Production of Alcaligenes xylosoxydans EMS33 in a Bench-scale Fermenter

Summary Optimization of medium composition and pH for chitinase production by the Alcaligenes xylosoxydans mutant EMS33 was carried out in the present study and the optimized medium composition and conditions were evaluated in a fermenter. The medium components screened initially using Plackett–Burman design were (NH₄)₂SO₄, MgSO₄ 7H₂O, KH₂PO₄, yeast extract, Tween 20 and chitin in shake flask experiments. The significant medium components identified by the Plackett–Burman method were MgSO₄ 7H₂O, Tween 20 and chitin. Central composite response surface methodology was applied to further optimize chitinase production. The optimized values of MgSO₄ 7H₂O, Tween 20, chitin and pH were found to be 0.6 g/l, 0.05 g/l, 11.5 g/l and 8.0, respectively. Chitinase and biomass production of Alcaligenes xylosoxydans EMS33, was studied in a 2-l fermenter containing (g/l): chitin, 11.5; yeast extract, 0.5; (NH₄)₂SO₄, 1; MgSO₄ 7H₂O, 0.6; KH₂PO₄, 1.36 and Tween 20, 0.05. The highest chitinase production was 54 units/ml at 60 h and pH 8.0 when the dissolved O₂ concentration was 60%, whereas the highest biomass production was achieved at 36 h and pH 7.5 without any dissolved O₂ control.     

Application of statistically-based experimental designs for the optimization of nisin production from whey

Statistically-based experimental designs were applied for the optimization of nisin production by Lactococcus lactis in a whey-based medium. Yeast extract, KH₂PO₄, and MgSO₄ were identified to have significant effects on nisin biosynthesis by a Plackett–Burman design. These three significant factors were subsequently optimized using central composite design, and the optimal conditions were determined to be 12.067 g l^–1 for yeast extract, 0.569 g l^–1 for KH₂PO₄, and 0.572 g l^–1 for MgSO₄. The validity of the optimal conditions was verified by a separate experiment.     

Use of response surface methodology to study the effect of media composition on aflatoxin production by Aspergillus flavus

Aflatoxins are one of the most important secondary metabolites. These extrolites are produced by a number of Aspergillus fungi. In this study, we demonstrate the effect of media components and enhanced aflatoxin yield shown by A. flavus using response surface methodology in response to different nutrients. Different components of a chemically defined media that influence the aflatoxin production were monitored using Plackett–Burman experimental design and further optimized by Box–Behnken factorial design of response surface methodology in liquid culture. Interactions were studied with five variables, namely sorbitol, fructose, ammonium sulfate, KH₂PO₄, and MgSO₄.7H₂O. Maximum aflatoxin production was envisaged in medium containing 4.94 g/l sorbitol, 5.56 g/l fructose, 0.62 g/l ammonium sulfate, 1.33 g/l KH₂PO₄, and 0.65 g/l MgSO₄·7H₂O using response surface plots and the point prediction tool of the DESIGN EXPERT 8.1.0 (Stat-Ease, USA) software. However, a production of 5.25 μg/ml aflatoxin production was obtained, which was in agreement with the prediction observed in verification experiment. The other component (MgSO₄.7H₂O) was found to be an insignificant variable.     

Production,Purification and Properties of Ribonuclease from Aspergillus niger

Aspergillus niger NRC–A–1–233 was cultivated by the shaking method. The optimal cultural conditions for ribonuclease (RNase) production were: composition of medium: sucrose, 15%; NH₄NO₃, 0.2%; KH₂PO₄, 0.1%; MgSO₄·7 aq., 0.025%; initial pH, 2.2; shaking conditions: 50 ml of medium /500 ml flask; cultivation time, 120 hr. The RNase was purified by acid clay treatment and chromatography on DEAE-cellulose and Sephadex G–75 columns. The purified RNase was homogeneous by ultracentrifuge and disc electrophoresis.

The molecular weight of the RNase was estimated to be 28,500 on SDS-polyacrylamide gel and its isoelectric point was 2.8 by Ampholine electrofocusing method. Digestion rate of RNA by the RNase was 100%. The RNase did not have an exact base specificity and produced four kinds of 3′-nucleotides from yeast RNA.     

Statistical optimization of medium components for improved synthesis of riboflavin by Eremothecium ashbyii

Statistical designs were used to determine optimum levels of medium nutrients for riboflavin production in shake flask fermentation. The medium components considered include molasses, sesame seed cake (SSC), yeast extract, KH₂PO₄, MgSO₄·7H₂O, NaCl and Tween-80. Information about the effects of different medium components on riboflavin production were investigated by using the Plackett-Burman experimental design. MgSO₄·7H₂O and NaCl were found to significantly influence the riboflavin production (confidence levels above 70%). For obtaining the mutual interaction between the variables required, for achieving the optimal concentrations, a 2²-factorial central composite design was employed. The optimal concentrations for the enhanced production of riboflavin were (g/l): molasses =50.0 (glucose equivalents); SSC =50.0; yeast extract =2.0; KH₂PO₄=2.0; MgSO₄·7H₂O=0.117 and NaCl =1.13.     

Growth and physiological characteristics of wasabi plantlets cultured by photoautotrophic micropropagation at different temperatures

Iranian seedless barberry is a very recalcitrant species in in vitro culture which does not show appropriate growth on standard culture media. Response surface methodology was employed to evaluate the effects of changing macronutrients concentrations on establishment and proliferation phases. KNO₃ and NH₄NO₃ macronutrients at 0.3 to 1.5?×?MS medium levels and CaCl₂, MgSO₄ and KH₂PO₄ macronutrients in a range of 0.5 to 1.5?×?MS medium concentrations were tested in a response surface design with 30 treatments. Many significant interactions were found among the macronutrients. High concentrations of KNO₃, NH₄NO₃ and CaCl₂ improved the growth rate in the establishment phase. The growth rate in media containing high KNO₃ and low CaCl₂ was high. Reduced concentrations of CaCl₂ and KNO₃ decreased hyperhydricity. The greatest hyperhydricity was induced when both NH₄NO₃ and CaCl₂ were used at 1.5?×?MS level. The number of hooked leaves decreased as KH₂PO₄ increased and MgSO₄ reduced. In the proliferation phase, there were many significant interactions among the macronutrients. Increased concentration of NH₄NO₃ and reduced concentration of KH₂PO₄ improved the growth rate. Proliferation rate increased in media containing high concentration of KNO₃ and low to moderate concentrations of NH₄NO₃. The greatest production of new tissues and organs was seen in media with high KNO₃ and moderate to high CaCl₂. High concentration of NH₄NO₃ and low concentration of KH₂PO₄ also increased production of new tissues and organs. No shoot apical meristem was seen when CaCl₂ level was high and KNO₃ level was low. Formation of shoot apical meristem required high KH₂PO₄ concentration and low CaCl₂ concentration. Finally, low concentration of KNO₃ and low to moderate concentrations of NH₄NO₃ increased phenol exudation.

     

Induction of somatic embryos by macrosalt stress from mature zygotic embryos of Panax ginseng

Mature zygotic embryos of ginseng (Panax ginseng C. A. Meyer) were germinated on a Murashige and Skoog medium lacking growth regulators. However, when the zygotic embryos were cultured on MS medium containing increased levels of macrosalts (NH₄NO₃, KNO₃, KH₂PO₄, MgSO₄, or CaCl₂) to result is a mild salt stress, growth of zygotic embryos was strongly suppressed and eventually browning occurred. Somatic embryos or embryogenic calli were formed directly from these abnormal stressed zygotic embryos. Cotyledons were the most competent tissue for somatic embryo production. The highest frequency of somatic embryo formation (56.3%) was observed on medium containing 61.8 mM of NH₄NO₃. The highest frequency of somatic embryo formation by five different macrosalt treatments occurred in the following order: NH₄NO₃> KNO₃> KH₂PO₄> MgSO₄> CaCl₂. Somatic embryos were regenerated into plants with a shoot and root, and the plants survived on soil in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Components of fermentation medium regulate bacteriocin synthesis by the recombinant strain Lactococcus lactis subsp. lactis F-116

The regulation of the synthesis of bacteriocin produced by the recombinant strain Lactococcus lactis subsp. lactis F-116 has been studied. The synthesis is regulated by the components of the fermentation medium, the content of inorganic phosphate (KH₂PO₄), yeast autolysate (source of amine nitrogen), and changes in carbohydrates and amino acids. The strain was obtained by fusion of protoplasts derived from two related L. lactis subsp. lactis strains, both exhibiting a weak ability to synthesize the bacteriocin nisin. Decreasing the content of KH₂PO₄ from 2.0 to 1.0 or 0.5% caused bacteriocin production to go down from 4100 to 2800 or 1150 IU/ml, respectively; the base fermentation medium contained 1.0% glucose, 0.2% NaCl, 0.02% MgSO₄, and yeast autolysate (an amount corresponding to 35 mg % ammonium nitrogen). The substitution of sucrose for glucose (as the source of carbon) increased the antibiotic activity by 26%, and the addition of isoleucine, by 28.5%. Elevation of the concentration of yeast autolysate in the low-phosphate fermentation medium stimulated both the growth of the lactococci and the synthesis of bacteriocin. Introduction of 1% KH₂PO₄, yeast autolysate (an amount corresponding to 70 mg % ammonium nitrogen), 2.0% sucrose, and 0.1% isoleucine increased the bacteriocin-producing activity of the strain by 2.4 times.     

Isolation of Myrosinase Producing Microorganism

Screening tests were undertaken to obtain microorganisms which produce myrosinase. One strain of microorganism indicated a strong capasity for producing myrosinase. The morphological and physiological characteristics of this strain were studied. The organism was identified as Enterobacter cloacae, no. 506.

Enzyme production was induced by the addition of 0.01% sinigrin and 6% mustard extract*² to the culture medium. The highest production was obtained after 36~40 hr cultivation when the strain was cultivated at 28°C in a medium containing 0.01% sinigrin, 6% Mustard ext., 0.1% KH₂PO₄, 0.1% NH₄Cl, 0.1% NaCl and 0.1% MgSO₄·7aq, (pH 7.0).     

Use of RSM and CHAID data mining algorithm for predicting mineral nutrition of hazelnut

Defining optimal mineral-salt concentrations for in vitro plant development is challenging, due to the many chemical interactions in growth media and genotype variability among plants. Statistical approaches that are easier to interpret are needed to make optimization processes practical. Response Surface Methodology (RSM) and the Chi-Squared Automatic Interaction Detection (CHAID) data mining algorithm were used to analyze the growth of shoots in a hazelnut tissue-culture medium optimization experiment. Driver and Kuniyuki Walnut medium (DKW) salts (NH₄NO₃, Ca(NO₃)₂·4H₂O, CaCl₂·2H₂O, MgSO₄·7H₂O, KH₂PO₄ and K₂SO₄) were varied from 0.5× to 3× DKW concentrations with 42 combinations in a IV-optimal design. Shoot quality, shoot length, multiplication and callus formation were evaluated and analyzed using the two methods. Both analyses indicated that NH₄NO₃ was a predominant nutrient factor. RSM projected that low NH₄NO₃ and high KH₂PO₄ concentrations were significant for quality, shoot length, multiplication and callus formation in some of the hazelnut genotypes. CHAID analysis indicated that NH₄NO₃ at ≤1.701× DKW and KH₂PO₄ at >2.012× DKW were the most critical factors for shoot quality. NH₄NO₃ at ≤0.5× DKW and Ca(NO₃)₂ at ≤1.725× DKW were essential for good multiplication. RSM results were genotype dependent while CHAID included genotype as a factor in the analysis, allowing development of a common medium rather than several genotype specific media. Overall, CHAID results were more specific and easier to interpret than RSM graphs. The optimal growth medium for Corylus avellana L. cultivars should include: 0.5× NH₄NO₃, 3× KH₂PO₄, 1.5× Ca(NO₃)₂.     

Optimization of cultivation medium composition for isoamylase production

Summary The medium composition for production of isoamylase by Pseudomonas amyloderamosa JD210 was optimized using response surface methodology. The factors chosen for optimization were maltose, soybean protein hydrolysate (SPH), isoleucine, proline, KH₂PO₄ and MgSO₄. Fractional factorial designs (FFD) and the path of steepest ascent were effective in searching for the major factors and optimum medium composition. By a 2^6–1 FFD, supplementary isoleucine was shown to have a negative effect on enzyme production. The effects of the other five factors were further investigated by a central composite design and the optimum composition was found to be 1.10% maltose, 0.13% SPH, 0.15% proline, 0.38% KH₂PO₄, and 0.05% MgSO₄. When the strain was cultivated in the optimum medium, enzyme production increased 60% compared with ordinary medium. Proline was verified as being a significant factor in promoting enzyme production.     

Inulinase production and mathematical modeling from carob extract by using Aspergillus niger

The main objectives of the study were to produce inulinase from carob extract by Aspergillus niger A42 (ATCC 204447) and to model the inulinase fermentation in the optimum carob extract-based medium. In the study, carob extract was used as a novel and renewable carbon source in the production of A. niger inulinase. For medium optimization, eight different variables including initial sugar concentration (°Bx), (NH₄)₂HPO₄, MgSO₄.7H₂O, KH₂PO₄, NH₄NO₃, yeast extract, peptone, and ZnSO₄.7H₂O were employed. After fermentations, optimum medium composition contained 1% yeast extract in 5°Bx carob extract. As a result of the fermentation, the maximum inulinase activity, maximum invertase-type activity, I/S ratio, maximum inulinase- and invertase-type activity rates, maximum sugar consumption rate, and sugar utilization yield were 1507.03 U/ml, 1552.86 U/ml, 0.97, 175.82 and 323.76 U/ml/day, 13.26 g/L/day, and 98.52%, respectively. Regarding mathematical modeling, the actual inulinase production and sugar consumption data were successfully predicted by Baranyi and Cone models based on the model evaluation and validation results and the predicted kinetic values, respectively. Consequently, this was the first report in which carob extract was used in the production of inulinase as a carbon source. Additionally, the best-selected models can serve as universal equations in modeling the inulinase production and sugar consumption in shake flask fermentation with carob extract medium.     

Modeling some mineral nutrient requirements for micropropagated wild apricot shoot cultures

A response surface methodology (RSM) experimental design was applied for improving micropropagation of a wild apricot, Prunus armeniaca Lam., from the mountains of Kazakhstan. In an initial study, woody plant medium (WPM) mineral nutrients [calcium nitrate, ammonium nitrate, mesos (calcium chloride, potassium phosphate and magnesium sulfate) potassium sulfate and minor nutrients] were tested in a response surface methodology (RSM) experiment. Shoot quality was the best when nitrogen and mesos (CaCl₂, MgSO₄, K₂SO₄, KH₂PO₄) compounds were altered. In this study an expanded mesos optimization experiment was run. Data taken included a subjective quality rating, shoot length, shoot number, leaf color and size, callus and physiological disorders. Data were analyzed by Classification and Regression Tree Analysis (CART), a data mining technique that provides specific cutoff values for data and easy to interpret data trees. The CART analysis indicated that the best quality would be with ≤2.4× WPM levels of KH₂PO₄ and ≤0.75× MgSO₄. Shoot length was affected by K₂SO₄, but most shoots were of good size at any concentration. Shoot multiplication was affected by KH₂PO₄, but there were >5 shoots at any concentration. Leaf color was best with ≤2.41× KH₂PO₄ and ≤1.22× K₂SO₄. Based on the CART analysis, a recommendation for improved mesos compounds was developed. Each of the individual trees was analyzed and the cutoff points determined so that all the growth characteristics could be considered in the final concentrations chosen. Using the combined results from the CART analysis, the suggested medium would include WPM with CaCl₂ 2.7×, MgSO₄ 2.7×, K₂SO₄ 0.8×, KH₂PO₄ 0.75×.     

Optimization of β-Glucosidase Production by Aspergillus terreus Strain EMOO 6-4 Using Response Surface Methodology Under Solid-State Fermentation

Forty-two morphologically different fungal strains were isolated from different soil samples and agricultural wastes and screened for β-glucosidase activity under solid-state fermentation. Eight species were chosen as the most active β-glucosidase producers and were subjected to primary morphological identification. β-Glucosidase was highly produced by Aspergillus terreus, which showed the highest activity, and was subjected to full identification using scanning electron microscopy and molecular identification. Initial screening of different variables affecting β-glucosidase production was performed using Plackett-Burman design and the variables with statistically significant effects were identified. The optimal levels of the most significant variables with positive effect and the effect of their mutual interactions on β-glucosidase production were determined using Box-Behnken design. Fifteen variables including temperature, pH, incubation time, inoculum size, moisture content, substrate concentration, NaNO₃, KH₂PO₄, MgSO₄ · 7H₂O, KCl, CaCl₂, yeast extract, FeSO₄ · 7H₂O, Tween 80, and (NH₄)₂SO₄ were screened in 20 experimental runs. Among the 15 variables, NaNO₃, KH₂PO₄ and Tween 80 were found as the most significant factors with positive effect on β-glucosidase production. The Box–Behnken design was used for further optimization of these selected factors for better β-glucosidase production. The maximum β-glucosidase production was 4457.162 U g^?1.     

Optimization of the fermentation media for sophorolipid production from Candida bombicola ATCC 22214 using a simplex centroid design

This article describes the use of a simplex centroid mixture experimental design to optimize the fermentation medium in the production of sophorolipids (SLs) using Candida bombicola. In the first stage, 16 media ingredients were screened for the ones that have the most positive influence on the SL production. The sixteen ingredients that were chosen are five different carbohydrates (fructose, glucose, glycerol, lactose, and sucrose), five different nitrogen sources (malt extract, peptone extract, soytone, urea, and yeast extract), two lipid sources (mineral oil and oleic acid), two phosphorus sources (K₂HPO₄ and KH₂PO₄), MgSO₄, and CaCl₂. Multiple regression analysis and centroid effect analysis were carried out to find the sugar, lipid, nitrogen source, phosphorus source, and metals having the most positive influence. Sucrose, malt extract, oleic acid, K₂HPO₄, and CaCl₂ were selected for the second stage of experiments. An augmented simplex centroid design for five ingredients requiring 16 experiments was used for the optimization stage. This produced a quadratic model developed to help understand the interaction amongst the ingredients and find the optimal media concentrations. In addition, the top three results from the optimization experiments were used to obtain constraints that identify an optimal region. The model together with the optimal region constraints predicts the maximum production of SLs when the fermentation media is composed of sucrose, 125 g/L; malt extract, 25 g/L; oleic acid, 166.67 g/L; K₂HPO₄, 1.5 g/L; and CaCl₂, 2.5 g/L. The optimal media was validated experimentally and a yield of 177 g/L was obtained. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Some Factors Influencing the Efficiency of Production of Acetylmethylcarbinol by Rhizopus nigricans

Incubation time had no effect on the efficiency ratio [dry weight-acetylmethylcarbinol (AMC)] during the incubation times tested. The mycelium was more efficient in synthetic media at pH 3 and 4 than at pH 5 and 6. Mycelium also produced AMC more efficiently in apple juice and in synthetic media containing yeast extract or vitamins, especially thiamine. KCl, MgSO₄, and KH₂PO₄ had significant suppressing effects on AMC production in a synthetic medium.     

Reduced major minerals and increased minor nutrients improve micropropagation in three apple cultivars

Mineral nutrient medium requirements for propagation of in vitro shoots of apple (Malus domestica Borkh) ‘Golden Delicious’, ‘Maksat’, and ‘Voskhod’ were studied using response surface methodology (RSM). The mineral nutritional factors evaluated were based on Murashige and Skoog (MS) mineral nutrients (NH₄NO₃, KNO₃, CaCl₂, KH₂PO₄, MgSO₄, and minor nutrients), with concentrations ranging from 0.5 to 3.0× the MS concentrations. Nine plant growth qualities were evaluated. The most significant factors were NH₄NO₃ at 0.5 to 1.0× MS, and minor nutrients at 2.0× MS. Most of the other factors were optimal at 0.5×. The quality rating was highest when minor nutrients were 2.0× MS, and most other nutrients were standard concentrations or lower. Increased KH₂PO₄ and minor nutrients were the most significant for improved multiplication, and higher KNO₃ for shoot length. Optimized media were developed for each cultivar based on these models. The cultivars were grown on the three individual optimized media, a general medium based on the three optimizations, and MS. The optimized medium for each cultivar was significantly better for shoot quality and shoot length of each cultivar than MS, but the generalized medium of minors at 2.0× and NH₄NO₃, CaCl₂, and MgSO₄ at 0.5× MS, was significantly better for two of the three cultivars and not significantly different for the third. The next step to develop a final optimized medium will require the evaluation of the minor nutrients, determination of optimal concentrations of each, and screening a wide range of Malus germplasm on the finalized medium.

     

Media optimization studies for glucose isomerase production by arthrobacter species

Media optimization studies are carried out with the objective of maximising glucose isomerase production by Arthrobacter sp. The recommended media consists of 1.0% (w/v) xylose, 1.0% peptone, 0.5% yeast extract, 0.025% MgSO₄·7H₂O, 0.6% (NH₄)₂HPO₄ and 0.2% KH₂PO₄. Activity of the enzyme produced in this media is 11.2 units/ml. Growth cycle for batch cultivation is studied and the lag period is 2 hours, followed by exponential phase extending upto 32 hours.     

Selection and characterization of a newly isolated thermotolerant Pichia kudriavzevii strain for ethanol production at high temperature from cassava starch hydrolysate

Pichia kudriavzevii DMKU 3-ET15 was isolated from traditional fermented pork sausage by an enrichment technique in a yeast extract peptone dextrose (YPD) broth, supplemented with 4 % (v/v) ethanol at 40 °C and selected based on its ethanol fermentation ability at 40 °C in YPD broth composed of 16 % glucose, and in a cassava starch hydrolysate medium composed of cassava starch hydrolysate adjusted to 16 % glucose. The strain produced ethanol from cassava starch hydrolysate at a high temperature up to 45 °C, but the optimal temperature for ethanol production was at 40 °C. Ethanol production by this strain using shaking flask cultivation was the highest in a medium containing cassava starch hydrolysate adjusted to 18 % glucose, 0.05 % (NH₄)₂SO₄, 0.09 % yeast extract, 0.05 % KH₂PO₄, and 0.05 % MgSO₄·7H₂O, with a pH of 5.0 at 40 °C. The highest ethanol concentration reached 7.86 % (w/v) after 24 h, with productivity of 3.28 g/l/h and yield of 85.4 % of the theoretical yield. At 42 °C, ethanol production by this strain became slightly lower, while at 45 °C only 3.82 % (w/v) of ethanol, 1.27 g/l/h productivity and 41.5 % of the theoretical yield were attained. In a study on ethanol production in a 2.5-l jar fermenter with an agitation speed of 300 rpm and an aeration rate of 0.1 vvm throughout the fermentation, P. kudriavzevii DMKU 3-ET15 yielded a final ethanol concentration of 7.35 % (w/v) after 33 h, a productivity of 2.23 g/l/h and a yield of 79.9 % of the theoretical yield.     

The effects of potassium and chloride ions on the ethanolic fermentation of sucrose by Zymomonas mobilis 2716

Summary The inclusion of specific salts in Zymomonas mobilis batch sucrose fermentations can limit by-product formation. Sorbitol and fructo-oligosaccharide formation can be reduced and ethanol production enhanced by manipulating mineral salt concentrations. Chloride salts reduced the production of biomass and sorbitol in favour of fructo-oligosaccharide formation at concentrations lower than 10 g NaCl/l or MgCl₂. Higher concentrations led to the accumulation of glucose and fructose. Low concentrations of KH₂PO₄ (<20 g/l) enhanced biomass formation, and the concomitant reduction in sorbitol and fructo-oligosaccharides favoured enhanced ethanol formation. At concentrations above 20 g/l, its effects were similar to those obtained with the chloride salts. Invertase addition at the start of fermentation increased sorbitol formation, whereas addition after the completion of sucrose hydrolysis resulted in the conversion of fructo-oligosaccharides formed into fructose or ethanol. Fermentation with 250 g/l of sugar-cane syrup ( = 130 g sucrose/l) in the presence of 8 g KH₂PO₄/l, with 0.05 g invertase/l added on the completion of sucrose hydrolysis, resulted in a conversion efficiency of 94% with complete carbon accountability, and only 7 g sorbitol/l. Offprint requests to: H. W. Doelle