Interrelationships between Growth Yield,ATPase and Adenylate Kinase Activities in Zymomonas mobilis

The presence of cytoplasmic and membrane‐bound adenylate kinase (EC 2.7.4.3) as well as inorganic pyrophosphatase (EC 3.6.1.1) was detected in Zymomonas mobilis ATCC 29191. An increase in the molar growth yield (Y_X/S) of Z. mobilis under aerobic growth conditions appeared to be in proportion to a reduction of membrane‐bound adenylate kinase (mAK) and ATPase activities and to an increase in cytoplasmic adenylate kinase (AK) activity. Significant (1 — P < 0.01) multiple regressions were observed between the values of Y_X/S (dependent variable), ATPase and AK or AK and mAK as independent variables, suggesting that a combined operation of these phosphohydrolases and phosphotransferases would be responsible for the biomass yield in Z. mobilis.     

An elevation of the molar growth yield of Zymomonas mobilis during aerobic exponential growth

Elevated values of molar growth yield (Y_x/s = 14–26 g mol^–1) were obtained during exponential growth (μ > 0.4 h^–1) of Zymomonas mobilis ATCC 29191 by using reduced concentrations of glucose (6.25–100 mM) and increased oxygen supply (E _h > 300 mV) in the growth medium, as compared to the Y_x/s of anaerobic exponential growth (8–10 g mol^–1). Aerobically grown cells showed an increased maximum growth rate (μ_max), and a reduced specific glucose consumption rate (q_s), and specific ethanol formation rate (q_p), thus demonstrating a more pronounced energy-coupling growth under oxic conditions. These results can be neither explained by the concept of a solely operating Entner-Doudoroff pathway as an ATP source in aerobically growing cultures of Z. mobilis nor considered to be consistent with existing data on the lack of the Pasteur effect in this bacterium. Therefore, the results rather give evidence for the essential contribution of aerobic ATP generation under the reported conditions. Received: 24 September 1996 / Accepted: 9 December 1996     

Influence of pH and initial lactate concentration on the growth of Lactobacillus plantarum

Summary Fermentation yields of Lactobacillus plantarum were measured at controlled pH between 4.0 and 8.0 and initial lactate concentrations of 0–90 g/l. Optimal growth conditions at pH 6.0 without addition of lactate gave a growth rate of 0.57 h^–1 and 20 g dry biomass/mol ATP formed (Y _ATP). The pH variations resulted in a decrease in growth rate but the effect on Y _ATPwas insignificant. The addition of lactate to the medium at 0 h resulted in linear decrease in the growth rate of the culture, and all the metabolic activities were completely inhibited at 110 g/l. The Y _ATPand biomass/ substrate yield (Y _X/S) remained fairly steady up to 33 g lactate/l, beyond which both yields decreased considerably. Offsprint requests to: M. Raimbault     

Energetics of Saccharomyces cerevisiae CBS 426: Comparison of anaerobic and aerobic glucose limitation

Saccharomyces cerevisiae CBS 426 was grown aerobically and anaerobically in a glucose-limited chemostat. The flows of biomass, glucose, ethanol, carbon dioxide, oxygen, glycerol, and the elemental composition of the biomass were measured. Models for anaerobic and aerobic growth are constructed. Values for Y_ATP and P/O are obtained from continuous culture data for aerobic growth; this Y_ATP value is compared with that obtained from the anaerobic growth results. The ratio between the heat produced and the oxygen consumed increases if more glucose in fermented to ethanol and carbon dioxide. An equation for ?_H/?_O as a function of the respiratory quotient is given.     

Batch fermentation kinetics of ethanol production by Zymomonas mobilis on cellulose hydrolysate

Summary Growth and ethanol production by three strains (MSN77, thermotolerant, SBE15, osmotolerant and wild type ZM4) of the bacterium Zymomonas mobilis were tested in a rich medium containing the hexose fraction from a cellulose hydrolysate (Aspen wood). The variations of yield and kinetic parameters with fermentation time revealed an inhibition of growth by the ethanol produced. This inhibition may result from the increase in medium osmolality due to ethanol formation from glucose.Nomenclature S glucose concentration (g/L) - C conversion of glucose (%) - t fermentation time (h) - q_S specific glucose uptake rate (g/g.h) - q_p specific ethanol productivity (g/g.h) - Q_p volumetric ethanol productivity (g/L.h) - Q_X volumetric biomass productivity (g/L.h) - Y_X/S biomass yield (g/g) - Y_p/S ethanol yield (g/g) - specific growth rate (h^-1)     

NADP+-dependent acetaldehyde dehydrogenase from Zymomonas mobilis

An NADP⁺-linked acetaldehyde dehydrogenase (EC 1.2.1.4) from the ethanol producing bacterium Zymomonas mobilis was purified 180-fold to homogeneity. The enzyme is a cytosolic protein with an isoelectric point of 8.0 and has an apparent molecular weight of 210000. It showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 55000, which indicates that it consists of four probably identical subunits. The apparent K _m values for the substrate acetaldehyde were 57 M and for the cosubstrate NADP⁺ 579 M. The enzyme was almost inactive with NAD⁺ as cofactor. Several other aldehydes besides acetaldehyde were accepted as a substrate but not formaldehyde or trichloroacetaldehyde. In anaerobically grown cells of Zymomonas mobilis the enzyme showed a specific activity of 0.035 U/mg protein but its specific activity could be increased up to 0.132 U/mg protein by adding acetaldehyde to the medium during the exponential growth phase or up to 0.284 U/mg protein when cells were grown under aeration. The physiological role of the enzyme is discussed.Abbreviations ALD-DH acetaldehyde dehydrogenases from Z. mobilis - DTT dithiothreitol - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - SDS sodium dodecylsulfate Dedicated to Prof. Dr. H.-G. Schlegel, Universität Göttingen, on the occasion of his 65th birthday     

Production of Acetaldehyde by Zymomonas mobilis

Mutants of Zymomonas mobilis were selected for decreased alcohol dehydrogenase activity by using consecutively higher concentrations of allyl alcohol. A mutant selected by using 100 mM allyl alcohol produced acetaldehyde at a level of 4.08 g/liter when the organism was grown in aerated batch cultures on a medium containing 4.0% (wt/wt) glucose. On the basis of the amount of glucose utilized, this level of acetaldehyde production represents nearly 40% of the maximum theoretical yield. Acetaldehyde produced during growth was continuously air stripped from the reactor. Acetaldehyde present in the exhaust stream was then trapped as the acetaldehyde-bisulfite addition product in an aqueous solution of sodium bisulfite and released by treatment with base. Acetaldehyde was found to inhibit growth of Z. mobilis at concentrations as low as 0.05% (wt/wt) acetaldehyde. An acetaldehyde-tolerant mutant of Z. mobilis was isolated after both mutagenesis with nitrosoguanidine and selection in the presence of vapor-phase acetaldehyde. The production of acetaldehyde has potential advantages over that of ethanol: lower energy requirements for product separation, efficient separation of product from dilute feed streams, continuous separation of product from the reactor, and a higher marketplace value.     

Sucrose medium osmolality as a regulator of anabolic and catabolic parameters in Zymomonas culture

This study focuses on the growth of Zymomonas mobilis strain 113 S and its ethanol and levan production under the conditions of increasing sucrose medium osmolality caused by NaCl, KCl, sorbitol or maltose. The increase in medium osmolality (700–1,500 mosml/kg) was accompanied by the inhibition of growth (growth rate, biomass yield) and ethanol production (specific productivity and yield) In contrast, levan synthesis was less affected or even stimulated and, as a consequence, levan specific productivity was increased significantly. A decrease in the anabolic growth parameters correlated with a parallel inhibition of glucose-6-P dehydrogenase and alcohol dehydrogenase (isoenzyme ADH II) activities. A significant inverse linear relationship (r = ? 0.932, 1 ? P = 0.01) was observed between the values of the specific productivities of ethanol and levan. This relationship was confirmed independently by a controlled reduction of growth and ethanol productivity (3.75–4.75 mM sodiumbisulphite as an acceptor of acetaldehyde formed in the pyruvate decarboxylase reaction). As further support of this relationship, a significant inverse correlation was observed between levan specific productivity and ATP concentration in Zymomonas mobilis cells, most probably demonstrating that a reduced level of energetic metabolism is favourable for levan production.     

Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli

A genomic library of Zymomonas mobilis DNA was constructed in Escherichia coli using cosmid vector pHC79. Immunological screening of 483 individual E. coli strains revealed two clones expressing pyruvate decarboxylase, the key enzyme for efficient ethanol production of Z. mobilis. The two plasmids, pZM1 and pZM2, isolated from both E. coli strains were found to be related and to exhibit a common 4.6 kb SphI fragment on which the gene coding for pyruvate decarboxylase, pdc, was located.The pdc gene was similarily well expressed in both aerobically and anaerobically grown E. coli cells, and exerted a considerable effect on the amount of fermentation products formed. During fermentative growth on 25 mM glucose, plasmid-free E. coli lacking a pdc gene produced 6.5 mM ethanol, 8.2 mM acetate, 6.5 mM lactate, 0.5 mM succinate, and about 1 mM formate leaving 10.4 mM residual glucose. In contrast, recombinant E. coli harbouring a cloned pdc gene from Z. mobilis completely converted 25 mM glucose to up to 41.5 mM ethanol while almost no acids were formed.     

The reduction of xylose to xylitol by Candida guilliermondii and Candida parapsilosis: Incidence of oxygen and pH

Summary The ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol was evaluated under different oxygen transfer rates in order to enhance the xylitol yield. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/l.h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmoles of oxygen/l.h. The response of the yeasts to anaerobic conditions has shown that oxygen was required for xylose metabolism.Nomenclature max maximum specific growth rate (per hour) - qSmax maximum specific rate of xylose consumption (g xylose per g dry biomass per hour) - qpmax maximum specific productivity of xylitol (g xylitol per g dry biomass per hour) - Qp average volumetric productivity of xylitol (g xylitol per liter per hour) - Y_P/S xylitol yield (g xylitol per g substrate utilized) - Y_P'/S glycerol yield (g glycerol per g substrate utilized) - Y_X/S biomass yield (g dry biomass per g substrate utilized)     

Levan production by Zymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculation Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethano fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan. The activation period of cells within wire spheres (WS) was 48 h in duration. During this time, cell growth stabilized at production levels of ethanol and levan of Q_eth = 1.238 g/l × h and q_eth = 0.47 g/l × h; Q_eth = 0.526 g/l × h and q_eth = 0.20 g/l × h. Five stable fermentation cycles were realized using one wire sphere inoculum, and maintaining a stable ratio of 2.4 of biomass suspended in the medium to biomass fixed in the sphere. Using fixed Z mobilis biomass in the WS, the total amount of inoculum could be reduced for batch fermentation. Large plaited wire spheres with biomass may have potential in fermentation in viscous systems, including levan production.     

Purification of two alcohol dehydrogenases from Zymomonas mobilis and their properties

Summary Two alcohol dehydrogenases (ADHI and ADHII, EC 1.1.1.1) were purified to homogeneity from the cell extract of Zymomonas mobilis. The subunit molecular weights of ADHI and ADHII were 40,000 and 38,000, respectively, and both enzymes were homologous dimers. The optimal pHs of ADHI in ethanol oxidation and acetaldehyde reduction reactions were 9.5 and 4.5, and those of ADHII were 9.5 and 6.5, respectively. The optimal temperatures of ADHI and ADHII were 55° C and 45° C, respectively. ADHI was heat-inactivated at 65° C at a 10-fold higher rate than ADHII. ADHI and ADHII were inhibited by 4 M and 1 mM p-chloromercuribenzoate, respectively, and the inhibitions were reversed by the addition of 70 mM 2-mercaptoethanol. ADHII activity was enhanced by 0.02 to 2 mM CoCl₂ and inhibited by 0.4 mM o-phenanthroline; and the activity of inactivated ADHII was restored by addition of 1 mM CoCl₂ or ZnCl₂.ADHI was active on most primary alcohols but not secondary alcohols. ADHII was active on only ethanol, n-propanol, allylalcohol, and furfuryl alcohol.In the anaerobic culture of Z. mobilis, ADHII activity accounted for more than 80% of total alcohol dehydrogenase activity. In aerobic culture, ADHII was the main enzyme but was produced only in the early growth phase.     

Studies on quantitative physiology of Trichoderma reesei with two-stage continuous culture for cellulose production

By employing a two-stage continuous-culture system, some of the more important physiological parameters involved in cellulose biosynthesis have been evaluated with an ultimate objective of designing an optimally controlled cellulose process. The two-stage continuous-culture system was run for a period of 1350 hr with Trichoderma reesei strain MCG-77. The temperature and pH were controlled at 32°C and pH 4.5 for the first stage (growth) and 28°C and pH 3.5 for the second stage (enzyme production). Lactose was the only carbon source for the both stages. The ratio of specific uptake rate of carbon to that of nitrogen, Q(C)/Q(N), that supported good cell growth ranged from 11 to 15, and the ratio for maximum specific enzyme productivity ranged from 5 to 13. The maintenance coefficients determined for oxygen, M_O, and for carbon source, M_C, are 0.85 mmol O₂/g biomass/hr and 0.14 mmol hexose/g biomass/hr, respectively. The yield constants determined are: Y_X/O = 32.3 g biomass/mol O₂, Y_X/C = 1.1 g biomass/g C or Y_X/C = 0.44 g biomass/g hexose, Y_X/N = 12.5 g biomass/g nitrogen for the cell growth stage, and Y_X/N = 16.6 g biomass/g nitrogen for the enzyme production stage. Enzyme was produced only in the second stage. Volumetric and specific enzyme productivities obtained were 90 IU/liter/hr and 8 IU/g biomass/hr, respectively. The maximum specific enzyme productivity observed was 14.8 IU/g biomass/hr. The optimal dilution rate in the second stage that corresponded to the maximum enzyme productivity was 0.026 ～ 0.028 hr^?1, and the specific growth rate in the second stage that supported maximum specific enzyme productivity was equal to or slightly less than zero.     

Growth yield from electrons available from substrates in aerobic- and photoheterotrophic cultures of Rhodopseudomonas sphaeroides S

A balance of electrons available from acetic acid consumed for growth and oxygen uptake in the aerobic- and photoheterotrophic growth of Rhodopseudomonas sphaeroides S on acetate-minimal medium could be realized the same as in the carbon balance. The unmeasured amounts of yeast extract consumed by the cells grown on propionate–yeast extract media were indirectly estimated from the balance equation of electrons available from carbon substrates. The specific consumption rate of the yeast extract increased with an increase in propionate consumption rate in aerobic and photoheterotrophic cultures. Growth yields from acetic acid and from propionic acid plus yeast extract were calculated on the electron level, i.e., Y_X/ave g cell produced/equivalent electrons available from substrate consumed. Y_X/ave values were 5.0 to 5.8 g cell/ave in photoheterotrophic cultures and 2.7 to 3.6 in aerobic–heterotrophic cultures regardless of different medium compositions.     

Ethanol fermentation using a fructose-negative mutant of Zymomonas mobilis

Summary The fermentation of an equimolar mixture of glucose and fructose into ethanol and sorbitol by a fructose negative mutant of Zymomonas mobilis is analysed using a recently described methodology (Ait-Abdelkader and Baratti, Biotechnol. Tech. 1993,329–334) based on polynomial fitting and calculation of instantaneous and overall parameters. These parameters are utilized to describe this mixed-substrate mixed-product fermentation.Nomenclature X biomass concentration, g/l - S total sugar concentration, g/l - Glu glucose concentration, g/l - Fru fructose concentration, g/l - Sor sorbitol concentration, g/l - P ethanol concentration, g/l - t fermentation time, h - specific growth rate, h^-1 - q_s specific sugar uptake rate, g/g.h - q_g specific glucose uptake rate, g/g.h - q_F specific fructose uptake rate, g/g.h - q_P specific ethanol productivity, g/g.h - q_Sor specific sorbitol productivity, g/g.h - Y_X/S biomass yield on total sugar, g/g - Y_P/S ethanol yield on total sugar, g/g - Y_Sor/S sorbitol yield on total sugar, g/g - Y_Sor/F sorbitol yield on fructose, (g/g) - Y_P/G ethanol yield on glucose, (g/g)     

abetd-Xylose metabolism in Aureobasidium pullulans: effects of aeration and vitamins

Summary The yeast-like organism Aureobasidium pullulans efficiently converted abetd-xylose to cell mass (Y _X/S=0.45 g·g^–1) with negligible production of polyols (Y _P/S=0.003 g·g^–1) under aerobic conditions. A. pullulans grown semiaerobically exhibited different fermentation capacities in seven basal (vitaminless) medium and medium containing a mixture of seven vitamins. It was found that under semiaerobic conditions a mixture of vitamins significantly enhanced production of ethanol from abetd-xylose, resulting in a 15-fold higher yield coefficient of ethanol (Y _E/S=0.22 g·g^–1) as compared to that achieved in vitaminless medium. This increase in ethanol production was accomplished at the expense of cell mass. A. pullulans produced extremely low amounts of polyols throughout all aerobic and semiaerobic experiments. A. pullulans displayed strictly NADPH-linked xylose reductase and NAD⁺-linked xylitol dehydrogenase activities.     

Modeling the Kinetics of Contaminant Biodegradation by a Mixed Microbial Consortium Based on Carbon Mass Balance

The fate of contaminant carbon was monitored during aerobic biodegradation in the presence of a mixed indigenous microbial consortium in order to calibrate a microbial-growth-based biokinetic model. The methodology simultaneously monitored mineralization, substrate depletion and microbial population evolution in biomass extract spiked with¹⁴C-labeled hexadecane. Hexadecane depletion and hexadecane-degrader population were monitored using sacrificed microcosms by centrifuging the extract so that the supernatant and the residue contained residual hexadecane and microbial population, respectively. This methodology allowed verification of the carbon mass balance (average¹⁴C-carbon recovery of 90.33 ± 1.62% for biotic microcosms) and calibration of a biokinetic model. Four biokinetic parameters and three yield coefficients were identified (Haldane kinetic parameters:μ_S = 1.3639 d^-1, K_s = 0.4295 mg-C, KI = 6.6457 mg-C; decay kinetic parameter:μ_d = 1.3.10² d^-1; substrate/biomass, carbon dioxide/ biomass during growth and carbon dioxide/biomass during decay yield coefficients: Y_s = 1.5948 mg-C/mg-C, Y_P ^g = 0.4554 mg-C/mg-C, Y_P ^d = 1.3263 mg-C/mg-C) and compared with the literature data. The methodology can facilitate the identification of biodegradation models by decoupling the intrinsic ability of microorganisms to degrade contaminant from restrictions imposed by limiting conditions.     

Effect of oxygen on the metabolism of Zymomonas mobilis

The specific growth rate of the ethanol producing bacterium Zymomonas mobilis was 25–40% lower in the presence of oxygen than under anaerobic conditions, provided the cultures were supplied with a low substrate concentration (20 g glucose/l). However, the molar growth yield of these cultures was not influenced by oxygen. With washed cell suspensions, an oxygen consumption could be initiated by the addition of either glucose, fructose, or ethanol. Cell extracts catalyzed the oxidation of NADH with oxygen at a molar ratio of 2:1. Further experiments showed that this NADH oxidase is located in the cell membrane. The specific oxygen consumption rates of cell suspensions correlated with the intracellular NADH oxidizing activities; both levels decreased with increasing concentrations of the fermentation end-product ethanol. The addition of 5 mM NaCN completely inhibited both the intracellular oxygen reduction and also the oxygen consumption of whole cells. Both catalase and superoxide dismutase were present even in anaerobically grown cells. Aeration seemed to have little effect on the level of catalase, but the superoxide dismutase activity was 5-fold higher in cells grown aerobically. Under aerobic conditions considerable amounts of acetaldehyde and acetic acid were formed in addition to the normal fermentation products, ethanol and carbon dioxide.Dedicated to Professor Dr. H. G. Schlegel on the occasion of his 60th birthday     

Effect of bioreactor configuration on substrate uptake by cell suspension cultures of the plant Eschscholtzia californica

Summary The uptake of carbohydrates and oxygen by cell suspension cultures of the plant Eschscholtzia californica (California poppy) was studied in relation to biomass production in shake flasks, a 1-1 stirred-tank bioreactor and a 1-1 pneumatically agitated bioreactor. The sequence of carbohydrate uptake was similar in all cases, with sucrose hydrolysis occurring followed by the preferential uptake of glucose. The uptake of fructose was found to be affected by the oxygen supply rate. Carbohydrate utilization occurred at a slower rate in the bioreactors. Apparent biomass yields, Y _X/S, ranged from 0.42 to 0.50 g biomass/g carbohydrate, while true biomass yields, Y _X/S, were about 0.69 g/g. The maintenance coefficient for carbohydrate, m _S, ranged between 0.002 and 0.008 g/dry weight (DW) per hour. The maximum measured specific oxygen uptake rate was 0.56 mmol O₂/g DW per hour and occurred early in the growth stage. The decline in specific uptake rate coincided with a decline in cell viability. The oxygen uptake rate was faster in shake flasks, corresponding to the higher growth rate obtained. The true growth yield on oxygen, YX/O₂, was calculated to range from 0.83 to 1.23 g biomass/g O₂, while the maintenance coefficient, mO₂, ranged from 0.15 to 0.25 mmol O₂/g DW per hour. The growth yields for oxygen determined from the stoichiometry of an elemental balance were within 10% of those calculated from experimental data. Offprint requests to: Raymond L. Legge     

Oxygen effect on batch cultures of Leuconostoc mesenteroides: relationship between oxygen uptake,growth and end-products

Growth and lactose metabolism of a Leuconostoc mesenteroides strain were studied in batch cultures at pH 6.5 and 30° C in 101 modified MRS medium sparged with different gases: nitrogen, air and pure oxygen. In all cases, growth occurred, but in aerobiosis there was oxygen consumption, leading to an improvement of growth yield Y _x/s and specific growth rate compared to anaerobiosis. Whatever the extent of aerobic growth, oxygen uptake and biomass production increased with the oxygen transfer rate so that the oxygen growth yield, Y _x/o2, remained at a constant value of 11 g dry weight of biomass/mol oxygen consumed. Pure oxygen had a positive effect on Leuconostoc growth. Oxygen transfer was limiting under air, but pure oxygen provided bacteria with sufficient dissolved oxygen and leuconostocs were able to consume large amounts of oxygen. Acetate production increased progressively with oxygen consumption so that the total molar concentration of acetate plus ethanol remained constant. Maximal Y _x/s was obtained with a 120 l/h flow rate of pure oxygen: the switch from ethanol to acetate was almost complete. In this case, a 46.8 g/mol Y _x/s and a 0.69 h^–1 maximal growth rate could be reached.