Continuous production ofl-lactic acid from whey permeate by immobilizedLactobacillus casei subsp.casei

Summary The production of l-lactic acid from whey permeate, a waste product of the dairy industry, by fermentation with the lactic acid bacterium Lactobacillus casei subsp. casei was investigated. A fermentation medium consisting of permeate and supplements, which enables exponential growth of the organisms, was developed. A fast method for determination of free and immobilized biomass in solid-rich media, based on measurement of cellular ATP, was evolved. Continuous fermentations in a stirred tank reactor (STR) and in a fluidized bed reactor (FBR) with immobilized biomass were compared. In the STR a volumetric productivity of 5.5 g/l per hour at 100% substrate conversion [dilution rate (D) = 0.22 h^–1] was determined. In the FBR porous sintered glass beads were used for immobilization and a maximum biomass concentration of 105 g/kg support was measured. A productivity of 10 g/l per hour was obtained at D = 0.4 h^–1 (substrate conversion 93%) and of 13.5 g/l per hour at D = 1.0 h^–1 (substrate conversion 50%). Offprint requests to: W. Krischke     

Continuous fermentation of sweet whey permeate for propionic acid production in a CSTR with UF recycle

Summary Propionic acid was produced byPropionibacterium acidi-propionici from sweet-whey permeate in a stirred tank reactor (CSTR) with cell recycle by ultrafiltration. The highest volumetric productivity achieved was 14.3 g.l^–1. h^–1, with a biomass of 100 g.l^–1 (dry weight). More concentrated product can be obtained by electrodialysis of the cell free fermentation medium.     

Propionic acid and biomass production using continuous ultrafiltration fermentation of whey

Summary This paper presents a study of propionic acid and propionibacteria production from whey by usingPropionibacterium acidi-propionici in continuous fermentation with cell recycle. The highest propionic acid volumetric productivity achieved was 5 g.l^–1.h^–1 with no biomass bleeding. A maximal biomass concentration of 130 g.l^–1 was achieved before initiating biomass bleeding to give a biomass volumetric productivity of 3.2 g.l^–1.h^–1 with a biomass of 75 g.l^–1 and a propionic acid productivity of 3.6 g.l^–1.h^–1 (for about 100 hours i.e. more than 50 residence times).     

Control of ethanol production and monitoring of membrane performance by mass-spectrometric gas analysis in the coupled fermentation-pervaporation of whey permeate

A coupled fermentation-pervaporation process was operated continuously with on-line mass spectrometric gas analysis monitoring of product accumulation on both the upstream and the downstream sides of the membrane. Efficient coupling of the fermentation with pervaporation was attained when a steady state of ethanol production and removal was achieved with whey permeate containing high concentrations of lactose (>8%) or by controlled lactose additions that also compensated for loss of liquid due to pervaporation. The combined system consists of a tubular membrane pervaporation module, directly connected to a stirred fermentor to form one circulation loop, kept at 38°C, with both units operating under computer control. Mass spectrometric gas analysis of the CO₂ gas evolved in the fermentor and the ethanol and water in the pervaporate on the downstream side of the membrane enabled us to follow the production of ethanol and its simultaneous removal. Membrane selectivity was calculated on-line and served to monitor the functioning of the membrane. Batch-wise-operated fermentation-pervaporation with Candida pseudotropicalis IP-513 yielded over 120 gl^–1 of concentrated ethanol solution using supplemented whey permeate containing 16% lactose. A steady state lasting for about 20 h was achieved with ethanol productivity of 20 g h^–1 (approx. 4 g l^–1 h^–1). Membrane selectivity was over 8. Controlled feeding of concentrated lactose suspension in the whey permeate (350 g l^–1) resulted in the continuous collection of 120–140 g l^–1 of ethanol pervaporate for 5 days, by which time salt accumulation hampered the fermentation. Medium refreshment restored the fermentative activity of the yeast cells and further extended the coupled process to over 9 days (200 h), when reversible membrane fouling occurred. The membrane module was exchanged and the combined process restarted. Correspondence to: Y. Shabtai     

Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions

Three 5 l working volume fermenters were used to investigate the growth of the yeast Kluyveromyces fragilis in acid cheese whey under ambient temperature in order to assess the specific growth rate and yield, the lactose and oxygen uptake rates during the various phases of batch culture, the effect of increasing temperature on the various kinetic parameters, and the need for a cooling unit for single cell production batch systems. The initial dissolved oxygen in the medium was 5.5 mg l^–1 and the pH was maintained at 4.5. The observed lag phase, specific growth rate and maximum cell number were 4 h, 0.2 h^–1 and 8.4 × 10⁸ cells ml^–1, respectively. About 99% of the lactose in cheese whey was utilized within 20 h, 85% during the exponential growth phase. The specific lactose utilization rates by K. fragilis were 0.20 × 10^–12, 1.457 × 10^–12, 0.286 × 10^–12 and 0.00 g lactose cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The dissolved oxygen concentration in the medium decreased as the cell number increased. The lowest oxygen concentration of 1.2 mg l^–1 was observed during the stationary phase. The volumetric oxygen transfer coefficient was 0.41 h^–1 and the specific oxygen uptake rates were 0.32 × 10^–12, 2.14 × 10^–12, 0.51 × 10^–12 and 0.003 × 10^–12 mg O₂ cell^–1 h^–1, for the lag, exponential, stationary and death phases, respectively. The maximum temperature recorded for the medium was 33 °C, indicating that a cooling unit for batch production of single cell protein at ambient temperature is not needed for this type of bioreactor. The increase in medium temperature affected the cell growth and the lactose and oxygen uptake rates.     

Selection of yeast strains for lactose hydrolysis in dairy effluents

The ability of eight selected yeast strains growing in unsupplemented whey permeate to abate its lactose concentration has been studied. All strains reached 1 × 10⁸ colony forming units (cfu) ml⁻¹ by 20h of culture. Rates of lactose disappearance in permeate were poorly correlated with intracellular lactase levels. Maximal lactase activity levels were registered by Candida kefir NCYC 143 and Kluyveromyces marxianus NCYC 1548 cells, which reached 200 nmol ONP min⁻¹ 10⁻⁷ cells. Large fluctuations in enzymatic activity were observed during incubation, presumably as a result of the reciprocal effects between lactase activity and both galactose (0–1.9 mM) and lactose (0–143 mM) concentrations in the permeate.     

The effect of yeast extract supplementation on the production of lactic acid from whey permeate byLactobacillus helueticus

Summary Batch and continuous two-stage cultures have been conducted in order to determine the effect of yeast extract (YE) on the homolactic fermentation of whey permeate byLactobacillus helveticus. Supplementation with YE had a significant effet on lactic acid concentration, volumetric productivity, and substrate conversion, but not on lactic acid yield. Volumetric productivity in the first stage increased from 2 to 9 g l^–1 per hour by increasing the YE concentration from 1.5 to 25 g l^–1 At the same time conversion improved from 22% to 93% at a dilution rate of 0.2 h^–1. The second stage demonstrated the effect of YE at a lower dilution rate (0.14 h^–1. A high system conversion (97%) and a high final lactic acid concentration (40 g l^–1) were achieved with 10 g l^–1 YE.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

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.     

Continuous lactic acid fermentation with concentrated product recovery by ultrafiltration and electrodialysis

Lactose of sweet whey permeate was converted into sodium lactate byLactobacillus helveticus. To increase the, productivity of the lactic acid fermentation and to reduce the amounts of effluents, the bioreactor was coupled with an ultrafiltration module and an electrodialysis unit. Without the electrodialyzer, with total cell recycling and at a dilution rate of 0.88 h^–1, a cellular concentration of 64 gl^–1 and a productivity of 22 gl^–1 h^–1 were obtained. When the electrodialysis unit is coupled, the outlet concentration of lactate was stabilized at 85±5 gl^–1.     

Protein production from whey usingPenicillium cyclopium; growth parameters and cellular composition

Summary The growth parameters ofPenicillium cyclopium have been evaluated in a continuous culture system for the production of fungal protein from whey. Dilution rates varied from 0.05 to 0.20 h^–1 under constant conditions of temperature (28°C) and pH (3.5). The saturation coefficients in the Monod equation were 0.74 g l^–1 for lactose and 0.14 mg l^–1 for oxygen, respectively. For a wide range of dilution rates, the yield was 0.68 g g^–1 biomass per lactose and the maintenance coefficient 0.005 g g^–1 h^–1 lactose per biomass, respectively. The maximum biomass productivity achieved was 2 g l^–1 h^–1 biomass at dilution rates of 0.16–0.17 h^–1 with a lactose concentration of 20 g l^–1 in the feed. The crude protein and total nucleic acid contents increased with a dilution rate, crude protein content varied from 43% to 54% and total nucleic acids from 6 to 9% in the range of dilution rates from 0.05 to 0.2 h^–1, while the Lowry protein content was almost constant at approximately 37.5% of dry matter.Nomenclature (mg l^–1) C_o initial concentration of dissolved oxygen - (h^–1) D dilution rate - (mg l^–1) K₀₂ saturation coefficient for oxygen - (g l^–1) K_s saturation coefficient for substrate - (g g^–1 h^–1) lactose per biomass) m maintenance energy coefficient - (mM g^–1 h^–1O₂ per biomass) Q₀₂ specific oxygen uptake rate - (g l^–1) S residual substrate concentration at steady state - (g l^–1) S_o initial substrate concentration in feed - (min) t_1/2 time when C_o is equal to C_o/2 - (g l^–1) X biomass concentration - (g l^–1) X biomass concentration at steady state - (g g^–1 biomass per lactose) Y_G yield coefficient for cell growth - (g g^–1 biomass per lactose) Y_x/s overall yield coefficient - (h^–1) specific growth rate     

Fed-batch fermentation for production of Kluyveromyces marxianusFII 510700 cultivated on a lactose-based medium

A strain of Kluyveromyces marxianus was grown in batch culture in lactose-based media at varying initial lactose concentrations (10–60 g L^–1) at 30°C, pH 5.0, dissolved oxygen concentrations greater than 20%. Increasing the concentration of mineral salts three-fold at 40 g L^–1 and 60 g L^–1 initial lactose concentration showed only a small increase in the yield of biomass, from 0.38 g g^–1 to 0.41 g g^–1, indicating that the initial batch cultures were not significantly nutrient- (mineral salts)-limited. A relatively high biomass concentration (105 g L^–1) was obtained in fed-batch culture following extended lactose feeding. An average specific growth rate (0.27 h^–1), biomass yield (0.38 g g^–1) and overall productivity (2.9 g L^–1 h^–1) were obtained for these fed-batch conditions. This fed-batch protocol provides a strategy for achieving relatively high concentrations and productivities of K. marxianus on other lactose-based substrate streams (e.g., whey) from the dairy industry.     

Valorization of Cheese and Tofu Whey through Enzymatic Synthesis of Lactosucrose

This work deals with the development of a new bioprocess for the efficient synthesis of lactosucrose, a potential prebiotic oligosaccharide with a high value-added, from two important and inexpensive agro-industrial by-products such as tofu whey and cheese whey permeate. The bioconversion is driven by the ability of the enzyme levansucrase SacB from Bacillus subtilis CECT 39 to transfructosylate lactose contained in the cheese whey permeate by using not only sucrose but also raffinose and stachyose, which are present in considerable amounts in the tofu whey, as suitable donors of fructosyl moieties. The maximum lactosucrose concentration obtained from both by-products was 80.1 g L^-1 after a short reaction time 120 min at 37°C, leading to productivity and specific productivity values of 40.1 g lactosucrose L^-1 h^-1 and 80.1 mg lactosucrose U enzyme⁻¹ h⁻¹, respectively. Findings contained in this work could provide a new strategy to valorize agro-industrial by-products as cheese whey permeate and, specially, tofu whey by means of their use as renewable resources in the enzymatic synthesis of bioactive oligosaccharides.     

Lactic acid from cheese whey permeate. Productivity and economics of a continuous membrane bioreactor

The economics of incorporating membrane modules in several steps in the conversion of whey permeate to lactic acid was studied. Membrane recycle fermenters operating at a cell concentration of 40 g l^–1 resulted in a productivity of 22.5 g l^–1h^–1 with a lactate concentration of 89 g l^–1 and a yield of 0.89. The membrane units (reverse osmosis for preconcentrating whey permeate, hollow-fiber ultrafiltration for clarification and for cell recycling) contribute about 28% of the total fixed capital costs and less than 5% of the operating cost. The two largest costs are whey transportation and yeast extract, contributing about 35% and 38% to the total product cost of US $ 0.98/kg 85% lactate. Without these two costs, unpurified lactate could be produced for $ 0.27/kg.     

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.     

Effect of benzoic acid on the ammonium transport system of Zygosaccharomyces bailii

Summary A study of the ammonium transport system of Zygosaccharomyces bailii was carried out using methylammonium as a non-metabolizable analogue. Benzoic acid in the growth medium decreased the capacity of the transport system from 1.46 ± 0.11 mmol.g^–1.h^–1 to 0.41±0.04 mmol.g^–1.h^–1, while the affinity for ammonium was not significatively affected. Although ammonium uptake was inhibited by benzoic acid, the ammonium transport system was still active at preservative concentrations which fully inhibited growth suggesting that inhibition of growth was not governed by the uptake of this nutrient.     

Metabolic,physiological and kinetic aspects of the alcoholic fermentation of whey permeate by Kluyveromyces fragilis NRRL 665 and Kluyveromyces lactis NCYC 571

Optimum growth conditions for the fermentation of non-concentrated whey permeate by Kluyveromyces fragilis NRRL 665 have been defined. Use of 3.75 g yeast extract l^?1, a growth temperature of 38°C and a pH of 4.0 allowed a maximum productivity of 5.23 g ethanol l^?1 h^?1 in continuous culture with a yield 91% of theoretical. Complete batch fermentation of permeate with 100 g lactose l^?1 was possible with a maximum specific growth rate of 0.276 h^?1 without any change in ethanol yield. Fermentation of concentrated permeate resulted, however, in a general decrease of specific substrate consumption rate, demonstrated by the inability to completely convert an initial 90 or 150 g lactose l^?1 in continuous culture, even at dilution rates as low as 0.05 and 0.08 h^?1, respectively. The decrease could be related to substrate inhibition, to an increase in osmotic pressure caused by lactose and salts, and to ethanol inhibition of both alcohol and biomass yield. The decrease in specific productivity could be counterbalanced by use of high cell density cultures, obtained by cell recycle of K. fragilis. Fermentation of a non-concentrated permeáte at a dilution rate of 1 h^?1 resulted in a productivity of 22 g l^?1 h^?1 at 22 g ethanol l^?1. Cell recycle using flocculating Kluyveromyces lactis NCYC 571 was also tested. With this strain a productivity of 9.3 g l^?1 h^?1 at 45 g product l^?1 was attained at a dilution rate of 0.2 h^?1, with an initial lactose concentration of 95 g l^?1.     

Batch propagation of Lactobacillus helveticus for production of lactic acid from lactose concentrated cheese whey with microaeration and nutrient supplementation

Continuous mix batch bioreactors were used to study the kinetic parameters of lactic acid fermentation in microaerated-nutrient supplemented, lactose concentrated cheese whey using Lactobacillus helveticus. Four initial lactose concentrations ranging from 50 to 150 g l^–1 were first used with no microaeration and no yeast extract added to establish the substrate concentration above which inhibition will occur and then the effects of microaeration and yeast extract on the process kinetic parameters were investigated. The experiments were conducted under controlled pH (5.5) and temperature (42 °C) conditions. The results indicated that higher concentrations of lactose had an inhibitory effect as they increased the lag period and the fermentation time; and decreased the specific growth rate, the maximum cell number, the lactose utilization rate, and the lactic acid production rate. The maximum lactic acid conversion efficiency (75.8%) was achieved with the 75 g l^–1 initial lactose concentration. The optimum lactose concentration for lactic acid production was 75 g l^–1 although Lactobacillus helveticus appeared to tolerate up to 100 g l^–1 lactose concentration. Since the lactic acid productivity is of a minor importance compared to lactic acid concentration when considering the economic feasibility of lactic acid production from cheese whey using Lactobacillus helveticus, a lactose concentration of up to 100 g l^–1 is recommended. Using yeast extract and/or microaeration increased the cell number, specific growth rate, cell yield, lactose consumption, lactic acid utilization rate, lactic acid concentration and lactic acid yield; and reduced the lag period, fermentation time and residual lactose. Combined yeast extract and microaeration produced better results than each one alone. From the results it appears that the energy uncoupling of anabolism and catabolism is the major bottleneck of the process. Besides lactic acid production, lactose may also be hydrolysed into glucose and galactose. The -galactosidase activity in the medium is caused by cell lysis during the exponential growth phase. The metabolic activities of Lactobacillus helveticus in the presence of these three sugars need further investigation.     

Lactic acid from acid whey permeate in a membrane recycle bioreactor

Whey permeate was obtained by ultrafiltration of cottage cheese whey and supplemented with yeast extract. The lactose in the permeate was converted into lactic acid by Lactobacillus bulgaricus in a high-performance membrane bioreactor configured in the cell recycle mode. At a cell concentration of 10 g l⁻¹, optimum productivity of lactic acid was 35 g l⁻¹ h⁻¹. Increasing the cell concentration to 30 g l⁻¹ enabled the use of a dilution rate of 1 h⁻¹ with complete substrate utilization. At 60 g l⁻¹, productivity was over 80 g l⁻¹ h⁻¹ with complete substrte utilization; this is vastly superior to conventional batch fermentations.     

Sophorolipid Production with High Yields on Whey Concentrate and Rapeseed Oil without Consumption of Lactose

Sophorolipids were produced by single-step batch cultivation of Candida bombicola ATCC 22214 on deproteinized whey concentrate and repeated feed of rapeseed oil. A mild sterilization method for whey was developed. High yields of 280 g dry sophorolipids l^–1 were obtained from deproteinized whey concentrate containing 100 g lactose l^–1 and 300 g rapeseed oil l^–1. Surprisingly, the whey lactose was not consumed by the organism. Growth only on the oil was assumed and a high lipase activity of 24 U per g cell dry weight resulted.