic, malonic, maleic, itaconic, adipic, tartaric and acetoacetic acids. Of all these acid esters, only glycollic, oxalic and glyoxylic acid esters, and itaconic and maleic acid esters, could not be separated from each other. Using the dual hydrogen flame ionization detector on a Beckman GC-M, a helium flow rate of 100 ml/min and temperature programming between 50 and 200° at a rate of 5°/min, were selected as optimum conditions for the separation and determination of the acid esters. Under these conditions, all seven tricarboxylic acid cycle acid esters with the exception of α-ketoglutarate and isocitrate, could be standardized down to 0.25 μg; α-ketoglutarate and isocitrate were standardizable to 25 μg. In addition, all of the esters could be estimated in amounts as low as 0.1 μg, with cis-aconitate estimable to 0.05 μg. The boron trifluoride-methanol esterification procedure was tested, and found to produce 90-100% recovery for the dimethyl esters, and 50-55% recovery for the methyl and trimethyl esters of the above-mentioned acids.

The production of ethanol from sucrose using Zymomonas mobilis

SummaryA new single-batch fermentation process for the commercial production of ethanol from refined sucrose, raw sugar, sugar cane juice and sugar cane syrup has been developed using a highly adapted and efficient strain of Zymomonas mobilis. The process gives a 94–98% sucrose hydrolysis efficiency and a 95–98% ethanol conversion efficiency. Within 24–30 h, 200 g/l sucrose is converted to produce 95.5 g/l ethanol. Reinoculation is carried out from the fermented broth without the need for centrifugation or membrane filtration.

Kinetic characteristics and regulatory mechanisms of glucokinase and fructokinase from Zymomonas mobilis

SummaryGlucokinase (EC 2.7.1.2) exhibits two pH optima (pH 7.0 and 8.2), gives hyperbolic saturation curves and reacts equally with ATP, UTP, GTP, ITP and CTP. Inhibition occurs with high concentrations of these nucleotides and in addition with ADP, AMP and glucose 6-phosphate. No inhibition was observed with sucrose, glucose, fructose (11 mM), ethanol (542 mM), mannose, ribose, galactose, deoxy-glucose, lactose and gluconate and no reaction except with glucose.Fructokinase (EC 2.7.1.4) exhibits one pH optimum (7.4), gives hyperbolic saturation curves and is highly specific towards ATP and fructose. Mannose, glucose, GTP and CTP do not react. Inhibition occurs with glucose, glucose 6-phosphate and mildly fructose 6-phosphate. ATP at high concentrations gives slight inhibition, ADP and AMP show differential effects, whereas all other above mentioned compounds do not inhibit.Regulatory mechanisms for sucrose, glucose and fructose metabolism are discussed.

The production of ethanol plus fructose sweetener using fructose utilization negative mutants of Zymomonas mobilis

SummaryTwo mutants, unable to utilize fructose (Fru−) as a sole source of carbon and energy, were isolated fromZymomonas mobilis following ethyl methane sulfonate (EMS) mutagenesis. The frequency of stable Fru− mutants among survivors of mutagenesis was 1 in 104. The two Fru− mutants were able to cleave sucrose to glucose and fructose, and then ferment only the glucose to ethanol while accumulating fructose close to the theoretical value. Under controlled fermentation conditions, sucrose was converted to ethanol plus 80% or higher purity fructose syrup in a single-stage batch fermentation process, improving the Sucrotech Process significantly.

Nutritional effects on the kinetics of ethanol production from glucose by Zymomonas mobilis

SummaryChanges in the nutritional media significantly affected specific glucose uptake rate (Qg), glucose utilization and ethanol production rate (Qp) of Zymomonas mobilis Z 10. Varying the yeast extract concentrations (pantothenate) between 0.15% and 1.0% in a 10% glucose system at μ=0.2 h−1, Qg values between 14.9 and 35.3 and Qp values between 7.4 and 14.6 were obtained. A concentration of 0.25% yeast extract gave highest results. High NH4+concentrations were detrimental to ethanol production, whereas phosphate, potassium and magnesium stimulated the catabolic activity. In the case of magnesium, glucose utilization increased from 2.8 to 92.2%. Batch and continuous cultures showed that by increasing catabolic activity, ethanol productivity can be increased irrespective of glucose concentration.