Pilot-scale culture of somatic embryos of Eleutherococcus senticosus in airlift bioreactors for the production of eleutherosides

Purpose of work

To establish pilot scale bioreactor cultures of somatic embryos of Siberian ginseng for the production of biomass and eleutherosides. Somatic embryos of Eleutherococcus senticosus were cultured in airlift bioreactors using Murashige and Skoog medium with 30 g sucrose l^?1 for the production of biomass and eleutherosides. Various parameters including the type of bioreactor, aeration volume, and inoculum density were optimized for 3 l capacity bioreactors. Balloon-type airlift bioreactors, utilizing a variable aeration volume of 0.1–0.3 vvm and an inoculum of 5 g l^?1, were suitable for biomass and eleutheroside production. In 500 l balloon-type airlift bioreactors, 11.3 g dry biomass l^?1, 220 µg eleutheroside B l^?1, 413 µg eleutheroside E l^?1, and 262 µg eleutheroside E1 l^?1 were produced.     

Integrated bioprocess for the simultaneous production of lactic acid and dairy sewage treatment

An integrated biological process was developed for the conversion of whey lactose to lactic acid. We report about the achievement of maximum COD reduction and thus a substantial unburdening of the environment, combined with the economic production of lactic acid, appropriate for industrial scale. The process – designed for continuous operation – consists of four main steps: (i) Protein recovery by ultrafiltration leading to the first product: protein concentrate. The resulting filtrate is the fermentation substrate acid whey permeate. (ii) Adjustment of the composition of the permeate in the medium preparation step in order to ensure the proper function of the following process steps. (iii) Conversion of the lactose to lactate by fermentation with lactic acid bacteria in a cell recycle reactor, using ceramic microfiltration membranes. (iiii) Conversion of the lactate in the cell-free permeate stream of the fermentation to free lactic acid by bipolar electrodialysis. A stable operation of the process was attained up to more than 2000?hours. Using a new selected strain of lactic acid bacteria, a lactic acid productivity of 17?g?l^?1?h^?1 is achieved at total lactose conversion without any nitrogen supplements like yeast extract. A lactic acid concentration of 190?g?l^?1 is obtained in the acidic cell of the electrodialysis unit and the COD of the remaining sewage is diminished by 92%. As an additional cost reduction item, the neutralization agent of the fermentation is recovered in the caustic cell of the bipolar electrodialysis unit. A cost evaluation for an industrial scale process (100?000?t of whey per year) resulted in a price of 0.66 $ per kg of lactic acid, which under present terms hits the goal of making this process economic for the large scale production of lactic acid as an attractive building block for various purposes in chemical industry.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

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.     

Optimization of nutritional supplements for enhanced lactic acid production utilizing sugar refinery by-products

The present study investigated the synergistic effect of nutritional supplements (amino acid and Tween 80) on lactic acid production by Lactobacillus delbruckii utilizing a sugar refinery by product (cane molasses) in a submerged fermentation process. Initially, the effect of individual factors on lactic acid yield was studied by supplementing amino acids and their combinations, Tween 80 and cane molasses at varying concentrations in production medium. A combination of l-phenylalanine and l-lysine gave a maximum lactic acid yield of 47.89?±?0.1 g/L on a dry cell weight basis at individual factor level. Similarly, maximum lactic acid yield was obtained by supplementing the production medium with 40.0 g/L and 2.0 g/L Tween 80 and cane molasses, respectively, at individual factor level. In order to further improve the lactic acid yield, nutritional supplements were optimized by central composite rotatable design (CCRD) using Minitab 15 software. Shake flask cultivation under optimized conditions, i.e., cane molasses (32.40 g/L), Tween 80 (2.0 g/L) and l-phenylalanine and l-lysine (34.0 mg/L) gave a lactic acid yield of 64.86?±?0.2 g/L, corresponding to 95.0 % of the predicted yield of 67.78?±?0.3 g/L. Batch cultivation performed in 7.5 L bioreactor (working volume: 3.0 L) under optimized conditions gave maximum lactic acid yield and productivity of 79.12?±?0.2 g/L and 3.40 g/L·h, which is higher than previous studies with reduced fermentation time. Screening of lactic acid producing bacteria and characterization of lactic acid was also done.     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Increased production of γ-lactones from hydroxy fatty acids by whole Waltomyces lipofer cells induced with oleic acid

Among several fatty acids tested, oleic acid was selected as the most efficient inducer for the production of 4-hydroxydodecanoic acid, a metabolite of β-oxidation, by Waltomyces lipofer. Cells were induced by incubation for 12 h in a medium containing 10 g l^?1 yeast extract, 10 g l^?1 peptone, 5 g l^?1 oleic acid, 1 g l^?1 glucose, and 0.05 % (w/v) Tween 80. The optimal reaction conditions for the production of γ-lactones by induced cells were pH 6.5, 35 °C, 200 rpm, 0.71 M Tris, 60 g l^?1 hydroxy fatty acid, and 20 g l^?1 cells. Non-induced cells produced 38 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 63 % (w/w) and a productivity of 1.3 g l^?1 h^?1 under the optimized conditions, whereas induced cells produced 51 g l^?1 γ-dodecalactone from 60 g l^?1 10-hydroxystearic acid after 30 h, with a conversion yield of 85 % (w/w) and a productivity of 1.7 g l^?1 h^?1. The conversion yield and productivity of induced cells were 22 % and 1.3-fold higher, respectively, than those of non-induced cells. Induced cells also produced 28 g l^?1 γ-decalactone and 12 g l^?1 γ-butyrolactone from 60 g l^?1 12-hydroxystearic acid and 60 g l^?1 10-hydroxydecanoic acid, respectively, after 30 h. The concentration, conversion yield, and productivity of γ-dodecalactone and γ-decalactone are the highest reported thus far. This is the first study on the biotechnological production of γ-butyrolactone.     

Production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells of recombinant Escherichia coli expressing the oleate hydratase gene of Stenotrophomonas maltophilia

Recombinant Escherichia coli, expressing the oleate hydratase gene of Stenotrophomonas maltophilia, was permeabilized by sequential treatments with 0.125 M NaCl and 2 mM EDTA. The optimal conditions for the production of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid from α-linolenic acid by permeabilized cells were 35 °C and pH 7.0 with 0.1 % (v/v) Tween 40, 50 g permeabilized cells l^?1, and 17.5 g α-linolenic acid l^?1. Under these conditions, permeabilized cells produced 14.3 g 10-hydroxy-12,15(Z,Z)-octadecadienoic acid l^?1 after 18 h, with a conversion yield of 82 % (g/g) and a volumetric productivity of 0.79 g l^?1 h^?1. These values were 17 and 168 % higher than those obtained by nonpermeabilized cells, respectively. The concentration, yield, and productivity of 10-hydroxy-12,15(Z,Z)-octadecadienoic acid obtained by permeabilized cells are the highest reported thus far.     

Nisin production in a chitin-including continuous fermentation system with Lactococcus lactis displaying a cell wall chitin-binding domain

The limiting factors in the continuous production of nisin are high amount of biomass loss and low dilution rate application. In this study, a chitin-including continuous nisin fermentation system (CICON-FER) was constructed for high volumetric nisin production using nisin producer L. lactis displaying cell wall chitin-binding domain (ChBD) together with chitin in the reactor. In this respect, the highest binding conditions of relevant L. lactis cells to chitin were determined. Then the chitin flakes carrying nisin-producing L. lactis cells were used within the CICON-FER system at different dilution rates (0.1–0.9 h^?1) and initial glucose concentrations (20–60 g l^?1). The results revealed that the pH 7 conditions and the use of 100 mM sodium phosphate buffer with 0.1 % Tween 20 and Triton X-100 significantly increased the binding capacity of ChBD displaying L. lactis cells to chitin. The constructed CICON-FER system maintained the presence of the ChBD surface displaying L. lactis cells in the reactor system until 0.9 h^?1 dilution rate that resulted in a considerably high level of volumetric nisin production and productivity (10,500 IU ml^?1 and 9,450 IU ml^?1 h^?1, respectively) with the combination of a 0.9-h^?1 dilution rate and a 40-g l^?1 initial glucose concentration. In conclusion, an innovative nisin fermentation system that yielded the highest nisin production thus far and that was feasible for industrial application was created.     

Production of a novel compound, 10,12-dihydroxystearic acid from ricinoleic acid by an oleate hydratase from Lysinibacillus fusiformis

A recombinant oleate hydratase from Lysinibacillus fusiformis converted ricinoleic acid to a product, whose chemical structure was identified as the novel compound 10,12-dihydroxystearic acid by gas chromatograph/mass spectrometry, Fourier transform infrared, and nuclear magnetic resonance analysis. The reaction conditions for the production of 10,12-dihydroxystearic acid were optimized as follows: pH?6.5, 30 °C, 15 g?l^?1 ricinoleic acid, 9 mg?ml^?1 of enzyme, and 4 % (v/v) methanol. Under the optimized conditions, the enzyme produced 13.5 g?l^?1 10,12-dihydroxystearic acid without detectable byproducts in 3 h, with a conversion of substrate to product of 90 % (w/w) and a productivity of 4.5 g?l^?1?h^?1. The emulsifying activity of 10,12-dihydroxystearic acid was higher than that of oleic acid, ricinoleic acid, stearic acid, and 10-hydroxystearic acid, indicating that 10,12-dihydroxystearic acid can be used as a biosurfactant.     

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.     

Construction of lactose-consuming Saccharomyces cerevisiae for lactose fermentation into ethanol fuel

Two lactose-consuming diploid Saccharomyces cerevisiae strains, AY-51024A and AY-51024M, were constructed by expressing the LAC4 and LAC12 genes of Kluyveromyces marxianus in the host strain AY-5. In AY-51024A, both genes were targeted to the ATH1 and NTH1 gene-encoding regions to abolish the activity of acid/neutral trehalase. In AY-51024M, both genes were respectively integrated into the MIG1 and NTH1 gene-encoding regions to relieve glucose repression. Physiologic studies of the two transformants under anaerobic cultivations in glucose and galactose media indicated that the expression of both LAC genes did not physiologically burden the cells, except for AY-51024A in glucose medium. Galactose consumption was initiated at higher glucose concentrations in the MIG1 deletion strain AY-51024M than in the corresponding wild-type strain and AY-51024A, wherein galactose was consumed until glucose was completely depleted in the mixture. In lactose medium, the Sp. growth rates of AY-51024A and AY-51024M under anaerobic shake-flasks were 0.025 and 0.067 h^?1, respectively. The specific lactose uptake rate and ethanol production of AY-51024M were 2.50 g lactose g CDW^?1 h^?1 and 23.4 g l^?1, respectively, whereas those of AY-51024A were 0.98 g lactose g CDW^?1 h^?1 and 24.3 g lactose g CDW^?1 h^?1, respectively. In concentrated cheese whey powder solutions, AY-51024M produced 63.3 g l^?1 ethanol from approximately 150 g l^?1 initial lactose in 120 h, conversely, AY-51024A consumed 63.7 % of the initial lactose and produced 35.9 g l^?1 ethanol. Therefore, relieving glucose repression is an effective strategy for constructing lactose-consuming S. cerevisiae.     

L-Lactic acid production from raw cassava starch in a circulating loop bioreactor with cells immobilized in loofa (Luffa cylindrica)

l-Lactic acid was produced from raw cassava starch, by simultaneous enzyme production, starch saccharification and fermentation in a circulating loop bioreactor with Aspergillus awamori and Lactococcus lactis spp. lactis immobilized in loofa sponge. A. awamori was immobilized directly in cylindrical loofa sponge while the L. lactis was immobilized in a loofa sponge alginate gel cube. In the loofa sponge alginate gel cube, the sponge serves as skeletal support for the gel with the cells. The alginate gel formed a hard outer layer covering the soft porous gel inside. By controlling the rate and frequency of broth circulation between the riser and downcomer columns, the riser could be maintained under aerobic condition while the downcomer was under anaerobic condition. Repeated fed-batch l-lactic acid production was performed for more than 400 h and the average lactic acid yield and productivity from raw cassava starch were 0.76 g lactic acid g^–1 starch and 1.6 g lactic acid l^–1 h^–1, respectively.     

Hollow fibre bioreactor for ethanol production: Application to the conversion of lactose by Kluyveromyces fragilis

Kluyveromyces fragilis cells have been packed into the shell side of an industrial size hollow fibre module. The feed was pumped through the tube side under pressure. During continuous, single-pass operation with a synthetic lactose medium containing 50 g l^?1lactose, ethanol productivity was 30–60 g l^?1h^?1at dilution rates of 1–4 h^?1. With 150 g l^?1lactose concentration, the productivity was 100–135 g l^?1h^?1. Productivity was generally lower when cottage cheese whey permeate (45 g l^?1lactose) was used as the feed. Long-term stability of the hollow fibre bioreactor was good, provided adequate care was taken to bleed the gas generated and restrict cell concentration in the shell side.     

Optimization of lactic acid production by immobilized <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1

Production of lactic acid from glucose by immobilized cells of Lactococcus lactis IO-1 was investigated using cells that had been immobilized by either entrapment in beads of alginate or encapsulation in microcapsules of alginate membrane. The fermentation process was optimized in shake flasks using the Taguchi method and then further assessed in a production bioreactor. The bioreactor consisted of a packed bed of immobilized cells and its operation involved recycling of the broth through the bed. Both batch and continuous modes of operation of the reactor were investigated. Microencapsulation proved to be the better method of immobilization. For microencapsulated cells at immobilized cell concentration of 5.3 g l⁻¹, the optimal production medium had the following initial concentrations of nutrients (g l⁻¹): glucose 45, yeast extract 10, beef extract 10, peptone 7.5 and calcium chloride 10 at an initial pH of 6.85. Under these conditions, at 37 °C, the volumetric productivity of lactic acid in shake flasks was 1.8 g l⁻¹ h⁻¹. Use of a packed bed of encapsulated cells with recycle of the broth through the bed, increased the volumetric productivity to 4.5 g l⁻¹ h⁻¹. The packed bed could be used in repeated batch runs to produce lactic acid.     

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.     

Production of δ-decalactone from linoleic acid via 13-hydroxy-9(Z)-octadecenoic acid intermediate by one-pot reaction using linoleate 13-hydratase and whole <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> cells

Objective

To produce δ-decalactone from linoleic acid by one-pot reaction using linoleate 13-hydratase with supplementation with whole Yarrowia lipolytica cells.

Results

Whole Y. lipolytica cells at 25 g l^?1 produced1.9 g l^?1 δ-decalactone from 7.5 g 13-hydroxy-9(Z)-octadecenoic acid l^?1 at pH 7.5 and 30 °C for 21 h. Linoleate 13-hydratase from Lactobacillus acidophilus at 3.5 g l^?1 with supplementation with 25 g Y. lipolytica cells l^?1 in one pot at 3 h produced 1.9 g l^?1 δ-decalactone from 10 g linoleic acid l^?1 via 13-hydroxy-9(Z)-octadecenoic acid intermediate at pH 7.5 and 30°C after 18 h, with a molar conversion yield of 31 % and productivity of 106 mg l^?1 h^?1.

Conclusion

To the best of our knowledge, this is the first production of δ-decalactone using unsaturated fatty acid.

     

Spirit-based distillers’ grain as a promising raw material for succinic acid production

Spirit-based distillers’ grain (SDG) is the main by-product of the Chinese liquor industry, with an annual output of approx. 100 million tons. The economical potential of fermentative production of succinic acid from SDG was investigated using Actinobacillus succinogens. Use of pretreated SDG (PSDG) as the sole source of C and N yielded succinic acid at 35.5 g l^?1 with a yield of 19.7 % (g per 100 g PSDG) after 48 h in a 3 l stirred bioreactor. SDG is thus a promising feedstock for the economical production of succinic acid.     

Production of optically pure <Emphasis Type="SmallCaps">l</Emphasis>(+)-lactic acid from waste plywood chips using an isolated thermotolerant <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> SI at a pilot scale

Utilization of renewable and low-cost lignocellulosic wastes has received major focus in industrial lactic acid production. The use of high solid loadings in biomass pretreatment potentially offers advantages over low solid loadings including higher lactic acid concentration with decreased production and capital costs. In this study, an isolated Enterococcus faecalis SI with optimal temperature 42 °C was used to produce optically pure l-lactic acid (>?99%) from enzyme-saccharified hydrolysates of acid-impregnated steam explosion (AISE)-treated plywood chips. The l-lactic acid production increased by 10% at 5 L scale compared to the similar fermentation scheme reported by Wee et al. The fermentation with a high solid loading of 20% and 35% (w/v) AISE-pretreated plywood chips had been successfully scaled up to process development unit scale (100 L) and pilot scale (9 m³), respectively. This is the first report of pilot-scale lignocellulosic lactic acid fermentation by E. faecalis with high lactic acid titer (nearly 92 g L^?1) and yield (0.97 kg kg^?1). Therefore, large-scale l-lactic acid production by E. faecalis SI shows the potential application for industries.     

Micropropagation of Cymbidium sinense using continuous and temporary airlift bioreactor systems

Airlift bioreactors were programmed for continuous and temporary immersion culture to investigate factors that affect the rhizome proliferation, shoot formation, and plantlet regeneration of Cymbidium sinense. During rhizome proliferation, the continuous immersion bioreactor system was used to explore the effects of activated charcoal (AC) in the culture medium, inoculation density, and air volume on rhizome differentiation and growth. The optimum conditions for obtaining massive health rhizomes were 0.3 g l^?1 AC in the culture medium, 7.5 g l^?1 inoculation density, and 150 ml min^?1 air. In addition, the temporary immersion bioreactor system was used for both shoot formation and plantlet regeneration. Supplementing 4 mg l^?1 6-benzylaminopurine and 0.2 mg l^?1 naphthalene acetic acid (NAA) to the culture medium promoted shoot induction from the rhizome. Cutting the rhizome explants into 1 cm segments was better for massive shoot formation than cutting into 0.25 and 0.5 cm explant segments. NAA promoted plantlet regeneration and the rooting rate (94.7 %), with whole plantlets growing well in culture medium containing 1.0 mg l^?1 NAA. Therefore, applying bioreactors in C. sinense micropropagation is an efficient way for scaling up the production of propagules and whole plantlets for the industrial production of high-quality seedlings.