The inhibitory effect of ethanol on ethanol production by Zymomonas mobilis

Summary In an effort to establish the reasons for the limitations in the final ethanol concentration of Zymomonas mobilis fermentation, the effects of CO₂ and ethanol on the fermentation were investigated using continuous and fed-batch cultivation systems. The nucleation and stripping out of CO₂ from the fermenter using diatomaceous earth or nitrogen gas or both exhibited a profound effect on the glucose uptake rate during the early stages of fed-batch fermentation, but did not improve final ethanol yields. The addition of ethanol together with above mentioned experiments confirmed conclusively that ethanol inhibition is responsible for the final ethanol concentration obtainable during Zymomonas mobilis fermentation. The final concentration lies between 90 and 110 gl⁻¹ or approximately 12–15% (v/v) ethanol.     

A combination of methods for the on-line determination of alcohols in microbial cultures

Summary A new method for the continuous on-line determination of methanol (range 0.2 to 10 gl^–1) and ethanol (0.2 to 120 gl^–1) is described. The rate limiting step is diffusion of the alcohol through the walls of a silicone tube immersed in the culture broth. A sintered SnO₂ sensor was used instead of a Flame Ionization Detector for alcohol determination. Measurement is not affected by bioreactor aeration or agitation rates, dissolved oxygen, carbon dioxide, ammonia or the concentration of cells in the medium. The assay system was tested in extended batch cultivation of Methylomonas sp. with methanol as the sole carbon source (final biomass concentration, 35 gl^–1). Sensor readings agreed well with simultaneous off-line gas chromatographic methanol determination.     

Aerobic microbial treatment of sugar cane stillage byCandida utilis andPaecilomyces variotii in two step continuous cultures

Summary A two step continuous aerobic process is proposed for treating cane sugar stillage. In the first step the original stillage is used for growing aCandida utilis and the supernatant of this process is used for feeding the second step where aPaecilomyces strain is grown without addition of nutrients. A total reduction of COD of 87% and a mixed biomass output of 2.3–2.6 gl^-1h^-1 are attained.     

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.     

D-xylulose fermentation by free and immobilizedSaccharomyces cerevisiae cells

Summary D-xylulose fermentation by freeSaccharomyces cerevisiae cells in batch fermentation and by alginate entrapped cells in continuous fermentation was studied. At high cell densities volumetric ethanol productivities of 0.18 and 0.27 gl^-1h^-1 were obtained by free and immobilized cells, respectively, under anaerobic conditions. This productivity could not, however, be maintained continuously due to the death of cells. The anaerobic columns stabilized at a productivity level of 0.15 gl^-1 h^-1 and the aerobic column at 0.25 gl^-1h^-1.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

In vitro growth response of Prunus cerasifera shoots as influenced by different light-dark cycles and sucrose concentrations

Trials were carried out to test if the higher growth response shown by shoot clusters of Mr. S. 2/5, a clonal selection of Prunus cerasifera, submitted to short and frequent light-dark regimes could be related to the amount of sucrose added to growth medium.The reduction of sucrose from 30 gl^-1 (control) to 22.5 gl^-1, 15 gl^-1 and 7.5 gl^-1 caused a progressive and remarkable inhibition of shoot tip growth. With 15 gl^-1 the value of some growth parameters was reduced by more than half. Under 16-h daylength, the best sucrose concentration was 30 gl^-1, while with 4-h light-2-h dark no statistical differences appeared between 30 gl^-1 and 22.5 gl^-1 sucrose. Compared to 16-h light-8-h dark, the 4-h light-2-h dark cycle at the three highest sucrose concentrations gave rise to higher values of fresh and dry weight as well as increasing the number of axillary shoots produced.The increment in growth response induced by the shorter light-dark regime decreased with diminishing growth capacity in the cultures when sucrose concentration was lowered, but it was still appreciable even with 7.5 gl^-1. Since the 4-h light-2-h dark cycle induced a favourable effect in culture growth with all sucrose concentrations, we conclude that the greater growth response observed with this light regime was not triggered by carbohydrate availability but by some other unknown factors.     

Alcoholic fermentation by Zymomonas mobilis: Effect of initial substrate on physiological parameters

Summary The influence of initial substrate concentration on fermentation kinetics of Zymomonas mobilis on glucose has been studied in batch cultures over the range 50–190 gl^-1 glucose. With increasing glucose, parameters relative to growth ( and R_GX) are more rapidly and more noticeably affected than those connected with ethanol production (p and R_GP). The water content of the cells is also affected.     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Effect of dilution rate on growth characteristics of Candida utilis in multistage culture systems with different interstage mixing

The effect of dilution rate on important process parameters of biomass production in two multistage culture systems with different interstage mixing has been examined. Experiments were performed in a multistage tower fermenter and in a cascade of fermenters. Measurements were made at steady-state of continuous culture under constant and identical values of ethanol concentration of 50 gl^?1 in the feed, temperature, OTR and pH in both culture systems used. The microorganism used was Candida utilis. Ethanol inhibition influenced cell growth rate due to the complete dissimilation of the restricted quantity of acetate to H₂O and CO₂, leading to insufficient energy generation. The value of ethanol concentration at which ethanol started to inhibit cell growth was a combined function of OTR, S_R and D. The presence of the interstage mixing resulted in more efficient ethanol conversion to biomass in the whole range of dilution rates and significantly lowered the risk of washing-out at high values of both S_R and D.     

Evaluation of the SCP from Opuntia juice

Summary In deseeded opuntia juice medium batch and continuous cultivation ofCandida utilis produced 12 and 19.9gl^–1 yield of the SCP respectively. Its biological value(72), digestibility coefficient(70) and protein score(58) indicated it to be a good protein.     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

The effect of micro-aerobic conditions on continuous ethanol production by Saccharomyces cerevisiae

Summary The effect of trace amounts of oxygen on the degree of ethanol inhibition in a continuous anaerobic culture of Saccharomyces cerevisiae was studied at the 100 gl ^–1 feed glucose concentration level. Results showed that the use of micro-aerobic conditions (0,5% of saturation) enhanced the utilisation of substrate by increasing the ethanol tolerance of the yeast without any significant decrease in the ethanol yield per unit substrate consumed. When the results were fitted to an equation of the form % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaqcLbyacaqG8o% GaaeypaiqabY7agaqcaiaab6cadaWcaaGcbaqcLbyacaqGdbWaaSba% aSqaaKqzagGaae4CaaWcbeaaaOqaaKqzagGaae4qamaaBaaaleaaju% gGbiaabohaaSqabaqcLbyacqGHRaWkcaqGlbWaaSbaaSqaaKqzagGa% ae4CaaWcbeaaaaqcLbyacaGGUaWaaSaaaOqaaKqzagGaae4samaaBa% aaleaajugGbiaabchaaSqabaaakeaajugGbiaabUeadaWgaaWcbaqc% LbyacaqGWbaaleqaaKqzagGaey4kaSIaaeywamaaBaaaleaajugGbi% aabchacaqGZbaaleqaaKqzagGaaiOlaiaacIcacaqGdbWaaSbaaSqa% aKqzagGaae4CaiaabAgaaSqabaqcLbyacqGHsislcaqGdbWaaSbaaS% qaaKqzagGaae4CaaWcbeaajugGbiaacMcaaaaaaa!6301!\[{\text{\mu = \hat \mu }}{\text{.}}\frac{{{\text{C}}_{\text{s}} }}{{{\text{C}}_{\text{s}} + {\text{K}}_{\text{s}} }}.\frac{{{\text{K}}_{\text{p}} }}{{{\text{K}}_{\text{p}} + {\text{Y}}_{{\text{ps}}} .({\text{C}}_{{\text{sf}}} - {\text{C}}_{\text{s}} )}}\]it was found that the values for % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabeiVdyaaja% aaaa!373F!\[{\text{\hat \mu }}\], K_s and Y_ps were the same as for the non-aerobic case while the ethanol inhibition constant, K_p , had increased from 5,2 to 14,0 gl ^–1.Notation C_sf feed substrate concentration - gl ^–1 - C_s substrate concentration gl ^–1 - C_p product concentration - gl ^–1 - C_x cell concentration - gl ^–1 - D dilution rate - h^-1 - K_s substrate saturation constant - gl ^–1 - K_p product inhibition constant - gl ^–1 - m maintenance coefficient - h^–1 - Y_ps product yield coefficient - g EtOH/g glucose - Y_xs cell yield coefficient - g cells/g glucose - specific growth rate - h^–1 - % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGabeiVdyaaja% aaaa!373F!\[{\text{\hat \mu }}\] maximum specific growth rate - h^–1     

An investigation of ethanol inhibition and other limitations occurring during the fermentation of concentrated whey permeate byKluyveromyces fragilis

Based on the well-known fact thatKluyveromyces fragilis strains show sub-optimal performance when grown in concentrated whey permeate, previously optimized medium was investigated for possible limitations appearing at high concentrations. Shaken flask cultures showed that no additional vitamin or mineral sources were required when the optimized amount of yeast extract was added to the concentrated permeate. Several aspects of the ethanol inhibition of the growth ofK. fragilis NRRL 665 were investigated in continuous culture. The maximum ethanol concentration tolerated by this yeast, i.e. 45 g/l, was much lower than commonly reported for other strains. Ethanol and biomass production were also influenced by the increased ethanol concentration of the medium. At 31 g/l of alcohol product yield was reduced to 0.23 g/g whereas biomass yield was 0.05 g/g. Some evidence suggested that residence time and residual lactose concentration played a significant role in modulating the toxic effect of ethanol.     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.     

Rheological behaviour of high density continuous cultures of Saccharomyces cerevisiae

By culture of Saccharomyces cerevisiae with cell recycle using tangential microfiltration, high cell concentrations are obtained (in the range of 0 to 345 gl⁻¹ dry-weight). The rheological properties of the cell suspension during the cell growth were studied. Over a wide range of biomass concentration (X<275 gl⁻¹D.W.) the power-law model was found adequate to describe the rheological behaviour of the broth. Pronounced non-Newtonian (pseudoplastic) behaviour occurred for X > 75 gl⁻¹. Experimental correlations for apparent viscosity (n_a, mPa.s) and power-law index vs. biomass concentration (X, gl⁻¹) were established: n_a = (1+0.012X)² suitable over the whole range of concentration up to 275 gl⁻¹ D.W. n_a = 1+0.04X in the low concentration range; X<100 gl⁻¹D.W. Beyond the cell concentration of 275 gl⁻¹ D.W. the viscosity increases suddenly.     

High productivity continuous ethanol fermentation with a flocculating mutant strain of Zymomonas mobilis

High fermenter (volumetric) ethanol productivities (80 g/lh^–1) were attained in a simple single-stage continuous-stirred-tank-reactor (CSTR) employing a flocculent mutant of Zymomonas mobilis with a feed containing 100g/l glucose. Under these conditions a final ethanol concentration of 47.6 g/l was obtained, representing a maximum conversion efficiency of 97% of theoretical.Nomenclature SR = Medium glucose concentration (g/l)X Biomass concentration (g/l) - P Ethanol concentration (g/l) - VP Volumetric productivity (g ethanol/l/h) - Yp/s Product yield coefficient (g ethanol/g glucose consumed) - Qp Specific rate of ethanol formation (g ethanol/g cells/h) - D Dilution rate (h^–1) - Dmax Maximum dilution rate: ie., highest dilution rate at which the effluent glucose concentration 4g/l (h^–1)     

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.     

Extractive fermentation of ethanol using membrane distillation

Summary During the anaerobic batch cultivation ofKluyveromyces fragilis on a complex medium containing glucose (100 gl^–1), ethanol was produced at levels resulting in the inhibition of growth and product (ethanol) formation. Continuous extraction of the ethanol using transmembrane distillation resulted in a 87% increase in ethanol productivity from 0.99 to 1.85 gl^–1h^–1.     

Channelling of glucose by methanol for citric acid production from Aspergillus niger

Citric acid produced by Aspergillus niger was increased from 4.6g l^-1 to 7.8gl^-1 by supplementing basal medium with methanol (30mll^-1). While stimulating citric acid production, methanol did not improve membrane permeability of the fungus for citric acid. Methanol inhibited the germination of Aspergillus spores. An increase in glucose concentration from 50gl^-1 to 100gl^-1 in the presence of methanol (30mll^-1) improved citric acid production (1.6-fold) while at higher levels of glucose concentration methanol had no effect on citic acid production.