Lactic acid production from cellulosic waste by immobilized cells of Lactobacillus delbrueckii

Industrial waste corn cob residue (from xylose manufacturing) without pretreatment was hydrolyzed by cellulase and cellobiase. The cellulosic hydrolysate contained 52.4 g l⁻¹ of glucose and was used as carbon source for lactic acid fermentation by cells of Lactobacillus delbrueckii ZU-S2 immobilized in calcium alginate gel beads. The final concentration of lactic acid and the yield of lactic acid from glucose were 48.7 g l⁻¹ and 95.2%, respectively, which were comparative to the results of pure glucose fermentation. The immobilized cells were quite stable and reusable, and the average yield of lactic acid from glucose in the hydrolysate was 95.0% in 12 repeated batches of fermentation. The suitable dilution rate of continuous fermentation process was 0.13 h⁻¹, and the yield of lactic acid from glucose and the productivity were 92.4% and 5.746 g l⁻¹ h⁻¹, respectively. The production of lactic acid by simultaneous saccharification and fermentation (SSF) process was carried out in a coupling bioreactor, the final concentration of lactic acid was 55.6 g l⁻¹, the conversion efficiency of lactic acid from cellulose was 91.3% and the productivity was 0.927 g l⁻¹ h⁻¹. By using fed-batch technique in the SSF process, the final concentration of lactic acid and the productivity increased to 107.6 g l⁻¹ and 1.345 g l⁻¹ h⁻¹, respectively, while the dosage of cellulase per gram substrate decreased greatly. This research work should advance the bioconversion of renewable cellulosic resources and reduce environmental pollution.     

Hydantoinase activity of immobilized non-growing Pseudomonas putida cells

Summary Hydantoinase (dihydropyrimidinase E.C. 3.5.2.2) activity of Pseudomonas putida DSM 84 was evaluated using cells immobilized in alginate beads and in a microporous hollow fibre bioreactor. Conversion of dihydrouracil into N-carbamyl--alanine was most efficient with alginate-immobilized cells. A 40 to 45% conversion was obtained in shake flasks and in continuous mode with packed bed columns. The highest volumetric productivity was obtained with a packed bed column operated at a dilution rate of 0.5 h^-1 (99 g of product. 100 l^-1 per hour). After 96 h the alginate beads began to swell and break apart; no free cells were detected however. Despite some initial loss of cells from the microporous hollow fibre bioreactor, a steady state was later established and maintained for 400 h at dilution rates of 0.1 and 0.25 h^-1.     

Production of ascorbic acid glucoside by alginate-entrapped mycelia of <Emphasis Type="Italic">Aspergillus niger</Emphasis>

The mycelia of Aspergillus niger, cultivated in a medium containing 45 g l⁻¹ maltose, 66 g l⁻¹ yeast extract, and 5 g l⁻¹ K₂HPO₄ at 30°C and 200 rpm, were used as a biocatalyst in the glucosylation of ascorbic acid. Free mycelia from 3-day-old culture, when used in a 6-h reaction with maltose as the acyl donor, gave 16.07 g l⁻¹ ascorbic acid glucoside corresponding to a volumetric productivity of 2.68 g l⁻¹ h⁻¹ and a conversion of 67%. Mycelia from 3-day-old cultures were entrapped in calcium alginate beads and used as a catalyst in the glucosylation of ascorbic acid. An ascorbic acid-to-maltose molar ratio of 1:9 was found to be optimum, and the conversion reached 75% after 12 h. The concentration of ascorbic acid glucoside produced at this molar ratio was 17.95 g l⁻¹, and the productivity was 1.5 g l⁻¹ h⁻¹. The biocatalyst was repeatedly used in a fixed bed bioreactor for the synthesis of ascorbic acid glucoside and approximately 17 g l⁻¹ of ascorbic acid glucoside corresponding to a volumetric productivity of 1.42 g l⁻¹ h⁻¹ was produced in each use. The conversion was retained at 70% in each use. The entrapped mycelia also exhibited exceptionally high reusability and storage stability. The product was purified to 85% by anion exchange and gel permeation chromatography with a final yield of 75%.     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Improved guggulsterone production from sugars, precursors, and morphactin in cell cultures of Commiphora wightii grown in shake flasks and a bioreactor

Cell cultures of Commiphora wightii (Arnott.) Bhandari were grown in shake flasks and a bioreactor and an increase in guggulsterone accumulation up to 18 μg l⁻¹ was recorded in cells grown in the production medium containing a combination of sucrose:glucose (4% total), precursors (phenylalanine, pyruvic acid, xylose, and sodium acetate), morphactin, and 2iP. A yield of 10 g l⁻¹ biomass and ∼200 μg l⁻¹ guggulsterone was recorded in a 3-l flask and in a 2-l stirred tank bioreactor compared with 6.6 g biomass and 67 μg l⁻¹ guggulsterone in 250-ml flasks. Increased vessel size was correlated with increased biomass and guggulsterone accumulation. 2iP alone was not effective for biomass and guggulsterone accumulation in cell cultures of C. wightii.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Improved production of biosurfactant by a <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> mutant using vegetable oil refinery wastes

Biosurfactant production by Pseudomonas aeruginosa EBN-8 mutant was studied in shake flasks on separate wastes from canola, soybean and corn oil refineries. Of the substrates tested, canola oil refinery waste (COD=20 g l⁻¹) supplemented with sodium nitrate (at COD/N=20) showed the best microbial growth (4.50 g l⁻¹) and rhamnolipid production (8.50 g l⁻¹), at 10 d of incubation with the specific growth rate of 0.316 h⁻¹ and specific product yield of 0.597 g g⁻¹ h. Its cell-free supernatant showed the critical micelle dilution (CMD) of 150 and surface tension (ST) of 28.5 mN m⁻¹.     

Production of 1,3-Propanediol by Clostridium butyricum VPI 3266 in continuous cultures with high yield and productivity

The effects of dilution rate and substrate feed concentration on continuous glycerol fermentation by Clostridium butyricum VPI 3266, a natural 1,3-propanediol producer, were evaluated in this work. A high and constant 1,3-propanediol yield (around 0.65 mol/mol), close to the theoretical value, was obtained irrespective of substrate feed concentration or dilution rate. Improvement of 1,3-propanediol volumetric productivity was achieved by increasing the dilution rate, at a fixed feed substrate concentration of 30, 60 or 70 g l⁻¹. Higher 1,3-propanediol final concentrations and volumetric productivities were also obtained when glycerol feed concentration was increased from 30 to 60 g l⁻¹, at D=0.05–0.3 h⁻¹, and from 60–70 g l⁻¹, at D=0.05 and 0.1 h⁻¹·30 g l⁻¹ of 1,3-propanediol and the highest reported value of productivity, 10.3 g l⁻¹ h⁻¹, was achieved at D=0.30 h⁻¹ and 60 g l⁻¹ of feed glycerol. A switch to an acetate/butyrate ratio higher than one was observed for 60 g l⁻¹ of feed glycerol and a dilution rate higher than 0.10 h⁻¹; moreover, at D=0.30 h⁻¹ 3-hydroxypropionaldehyde accumulation was observed for the first time in the fermentation broth of C. butyricum.     

Factors affecting the production of <Emphasis Type="SmallCaps">l</Emphasis>-xylulose by resting cells of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l⁻¹. A specific productivity of 1.09 ± 0.10 g g⁻¹ h⁻¹ was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the operation of the process in a bioreactor with reasonable accuracy.     

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.     

Production of nisin Z using <Emphasis Type="Italic">Lactococcus lactis</Emphasis> IO-1 from hydrolyzed sago starch

A membrane bioreactor for production of nisin Z was constructed using Lactococcus lactis IO-1 in continuous culture using hydrolyzed sago starch as carbon source. A strategy used to enhance the productivity of nisin Z was to maintain the cells in a continuous growth at high cell concentration. This resulted in a volumetric productivity of nisin Z, as 50,000 IU l⁻¹ h⁻¹ using a cell concentration of 15 g l⁻¹, 30^°C, pH 5.5 and a dilution rate of 1.24 h⁻¹. Adding 10 g l⁻¹ YE and 2 g l⁻¹ polypeptone, other inducers were unnecessary to maintain production of nisin. The operating conditions of the reactor removed nisin and lactate, thus minimizing their effects which allowed the maintenance of cells in continuous exponential growth phase mode with high metabolic activity.     

Taxane Production in Suspension Culture of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">Media</Emphasis> var. <Emphasis Type="Italic">Hicksii</Emphasis> Carried Out in Flasks and Bioreactor

Paclitaxel and 10-deacetylbaccatin III (10-DAB III) were produced in suspension cultures of Taxus × media var. Hicksii grown in shake-flasks and in a 7-l bioreactor reaching, in the bioreactor, 4.4 mg l⁻¹ (on day 14) and 37.5 mg l⁻¹ (on day 11). In shake-flasks the highest total content of paclitaxel and 10-DAB III was 7.3 mg l⁻¹ (on day 4) and 8.8 mg l⁻¹ (on day 18). Phenylalanine, at 0.05 mM, increased paclitaxel accumulation in cells cultivated in bioreactor and flasks 30-fold and 9-fold (from 0.02 mg l⁻¹ to 0.6 mg l⁻¹ and to 0.2 mg l⁻¹, respectively). The 10-DAB III content in cells from flasks was increased from 0.4 mg l⁻¹ to 1.6 mg l⁻¹.     

β-Galactosidase from Talaromyces thermophilus immobilized on to Eupergit C for production of galacto-oligosaccharides during lactose hydrolysis in batch and packed-bed reactor

The β-galactosidase from Talaromyces thermophilus CBS 236.58 immobilized onto Eupergit C produced galacto-oligosaccharides (GalOS) in batchwise and continuous packed-bed mode of operation. A maximum yield of GalOS of 12, 39 and 80 g l⁻¹ was obtained for initial lactose concentrations of 50, 100 and 200 g l⁻¹, respectively, for batch conversion experiments. The immobilized enzyme could be re-used for several cycles for lactose hydrolysis and transformation. The maximum GalOS concentration of approximately 50 g l⁻¹ was obtained with the dilution rate of 0.375 h⁻¹ in a packed-bed reactor, when using an initial lactose concentration of 200 g l⁻¹. Continuous conversion of lactose in the packed-bed reactor resulted in the formation of relatively more trisaccharides than when employing the immobilized enzyme in discontinuous mode of operation.     

Repeated fed-batch lactic acid production in a packed bed-stirred fermentor system using a pH feedback feeding method

Lactic acid production by repeated fed-batch fermentation using free and immobilized cells of Lactobacillus lactis-11 in a packed bed-stirred fermentor (PBSF) system filled with different support materials including ceramic beads, macro-activated carbon cylinders and glass fiber balls was investigated. The results showed that the optimal support materials were the ceramic beads with diameters of 1–2 mm. Compared with the free cell fermentation system, lactic acid production and volumetric productivity in the PBSF system increased by 16.6 and 12.5%, respectively. Though the concentration of free cells decreased sharply, lactic acid production remained stable in five consecutive fed-batch runs using the PBSF system. pH gradients, immobilized cell concentration and mass diffusion in the packed bed were all affected by the recirculation rate of the culture broth. Maximum lactic acid production, productivity and yield occurred at a recirculation rate of 50 mL min⁻¹.     

Process optimization of constitutive human granulocyte–macrophage colony-stimulating factor (hGM-CSF) expression in Pichia pastoris fed-batch culture

Human granulocyte–macrophage colony-stimulating factor (hGM-CSF) is a therapeutically important cytokine that is poorly expressed because of its toxic effects on the host cells. Extracellular expression of hGM-CSF was obtained by cloning its gene in Pichia pastoris under the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter with an N-terminal α peptide sequence for its extracellular production. The clones obtained were screened for a hyper producer following which media and cultivation conditions were optimized in shake flasks. Batch and fed-batch studies were performed in a bioreactor where different feed compositions were fed exponentially to obtain high biomass concentrations. Feeding of complex media allowed us to maintain a high specific growth rate of 0.2 h⁻¹ for the longest time period, and a final biomass of 98 g DCW/l was obtained in 34 h. Product formation was found to be growth associated, and the product yield with respect to biomass (Y _P/X) was ∼2.5 mg/g DCW. The above fed-batch strategy allowed us to obtain fairly pure glycosylated hGM-CSF at a final product concentration of 250 mg/l in the culture supernatant with a high volumetric productivity of 7.35 mg l⁻¹ h⁻¹.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.     

Mineralization of diuron [3-(3,4-dichlorophenyl)-1, 1-dimethylurea] by co-immobilized <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. and <Emphasis Type="Italic">Delftia acidovorans</Emphasis>

Mineralization of diuron has not been previously demonstrated despite the availability of some bacteria to degrade diuron into 3,4-dichloroaniline (3,4-DCA) and others that can mineralize 3,4-DCA. A bacterial co-culture of Arthrobacter sp. N4 and Delftia acidovorans W34, which respectively degraded diuron (20 mg l⁻¹) to 3,4-DCA and mineralized 3,4-DCA, were able to mineralize diuron. Total diuron mineralization (20 mg l⁻¹) was achieved with free cells in co-culture. When the bacteria were immobilized (either one bacteria or both), the degradation rate was higher. Best results were obtained with free Arthrobacter sp. N4 cells co-cultivated with immobilized cells of D. acidovorans W34 (mineralization of diuron in 96 h, i.e., 0.21 mg l⁻¹ h⁻¹ vs. 0.06 mg l⁻¹ h⁻¹ with free cells in co-culture).     

Production of 1,3-propanediol by Klebsiella pneumoniae from glycerol broth

Broth containing 152 g glycerol l⁻¹ from Candida krusei culture was converted to 1,3-propanediol by Klebsiella pneumoniae. Residual glucose in the broth promoted growth of K. pneumoniae while acetate was inhibitory. After desalination treatment of glycerol broth by electrodialysis, the acetate in the broth was removed. A fed-batch culture with electrodialytically pretreated broth as␣substrate was developed giving 53 g 1,3- propanediol l⁻¹ with a yield of 0.41 g g⁻¹ glycerol and a productivity of 0.94 g l⁻¹ h⁻¹.     

Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici

Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose. A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C. This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l^–1 h^–1). The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l^–1 h^–1 of at 30 °C, the optimal temperature. Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.     

Improved production of pristinamycin coupled with an adsorbent resin in fermentation by Streptomyces pristinaespiralis

Batch fermentation by Streptomyces pristinaespiralis with the addition of adsorbent resins was used to increase the production of pristinamycin. In consideration of the adsorption capacity and the desorption ability, a polymeric resin, JD-1, was finally selected. The maximum production of pristinamycin in Erlenmeyer flasks went up to 1.13 from 0.4 g l⁻¹, by adding 12% (w/v) resin JD-1 into the culture broth at 20 h after inoculation. In a 3 l bioreactor, pristinamycin fermentation with the addition of 12% (w/v) resin JD-1 at 20 h after inoculation reached 0.8 g l⁻¹, which was a 1.25-fold increase over fermentation without resin.