Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Efficient production by Escherichia coli of recombinant protein using salting-out effect protecting against proteolytic degradation

To produce glucoamylase efficiently as a recombinant protein, E. coli was grown with 20 g (NH₄)₂SO₄ l^–1 which removed proteolytic activity but did not effect cell growth. Growth in M9 medium with 20 g (NH₄)₂SO₄ l^–1 produced 11 U glucoamylase ml^–1 compared to 7 U ml^–1 without addition. Furthermore, the glucoamylase activity was maintained at about 9 U ml^–1.     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Arrowroot (Marantha arundinacea) starch as a new low-cost substrate for alkaline protease production

The feasibility of arrowroot (Marantha arundinacea) starch for alkaline protease production using an alkalophilic Bacillus lentus isolate was evaluated in batch fermentations in shake flasks and in a bioreactor under a range of conditions. A new arrowroot starch-casein medium (pH 10.2) was formulated having a composition (%, w/v): arrowroot starch 1, casein 1, sodium succinate 0.25, NH₄Cl 0.05, NaCl 0.05, KH₂PO₄ 0.04, K₂HPO₄ 0.03, MgCl₂ 0.01, yeast extract 0.01 and Na₂CO₃ 1.05. The isolate produced a maximum protease yield (6754.7 U ml^–1) in this medium when grown for 72 h at 250 rev/min and 37 °C. Scaling-up studies in a bioreactor showed a 5-fold increase in alkaline protease yields (31899 U ml^–1) at a lower production time of 45 h, aeration of 1 v/v/m and agitation of 400 rev/min at 37 °C.     

Production of S-adenosyl- L-methionine by a mutant strain of Kluyveromyces lactis

S-Adenosyl-l-methionine (AdoMet) was produced by a mutant strain Kluyveromyces lactis AM-65 grown on whey. A full factorial design method of three factors – (NH₄)₂SO₄ (factor x ₁), corn steep liquor (factor x ₂) and l-methionine (factor x ₃) on three levels – was used to determine the optimal medium conditions for the production of AdoMet. A time course shake-flask experiment in optimal whey medium (x ₁=3.1 g l^–1, x ₂=12.7 g l^–1, x ₃=4.6 g l^–1) was also carried out and the results confirmed the results of the factorial design and subsequent quadratic modelling and optimization of AdoMet production which reached 90 mg g^–1 cell dry wt.     

Mathematical description of bikaverin production in a fluidized bed bioreactor

Summary  Growth of Gibberella fujikuroi in submerged cultures occurs as micelles or filamentous hyphae dispersed in fluid and pellets or stable, spherical agglomerations. Gibberella fujikuroi growth, substrate consumption and bikaverin production kinetics obtained from submerged batch fermentation were fitted to three different sigmoid models: two and three-parameter Gompertz models and one Logistic model. Growth fitting was used to compare between models and select the best one by means of an F test. The best model for describing growth was the two-parameter Gompertz model and was used for glucose consumption and bikaverin production fitting. Data from eight different schemes of fermentations were analysed and parameter estimation was carried out by means of minimization of residual sum of squares. Some characteristic values obtained with the two-parameter Gompertz model fit are: μ=0.028 h⁻¹, Y_x/s=0.1089 g substrate/g biomass, α =0.1384 g product/g biomass.     

Biooxidation of ferrous iron by immobilized Acidithiobacillus ferrooxidans in poly(vinyl alcohol) cryogel carriers

PVA-cryogels entrapping about 10⁹ cells of Acidithiobacillus ferrooxidans per ml of gel were prepared by freezing-thawing procedure, and the biooxidation of Fe²⁺ by immobilized cells was investigated in a 0.365 l packed-bed bioreactor. Fe²⁺ oxidation fits a plug-flow reaction model well. A maximum oxidation rate of 3.1 g Fe²⁺ l^–1 h^–1 was achieved at the dilution rate of 0.4 h^–1 or higher, while no obvious precipitate was determined at this time. In addition, cell-immobilized PVA-cryogels packed in bioreactor maintained their oxidative ability for more than two months under non-sterile conditions. Nomenclature: C _A0 – Concentration of Fe²⁺ in feed stream (g l^–1) C _A – Concentration of Fe^{2 +} in outlet stream (g l^{– 1}) D – Dilution rate of the packed-bed bioreactor (h^–1) F – Volumetric flow rate of iron solution (l h^–1) F _A0 – Mass flow rate of Fe²⁺ in the feed stream (g h^–1) K – Kinetic constant (l l^–1 h^–1) r _A – Oxidation rate of Fe²⁺ (g l^–1 h^–1) V – Volume of packed-bed bioreactor (l) X _A – Conversion ratio of Fe²⁺ (%)     

Glucose oxidase and catalase activities ofPenicillium variabile P16 immobilized in polyurethane sponge

Conidia ofPenicillium variabile P16 were immobilized in polyurethane sponge and used in repeated-batch processes in a fluidized-bed reactor. Optimal conditions for production of glucose oxidase and catalase were: inoculum size, 10%; glucose concentration, 80 g L^–1; Ca-carbonate concentration, 15 g L^–1; temperature, 28°C and aeration rate, 4 VV^–1 min^–1. In an extended repeated-batch process, glucose oxidase activity was highest after the fourth batch and catalase activity was highest after the fifth batch. Scanning electron microscopy showed that the fungus grew only in the interior of carrier particles.     

Indigo production in hairy root cultures of Polygonum tinctorium Lour.

The transformed root culture of Polygonum tinctorium Lour. was established by infecting leaf explants with Agrobacterium rhizogenes A4. These cultures were examined for their growth and indigo content under various culture conditions. Among the four different culture media tested, SH medium showed the highest yield for root growth (28 mg dry wt/30 ml) and indigo production (152 g/dry wt). In SH medium, 30 g sucrose l^–1, 2500 mg KNO₃ l^–1, 300 mg NH₄H₂PO₄ l^–1 were the best conditions for indigo production at pH 5.7. The production of indigo in hairy roots slightly increased with the addition of 200 mg chitosan l^–1 (186 g/dry wt) and 20 U pectinase l^–1 (181 g/dry wt).     

Biotransformation of oleic acid into 10-hydroxy-8E-octadecenoic acid by Pseudomonas sp. 42A2

Pseudomonas sp. 42A2 when incubated for 36 h with oleic acid (20 g l^–1) in a stirred bioreactor, accumulated 10-hydroxy-8E-octadecenoic acid. Production in a 2 l bioreactor with 1.4 l of working volume, was increased from 0.65 g l^–1 to 7.4 g l^–1 with K _L a values ranging between 15 and 200 h^–1. A linear relationship was found between volumetric productivity and oxygen transfer rates and an exponential relation between the specific rate of product formation and specific growth rate.     

Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations

Addition of 40 g NaCl l^–1 to a chemically defined medium containing 140 g glucose l^–1 in shake-flask culture improved glycerol production by Candida krusei from 16.5 g l^–1 to 47.7 g l^–1. With 40 g NaCl l^–1 at a dilution rate of 0.065 h^–1, glycerol concentration, glycerol yield (based on glucose consumed), and productivity in a four-stage cascade bioreactor were higher by 240%, 27% and 28%, respectively, than in a single-stage continuous culture system.     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.     

Removal of inorganic nitrogen sources from water by the algal biofilm of the aerial microalga Trentepohlia aurea

Removal of inorganic nitrogen sources by cells of the aerial microalga Trentepohlia aurea grown on the surface of substrate, such as filter paper, has been investigated in a batch system. When the alga grew on the paper dampened with medium, it actively ingested inorganic nitrogenous compounds in the medium. Immobilized cells on the filter papers were called algal biofilm in this study. When the algal biofilms were soaked in modified Bold's Basal medium (using 1 g NH₄Cl l^–1 as a N source), the removal rate was 4.25 mg ammonium-N l^–1 day^–1 in 40 days. In modified medium with added 26 mg nitrite-N, the removal rate of the total inorganic N ion by the biofilms reached 5.11 mg N l^–1 day^–1. This removal rate of total N ion was higher than that in the medium by addition of 26 mg nitrate-N. In addition, we tried to examine simultaneous removal of ammonium, nitrate, and nitrite ions and growth inhibition of cyanobacteria in the medium by using the algal biofilms. Consequently, it was demonstrated that the algal biofilms of T. aurea could be utilized as a biofunctional material for the purification of wastewater.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

High density cell culture of Panax notoginseng for production of ginseng saponin and polysaccharide in an airlift bioreactor

High cell density of Panax notoginseng in a 17 l airlift bioreactor was achieved in batch cultivation using a modified MS medium. The dry cell weight, ginseng saponin and polysaccharide reached 24, 1.7 and 2.8 g l^–1, respectively, after 15 d. A strategy of sucrose feeding based on changes in the specific O₂ uptake rate was applied to the cell cultures, which increased these respective yields to 30, 2.3 and 3.2 g l^–1.     

Poly(3-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> in fed-batch culture

High poly(3-hydroxybutyrate) (PHB) content and volumetric productivity were achieved by fed-batch culture of Halomonas boliviensis using a defined medium. Initial shake flask cultivations in a minimal medium revealed that the growth of H. boliviensis was supported only when the medium was supplemented with aspartic acid, glycine, or glutamine. Addition of 0.1% (w/v) glutamine in the medium resulted in the highest cell dry weight (CDW; 3.9 g l⁻¹). Glutamine was replaced by the less expensive monosodium glutamate (MSG) in the medium without any notable change in the final cell density. Effect of initial concentrations of NH₄Cl and K₂HPO₄ on cell growth and PHB accumulation by H. boliviensis was then analyzed using a fed-batch fermentation system. The best conditions for PHB production by H. boliviensis were attained using 0.4% (w/v) NH₄Cl and 0.22% (w/v) K₂HPO₄ and adding MSG intermittently to the fermentor. Poly(3-hydroxybutyrate) content and CDW reached 90 wt.% and 23 g l⁻¹, respectively, after 18 h of cultivation. In order to increase CDW and PHB content, MSG, NH₄Cl, and K₂HPO₄ were initially fed to the fermentor to maintain their concentrations at 2%, 0.4%, and 0.22% (w/v), respectively, and subsequently their feed was suppressed. This resulted in a CDW of 44 g l⁻¹, PHB content of 81 wt.%, and PHB volumetric productivity of 1.1 g l⁻¹ h⁻¹.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Reducing by-product formation in L-lactic acid fermentation by Rhizopus oryzae

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l^–1 to 0.6 g l^–1 KH₂PO₄ reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l^–1 to 71 g l^–1 and from 1.36 g l^–1 to 0.18 g l^–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l^–1 KH₂PO₄ was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.     

Influence of medium composition and aeration on the synthesis of biosurfactants produced by Candida antarctica

Candida antarctica synthesised surface-active mannosylerythritol lipids at 46 g l^–1 by adding 80 g soybean oil l^–1 to the medium and maintaining the pO₂ at 50% with an air flow rate 1 vvm. Two-stage culturing of C. antarctica avoided medium foaming but the yield of biosurfactants synthesis was 28 g l^–1. The biosurfactants decreased the surface tension of water to 35 mN m^–1.     

Mixotrophic growth ofChlorella sorokiniana in outdoor enclosed photobioreactor

Chlorella sorokiniana was cultured in heterotrophic or mixotrophic mode in outdoor enclosed tubular photobioreactor. The culture temperature was maintained at 32–35 °C. At night, theChlorella culture grew heterotrophically, and 0.1 M glucose was completely consumed. The biomass growth yield of glucose was 0.35 ± 0.001 g-biomass g-glucose^–1. During the day, the algal culture grew mixotrophically and the biomass growth yield was 0.49 g-biomass g-glucose^–1 in low density culture (initial biomass concentration, X_o = 2 g l^–1), 0.56 g-biomass g-glucose^–1 in medium density culture (X_o = 4 g l^–1) and 0.46 g-biomass g-glucose^–1 in high density culture (X_o = 7 g l^–1). The daily area productivity of the culture, with X_o = 4 g l^–1 corresponded to 127 g-biomass m^–2 d^–1 during the day and 79 g-biomass m^–2 d^–1 during the night. In all the cultures, the dissolved O₂ concentration increased in the morning, reached the maximum value at noon, and then decreased in the afternoon. The dissolved CO₂ concentration remained at 3 mBar in the morning and increased in the afternoon. Glycolate was not found to accumulate in culture medium.