Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

Continuous butanol production from whey permeate with immobilized Clostridium beyerinckii LMD 27.6

Summary The aim of this study was to find the conditions necessary for the continuous butanol production from whey permeate with Clostridium beyerinckii LMD 27.6, immobilized in calcium alginate beads. The influence of three parameters on the butanol production was investigated: the fermentation temperature, the dilution rate (during start-up and at steady state) and the concentration of calcium ions in the fermentation broth. It was found that both a fermentation temperature of 30° C and a dilution rate of 0.1 h^-1 or less during the start-up phase are required to achieve continuous butanol production from whey permeate. Butanol can be produced continuously from whey permeate in reactor productivities sixteen times higher than those found in batch cultures with free C. beyerinckii cells on whey media.     

Yeast biomass production from acid whey permeate

Summary The yield ofKluyveromyces marxianus Y-113 grown on sulphuric acid casein whey permeate was decreased at high lactose or ethanol concentrations and slightly increased with high agitation and supplementation of the permeate with nutrients. The maximum specific growth rate was most influenced by the stirrer speed, ethanol concentration and nutrient supplementation.     

Microbial production of 2,3-butanediol from whey permeate

Summary Of four organisms tested in semi-synthetic medium for the production of 2,3-butanediol from lactose, Klebsiella pneumoniae N.C.I.B. 8017 proved to be the most promising. When tested using rennet whey permeate as substrate, a butanediol concentration of 7.5 g/l, representing a yield of 0.46 g/g lactose utilized, was observed after 96 h incubation. In whey permeate where the lactose had been hydrolysed enzymatically prior to the fermentation, a butanediol concentration of 13.7 g/l, representing a yield of 0.39 g/g sugar utilized was obtained. These results indicate that lactose utilization may be a limiting step in the fermentation process.     

Continuous solvent production from whey permeate using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

Cells of Clostridium acetobutylicum were immobilized by adsorption onto bonechar and used in a packed bed reactor for the continuous production of solvents from whey permeate. A maximum solvent productivity of 4.1 g l⁻¹ h⁻¹, representing a yield of 0.23 g solvent/g lactose utilized, was observed at a dilution rate of 1.0 h⁻¹. The reactor was operated under stable conditions for 61 days. High concentrations of lactose in the whey permeate favored solventogenesis, while low concentrations favored acidogenesis.     

Performance of the continuous glucose isomerase reactor system for the production of fructose syrup

Glucose isomerase in the form of heat-treated whole-cell enzyme prepared from Streptomyces phaeochromogenus follows the reversible single-substrate reaction kinetics in isomerization of glucose to fructose. Based on the Kinetic constants determined and the mathematical model of the reactor system developed, the preformance of a plug-flow-type continuous-enzyme reactor system was studied experimentally and also simulated with the aid of a computer for the ultimate objective of optimization of the glucose isomerase reactor system. The enzyme decay function for both the enzyme storage and during the use in the continuous reactor, was found to follow the first-order decay kinetics. When the enzyme decay function is taken into consideration, the ideal homogeneous enzyme reactor kinetics provided a satisfactory working model without further complicatin of the mathematical model, and the results of computer simulation were found to be in good agreement with the experimental results. Under a given set of constraints the performance of the continuous glucose isomerase reactor system can be predicted by using the computer simulation method described in this paper. The important parameters studied for the optimization of reactor operation were enzyme loading, mean space time of the reactor, substrate feed concentration, enzyme decay constants, and the fractional conversion, in addition to the kinetic constants. All these parameters have significant effect on the productivity. Some unique properties of the glucose isomerization reaction and its effects on the performance of the continuous glucose isomerase reactor system have been studied and discussed. The reaction kinetics of glucose isomerase and the effects of both the enzyme loading and the changes in reaction rate within a continuous reactor on the productivity are all found to be of particular importance to this enzyme reactor system.     

Model for continuous production of solvents from whey permeate in a packed bed reactor using cells of Clostridium acetobutylicum immobilized by adsorption onto bonechar

Summary A mathematical model has been developed to describe the operation of a packed bed reactor for the continuous production of solvents from whey permeate. The model has been used to quantitate the amounts of different physiological/ morphological types of biomass present in the reactor. The majority of biomass is inert, i.e. it neither grows nor produces solvent. Only relatively small amounts of biomass actively grow (vegetative, non-solvent-producing cells), while even smaller amounts are responsible for solvent production (clostridial, solvent-producing cells).     

Comparison between immobilized Kluyveromyces fragilis and Saccharomyces cerevisiae coimmobilized with beta-galactosidase, with respect to continuous ethanol production from concentrated whey permeate

Kluyveromyces fragilis immobilized in calcium alginate gel was compared to Saccharomyces cerevisiae coimmobilized with beta-galactosidase, for continuous ethanol production from whey permeate in packed-bed-type columns. Four different whey concentrations were studied, equivalent to 4.5, 10, 15, and 20% lactose, respectively. In all cases the coimmobilized preparation produced more ethanol than K. fragilis. The study went on for more than 5 weeks. K. fragilis showed a decline in activity after 20 days, while the coimmobilized preparation was stableduring the entrire investigation. Under experimental conditions theoretical yields of ethanol were obtained from 4.5 and 10% lactose substrates with the coimmobilized system. Using 15% lactose substrate, theoretical yields were only obtained when a galactose-adapted immobilized S. cerevisiae column was run in series with the coimmobilized column. Then a maximum of 71 g/L ethanol was produced with a productivity of 2.5 g/L h. The coimmobilized column alone gave a maximum ethanol concentration of 52 g/L with a productivity of 4.5 g/L h, whereas immobolized K. fragilis only produced 13 g/L ethanol with a productivity of 1.1 g/L h. It was not possible to obtain theoretical yields of ethanol from the highest substrate concentration.     

Continuous production of protein hydrolysates in immobilized enzyme reactors

The digestion of several proteins, casein, α-lactalbumin, human serum albumin and a mixture of whey proteins by immobilized pronase, thermitase and leucine aminopeptidase was studied on various conditions in five types of enzyme reactors. Reactors and operating conditions were designed to maximize the extent of hydrolysis and to minimize the adverse effects of the macromolecular nature of the substrates. A simple analytical method was developed to follow routinely the extent of hydrolysis. Substrate proteins were subjected to various pretreatments intended to disturb their native structure. The maximum feasible extent of hydrolysis in the reactor effluent, which is an average quantity, clustered around the magic figure of 33% in all systems studied. Protein digestion in bubbled column reactors charged with the polyaminomethylstyrene-fixed thermostable proteinase “thermitase” and operated at 50 to 60°C turned out to be the most efficient setup to produce continuously amino acid/peptide mixtures.     

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]     

Use ofClostridium acetobutylicum P262 for production of solvents from whey permeate

Summary The production of solvents from whey permeate in batch fermentation usingClostridium acetobutylicum P262 was examined. An overall reactor productivity of 0.24 g/l.h was observed, representing a marked improvement over reports using other strains of clostridia. Using a semi-synthetic medium galactose was shown to be as effective a substrate as glucose. When whey permeate was used in which the lactose was hydrolysed prior to fermentation, preferential uptake of glucose over galactose was observed, and such hydrolysis provided no advantage to the fermentation process.     

A kinetic model for methanogenesis from whey permeate in a packed bed immobilized cell bioreactor

The fermentation kinetics of methane production from whey permeate in a packed bed immobilized cell bioreactor at mesophilic temperatures and pHs around neutral was studied. Propionate and acetate were the only two major organic intermediates found in the methanogenic fermentation of lactose. Based on this finding, a three-step reaction mechanism was proposed: lactose was first degraded to propionate, acetate, CO(2), and H(2) by fermentative bacteria; propionate was then converted to acetate by propionate-degrading bacteria; and finally, CH(4) and CO(2) were produced from acetate, H(2), and CO(2) by methanogenic bacteria. The second reaction step was found to be the rate-limiting step in the overall methanogenic fermentation of lactose. Monod-type mathematical equations were used to model these three step reactions. The kinetic constants in the models were sequentially determined by fitting the mathematical equations with the experimental data on acetate, propionate, and lactose concentrations. A mixed-culture fermentation model was also developed. This model simulates the methanogenic fermentation of whey permeate very well.     

Mead production by continuous series reactors using immobilized yeast cells

Summary Two series reactors separately packed with immobilized cells ofSaccharomyces cerevisiae BRL-7 producing alcohol andHansenula anomala producing ethyl acetate were used to produce meads of controlled quality. The rate of alcohol production and the amount of ethyl acetate produced were 6.13 g/h and 61.6 mg/100 ml, respectively, at a dilution rate of 1.36 h⁻¹. Coimmobilized cells ofS. cerevisiae BRL-7 andH. anomala produced alcohol at the rate of 8.02 g/h with 40 mg/100 ml ethyl acetate content at a dilution rate of 2.15 h⁻¹. The process of immobilization, use of dual cultures and series reactors reduced the time period of mead production and eliminated the costlier aging process.     

Enhancing bioethanol production from delactosed whey permeate by upstream desalination techniques

Industrial cheese whey processing comprises generally the isolation of proteins and lactose, but the economic use for the residual molasses, the so‐called delactosed whey permeate (DWP), is still to be improved. One possibility to maximize valorization and to minimize waste water treatment is the conversion of the remaining lactose in the DWP to ethanol by the yeast Kluyveromyces marxianus. This fermentation process depends strongly on the total ash content of the DWP, as high salt concentrations inhibit yeast metabolism. Here, three different approaches were tested to lower the DWP salt content: (i) simple dilution; (ii) nanofiltration; and (iii) electrodialysis. Lactose consumption, ethanol production and time‐dependent yields were compared between the three methods. A dilution of DWP to 60% v/v led to fermentation taking less than 80 h and yield above 7% AbV (alcohol by volume). After nanofiltration, 7.5% AbV was produced in about 80 h, and after electrodialysis, 11% AbV was produced in about 52 h. On the one hand the technical treatments (nanofiltration and electrodialysis) led to enhanced productivity in the fermentations, but, on the other hand, elaborate and extensive preprocessing is needed. Overall, ethanol production from DWP could be enhanced by prior partial desalination.     

Packed-bed immobilized enzyme reactors for complex processes

Utilization of enzymic reactors for biotechnological-biomedical applications is currently developing at a sustained pace.Our present study concentrates on development of procedures for describing the performance of devices where enzyme-catalyzed reactions between two substrates take place, and for the rational design and optimization of the reactors considered. Within this context, an analytical model was developed for immobilized enzyme packed-bed reactors; it takes into account internal diffusion limitations for the cosubstrates, and hydrodynamic backmixing effects. In order to overcome the complex mathematical problems involved, the compartmental analysis approach was employed.Using this model, performance was simulated for various configurations of the enzymic unit, i.e. from a continuously operated stirred tank reactor (CSTR) to an essentially plug flow type. In addition, an experimental method is described for quantitatively assessing the backmixing effects prevailing in the reactor.The procedures established also provide the ground for further developments, particularly for systems where, in parallel to the enzymic reaction, additional processes (e. g. complexation) take place.List of Symbols C _j,i mM Concentration of substrate j in the pores of stage - iD _j cm²/s Internal (pore) diffusion coefficient of substrate j; defined in Eq. (7) - D _e cm²/s Axial dispersion diffusion coefficient - D _j, cm²/s cm²/s Bulk diffusion coefficient for substrate j - E mM Enzyme concentration inside the catalytic pores - J _j,immol/s/cm² Net flux of substrate j taking place from the bulk of stage i into the corresponding pores; defined in Eq. (6) - K _m,1, K _m,2 mM Michaelis-Menten constants for cosubstrates 1 and 2, respectively - k s ^–1 Catalytic constant - k _s cm/s Catalytic constant - n Total number of elementary stages in the reactor - Q cm³/s Volumetric flow rate throught the reactor - r cm Radius of the pore - R _j,i mM/s Reaction rate of substrate j in stage i, in terms of volumetric units - S cm² Internal surface of a pore - S _j,0 mM Concentration of substrate j in the reactor feed - S _j,i–1, S _j,i mM Concentration of substrate j in the bulk phase leaving stages i — 1 and i, respectivley - V _i cm³ Total volume of stage i (bulk phase + pore phase + inert solid carrier) - V cm³ Total volume of the reactor - V _m ^* mmol/s/cm² Maximal reaction rate in terms of surface units; defined in Eq. (8) - V _m mM/s Maximal reaction rate in terms of volumetric units; defined in Eq. (8) - V _p cm³ Volume of one pore - y cm Axial coordinate of the pores - y ₀ cm Depth of the pores - Z cm Axial coordinate of the reactor - Z ₀ cm Length of the reactor - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - ₁ Dimensionless parameter; defined in Eq. (27) - ₂ Dimensionless parameter; defined in Eq. (27) - Ratio between the radius of the enzyme molecule and the radius of the pore (dimensionless) - V₁ Dimensionless parameter; defined in Eq. (21) - v₂ Dimensionless parameter; defined in Eq. (21) - Q Volumetric packing density of catalytic particles (dimensionless) - Ø Porosity of the catalytic particles (dimensionless) - Ø Dimensionless concentration of substrate j in pores of stage i; defined in Eq. (16) - _j,i-1,_j,i Dimensionless concentration of substrate j in the bulk phase of stage i; defined in Eq. (18) - Dimensionless position; defined in Eq. (16) - ² s² Variance; defined in Eq. (33) - Mean residence time in the reactor; defined in Eq. (33)     

Continuous mixed strain mesophilic lactic starter production in supplemented whey permeate medium using immobilized cell technology

The aim of this work was to study a new process for the continuous production of mixed-strain lactic acid bacteria starters using immobilized cells. Three strains of Lactococcus (two Lactococcus lactis subsp. lactis: KB and KBP, and one Lactococcus lactis subsp. lactis biovar diacetylactis: MD) were immobilized separately in kappa-carrageenan-locust bean gum gel beads. Continuous fermentations were carried out in a 1 L pH-controlled stirred tank reactor with a 30% (v/v) bead inoculum (strain ratio 1:1:1), continuously fed with a whey UF permeate medium, supplemented with 1.5% yeast extract and 0.1M KCl. The effects of three parameters-pH, temperature (T), dilution rate (D), and their interactions on the composition and activity of the culture in the effluent at pseudosteady state were studied according to a rotatable central composite design, during a 53-day fermentation. The process showed a high biological stability and no strain became dominant, or was eliminated from the bioreactor. The statistical analysis showed that the three strains were differently affected by the studied parameters, and that a large range of effluent starter composition can be achieved by varying D, pH, and T. However, the acidifying characteristics were not affected by the culture conditions. A cross-contamination from other strains of the mixed culture was observed in gel beads entrapping a pure culture at the fermentation onset, and led to a biomass redistribution within the beads. However, the strain ratio (KB:KBP:MD) observed after the 53-day experiment (1:2:2) was close to the initial bead ratio (1:1:1). The beads demonstrated a high mechanical stability throughout the 53-day continuous fermentation. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 502-516, 1997.     

Anaerobic digestion of whey and whey permeate with suspended and immobilized complex and defined consortia

Summary Whey could be anaerobically digested at space loadings up to 36 kg COD/m³·d in an upflow digester containing porous clay beads for immobilization of microorganisms. In a parallel fermenter without immobilization a space loading of only 8 kg/m³·d was reached. The start-up time was very much reduced by the support material. The COD-reduction in both reactors was 95% and volatile fatty acids in the effluent were below 10 mmol/l. During the digestion of whey a thick layer of Methanothrix soehngenii and occasionally Methanobrevibacter arboriphilus was immobilized on the clay beads. The Methanothrix soehngenii layer disappeared, when whey permeate was fed. Methanosarcina spec. became the predominant acetotrophic methanogen, probably due to the lower pH resulting from digestion of whey permeate. Methanosarcina spec., however, was suspended and only occasionally trapped in the pores of the clay beads. No significant adhesion of other bacteria occurred.In a chemostat a consortium of 5 isolates from digested whey and a strain of Methanosarcina barkeri was able to degrade all components of whey, although at a slightly lower conversion rate than by the complex natural consortium. The total population in the whey digester was more than twice as numerous as that in the whey permeate digester. The lower number of acetotrophic methanogens seemed to be the rate-limiting step in the whey permeate digester and seemed to be responsible for the lower overall conversion rates.Dedicated to Professor Dr. H. J. Rehm on the occasion of his 60th birthday     

Ethanol production from whey with immobilized living yeast

Summary Living Kluyveromyces fragilis yeast cells were succesfully entrapped in calcium alginate gel beads at cell loadings of 4 to 16 g yeast (0.8 to 3.2 g d.m.) per 1 g of sodium alginate. In batch systems, about 90 % conversion in 48 h was obtained both with free and immobilized yeast using demineralized whey of 5 to 10 % lactose content as substrate. In continuous packed-bed column operation nearly a constant 2 % product ethanol concentration could be maintained at 5 % substrate lactose level for at least one month.