Extractability of polyhydroxyalkanoate synthesized by <Emphasis Type="Italic">Bacillus flexus</Emphasis> cultivated in organic and inorganic nutrient media

Bacillus flexus was isolated from local soil sample and identified by molecular methods. In inorganic nutrient medium (IM) containing sucrose as carbon source, yield of biomass and polyhydroxyalkanoate (PHA) were 2 g/l and 1 g/l (50% of biomass), respectively. Substitution of inorganic nitrogen by peptone, yeast extract or beef extract resulted in biomass yields of 4.1, 3.9 and 1.6 g/l, respectively. Corresponding yields of PHA in biomass was 30%, 40% and 44%. Cells subjected to change in nutrient condition from organic to inorganic, lacked diaminopimelic acid in the cell wall and the concentration of amino acids also decreased. Under these conditions the extractability of the polymer from the cells by hot chloroform or mild alkali hydrolysis was 86–100% compared to those grown in yeast extract or peptone (32–56%). The results demonstrated that growth, PHA production and the composition of cell wall of B. flexus are influenced by the organic or inorganic nutrients present in the growth medium. Cells grown in inorganic medium lysed easily and this can be further exploited for easier recovery of the intracellular PHA.     

High yield mixotrophic cultures of the marine microalga Tetraselmis suecica (Kylin) Butcher (Prasinophyceae)

The effects of three organic compounds were tested on one of the most used marine micro-algae in the aquaculture of molluscs and crustaceans, Tetraselmis suecica. Studies were made in axenic conditions with yeast extract, peptone and glucose added to the culture medium, each alone, in combinations of two or all together. Medium without any organic compound was used for the control. Cultures containing yeast extract grew best, reaching maximum cell density of 3.79 × 10⁶ and 3.84 × 10⁶ cells ml⁻¹. The organic carbon source affected the biochemical composition. The components most affected were the carbohydrates, with values between 6.5 pg cell⁻¹ in control cultures and 48.5 pg cell⁻¹ in glucose cultures. Protein content ranged between 27.5 pg cell⁻¹ in control cultures and 88.6 pg cell⁻¹ in yeast + glucose + peptone cultures. The lipid content changed little. Maximum protein yields were reached in cultures with yeast + glucose and with yeast - glucose - peptone, with values of 24.6 and 28.2 mg 1⁻¹ d⁻¹, respectively. These values are 22 and 25 times those in control cultures. A maximum carbohydrate yield of 7.9 mg carbohydrate per litre per day was obtained in yeast + glucose + peptone cultures, 27 times that in the control cultures. The maximum lipid yield was obtained with yeast + glucose + peptone and yeast + glucose. Maximum energy values were 308 kcal 1⁻ in yeast extract - glucose - peptone cultures and 279 kcal 1⁻¹ in yeast extract + glucose cultures. Gross energy values in control cultures were 24.5 kcal 1⁻¹, but peptone cultures presented the minimum energy value, 22 kcal 1⁻¹. The yeast extract: glucose ratio in the culture medium was optimized. A ratio 2:1 produced the best yields in cells, protein, carbohydrate and gross energy.     

Effect of the levels of dissolved oxygen on the expression of recombinant proteins in four recombinantEscherichia coli strains

Summary Four recombinant strains ofEscherichia coli were examined for the effects of the dissolved oxygen level on the level of biomass, the plasmid content, and the level of recombinant protein at the stationary phase of batch growth. Strains JM101/pYEJ001, and TB-1/pYEJ001 (encoding chloramphenicol acetyltransferase), and strain TB-1/p1034, and TB-1/pUC19 (encoding -galactosidase) were grown at the constant dissolved oxygen levels of 0, 50, and 100% air saturation, as well as in the absence of dissolved, oxygen control. The biomass of all strains under constant aerobic conditions was 12–36 times higher than that under anaerobic conditions, but was the same as or slightly higher than that without dissolved oxygen control. The plasmid content in all strains under anaerobic conditions was 2.9–11.7 times higher than that under aerobic conditions. The optimal dissolved oxygen concentration for the specific activity of recombinant proteins was dependent upon the strain. In no strain were constant aerobic conditions optimal. However, because of the effect on biomass, controlled aerobic conditions were optimal for the volumetric activity of recombinant protein in all but one strain.     

Production of lactic acid and byproducts from waste potato starch by <Emphasis Type="Italic">Rhizopus arrhizus</Emphasis>: role of nitrogen sources

Summary Lactic acid was produced by Rhizopus arrhizus using waste potato starch as the substrate. The aim of this study was to identify the role of nitrogen sources and their impact on the formation of lactic acid and associated byproducts. Ammonium sulphate, ammonium nitrate, urea, yeast extract and peptone were assessed in conjunction with various ratios of carbon to nitrogen (C:N). Fermentation media with a low C:N ratio enhanced the production of lactic acid, biomass and ethanol, while a high C:N ratio favoured the production of fumaric acid. Ammonium nitrate appeared to be the most suitable nitrogen source for achieving a high and stable lactic acid yield, and minimizing the production of byproducts such as biomass and ethanol, while urea proved to be the least favourable nitrogen source. Yeast extract and peptone appeared to improve fungal cell growth. The kinetics data revealed that a high concentration of ammonium nitrate enhanced the lactic acid productivity. The maximum lactic acid concentration of 36.4 g/l, representing a yield of 91%, was obtained with addition of 0.909 g/l ammonium nitrate in 32 h.     

Doublet cells inTetrahymena as indicators of culture media composition

Stomatogenesis in ciliates is a complex and carefully orchestrated event. The exo⁻ mutant SB255 ofTetrahymena thermophila has defects in mucocyst formation and docking and can also have one or two mouths. Three common culture media (proteose peptone, Medium 357, and yeast extract) were analyzed for total C, N, and inorganic elements and then tested for their effect on the number of mouths present in SB255. Cultures of SB255 grown in Medium 357 consisted of a mixed population of cells with either two mouths (doublet) or one mouth. Cultures from the same original stock grown in Medium 357 (SBm) and in 1% proteose peptone (SBpp) had different percentages of doublet cells in 1-, 2-, 3-, and 4-d-old cultures. When transferred to and grown in 1% yeast medium, both SBpp and SBm cultures had increased percentages of doublets over a 4-d culture period. When grown in 0.1, 0.5, or 1% yeast medium for 2 d, both SBpp and SBm cultures had more doublets in 1% than in either 0.1 or 0.5% yeast medium. Cultures of SBm grown in Medium 357. or 1% yeast medium for 2 d had a 10-fold increase in doublet cells compared to the inoculum. After 2 d in 1% proteose peptone, SBm cultures had percentages of doublet cells almost equal to that of the inoculum. Immunofluorescence and scanning electron microscopy (SEM) were used to examine cellular morphology of the doublet cells. These findings suggest that enriched media promote the growth of doublet cells. Furthermore, these doublets could prove to be a useful model system for the study of biological roles of trace elements.     

Optimization of critical medium components using response surface methodology for biomass and extracellular polysaccharide production by <Emphasis Type="Italic">Agaricus blazei</Emphasis>

Response surface methodology (RSM) was applied to optimize the critical medium ingredients of Agaricus blazei. A three-level Box–Behnken factorial design was employed to determine the maximum biomass and extracellular polysaccharide (EPS) yields at optimum levels for glucose, yeast extract (YE), and peptone. A mathematical model was then developed to show the effect of each medium composition and its interactions on the production of mycelial biomass and EPS. The model predicted the maximum biomass yield of 10.86 g/l that appeared at glucose, YE, peptone of 26.3, 6.84, and 6.62 g/l, respectively, while a maximum EPS yield of 348.4 mg/l appeared at glucose, YE, peptone of 28.4, 4.96, 5.60 g/l, respectively. These predicted values were also verified by validation experiments. The excellent correlation between predicted and measured values of each model justifies the validity of both the response models. The results of bioreactor fermentation also show that the optimized culture medium enhanced both biomass (13.91 ± 0.71 g/l) and EPS (363 ± 4.1 mg/l) production by Agaricus blazei in a large-scale fermentation process.     

Optimization of an ethanol production medium in very high gravity fermentation

Concentrations of Mg²⁺, glycine, yeast extract, biotin, acetaldehyde and peptone were optimized by a uniform design process for ethanol production by Saccharomyces cerevisiae. Using non-linear step-wise regression analysis, a predictive mathematical model was established. Concentrations of Mg²⁺ and peptone were identified as the critical factors: 50 mM Mg²⁺ and 1.5% (w/v) peptone in the medium increased the final ethanol titre from 14.2% (v/v) to 17% (v/v) in 48 h.     

Application of a simple yeast extract from spent brewer's yeast for growth and sporulation of Bacillus thuringiensis subsp. kurstaki: a Physiological Study

The suitability of using a simple brewer's yeast extract (BYE), prepared by autolysis of complete beer slurry, for growth and sporulation of Bacillus thuringiensis kurstaki was studied in baffled shake flasks. In a standard buffered medium with 2.5% (w/v) glucose and 1% (w/v) brewer's yeast extract, growth of B. t. kurstaki resulted in a low biomass production with considerable byproduct formation, including organic acids and a concomitant low medium pH, incomplete glucose utilization and marginal sporulation, whereas growth in the same medium with a commercial laboratory-grade yeast extract (Difco) resulted in a high biomass concentration, complete glucose utilization, relatively low levels of byproducts and complete sporulation (2.6 × 10⁹ spores/ml). When glucose was left out of the medium, however, growth parameters and sporulation were comparable for BYE and commercial yeast extract, but absolute biomass levels and spore counts were low. Iron was subsequently identified as a limiting factor in BYE. After addition of 3 mg iron sulphate/l, biomass formation in BYE-medium more than doubled, low byproduct formation was observed, and complete sporulation occurred (2.8 × 10⁹spores/ml). These data were slightly lower than those obtained in media with commercial yeast extract (3.6 × 10⁹spores/ml), which also benefited, but to a smaller extent, from addition of iron.     

Cost-effective multiplication of the entomopathogenic fungus Nomuraea rileyi (F) Samson

Cost-effective and rapid multiplication of Nomuraea rileyi is reported. The spore yields in semi-synthetic media were comparable or significantly higher to the standard medium. Maltose and peptone, carbon and nitrogen sources could be effectively replaced with 2% barley extract and 1% soybean extract respectively. However, replacement of yeast extract with dry yeast resulted in lower spore yields. Sporulation of the fungus multiplied on solid substrate was possible only when the bags used had a 0.2 m filter to facilitate passive exchange of sterile air. A high spore yield of 2.8 × 10⁹/g of substrate was realized on crushed sorghum.This revised version was published online in October 2005 with corrections to the Cover Date.     

Phytase production by high cell density culture of recombinant Bacillus subtilis

Bacillus subtilis BD170, harboring a plasmid pGT44[phyC] carrying the phytase gene (phyC) and a phosphate-depletion inducible pst-promoter, was grown in a 2 l bioreactor. Using a controlled feeding of glucose, high cell densities of 32 and 56 g dry cell weight l^–1 were achieved with peptone and yeast extract, respectively, as the complex nitrogen sources in a semi-defined growth medium. The fed-batch protocol was applied to production of recombinant phytase and a high extracellular phytase activity (48 U ml^–1) was reached with peptone. Although the yeast extract feeding resulted in a higher cell density, it was unsuitable as a medium component for phytase expression due to its relatively high phosphate content.     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

Statistical medium optimization and DO-STAT fed-batch fermentation for enhanced production of tyrosine phenol lyase in recombinant Escherichia coli

Abstract

Tyrosine phenol lyase (TPL) is a robust biocatalyst for the production of L-dihydroxyphenylalanine (L-DOPA). The improvement of TPL production is conducive to the industrial potential. In this study, the optimization of culture medium of recombinant Escherichia coli harboring TPL from Fusobacterium nucleatum (Fn-TPL) was carried out. Sucrose and combination of yeast extract and peptone were selected as carbon and nitrogen source, respectively. Their optimal concentrations were determined by Box-Behnken design and the synergistic effect between yeast extract and peptone was found to be significant, with p-value < 0.05. The DO-STAT fed-batch fermentation under optimized culture condition was established and the oxygen level was fixed at 20%. Both the biomass and Fn-TPL activity were significantly increased, which were 35.6 g dcw/L and 12292 U/L, respectively. The results obtained significantly promote the industrial production of L-DOPA production.     

Optimization of culture conditions for the production of rifamycin oxidase by Curvularia lunata

Maximum activity (8.9 IU/ml) of rifamycin oxidase in Curvularia lunata, grown in shake-flask culture at 28°C and pH 6.5, was after 96 h. Nearly all the glucose was used in 72 h. An initial culture pH of 6.5 and 28°C were optimum for the growth and enzyme production. Among various carbon and organic nitrogen sources, carboxymethylcellulose and peptone were the most effective for enzyme yield. The rate of enzyme production was enhanced when yeast extract was also added to the medium. The optimum medium for the production of rifamycin oxidase contained 10 g each of yeast extract, peptone and carboxymethylcellulose/l and 0.04% (NH₄)₂SO₄.The author is with the Biochemical Engineering Research and Process Development Centre, Institute of Microbial Technology, Post Box 1304, Sector 39-A, Chandigarh 160 014, India     

Enhanced production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer and antimicrobial yellow pigmentation from Cupriavidus sp. USMAHM13 with antibiofilm capability

Antibiofilm polymers have the ability to inhibit bacterial biofilm formation, which is known to occur ubiquitously in the environment and pose risks of infection. In this study, production of P(3HB-co-4HB) copolymer and antimicrobial yellow pigment from Cupriavidus sp. USMAHM13 are enhanced through medium optimization. Before the improvement of yellow pigment production, screening for the best additional supplement was performed resulting in high-yield yellow pigmentation using yeast extract with optimum concentration of 2?g/L. Effects of different concentrations of 1,4-butanediol, ammonium acetate, and yeast extract were studied using central composite design. Under optimal conditions, 53?wt% of polyhydroxyalkanoate (PHA) content, 0.35?g/L of pigment concentration, and 5.87?g/L of residual biomass were achieved at 0.56?wt% C of 1,4-butanediol, 1.14?g/L of ammonium acetate, and 2?g/L of yeast extract. Antibiofilm tests revealed that the yellow pigment coated on P(3HB-co-4HB) copolymer had significant effect on the inhibition of bacteria proliferation and colonization from 6?hr onward reaching 100% inhibition by 12?hr, hence effectively inhibiting the biofilm formation.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Control of xylanase production without protease activity in Bacillus sp. by selection of nitrogen source

Maximum xylanase activity, of 380 IU ml^–1, with negligible protease activity, occurred when Bacillus SSP-34 was grown for 96 h with yeast extract and peptone each at 0.25%. Other concentrations of the combination gave xylanase activities less than 66% of that with the optimum nitrogen source concentration and protease activities in the range of 0.01–0.045 IU ml^–1.     

Secretion of Escherichia coli beta-galactosidase in Saccharomyces cerevisiae using the signal sequence from the glucoamylase-encoding STA2 gene

The budding yeast Saccharomyces cerevisiae is a safe and widely used host for the production of recombinant DNA-derived proteins. We have used the signal sequence from the S. diastaticus STA2 gene, encoding glucoamylase II, to secrete Escherichia coli beta-galactosidase, encoded by the lacZ gene. In frame STA2/lacZ gene fusions have been constructed and expressed in S. cerevisiae under the control of either the STA2 or the galactose inducible GAL1-10 upstream promoters. Fairly high amounts of the enzyme (up to 76% of total activity, depending on the growth conditions) are secreted in the periplasmic space. Adding yeast extract and peptone to the growth medium results in a dramatic increase in both synthesis and secretion of beta-galactosidase.     

Anaerobic degradation of chlorophenol by an enrichment culture

Summary An anaerobic mixed culture from sewage sludge was enriched in a yeast extract and peptone-containing medium; it was able to degrade 2-cholorophenol completely to methane and CO₂. Degradation rates of 2-chlorophenol of up to 0.18 g/l per day were observed in suspended cultures without biomass retention and of 0.375 g/l per day in cultures immobilized on Liapor clay beads. Attempts to isolate the dechlorinating organism failed. The mixed culture was reduced to three morphologically distinctive microorganisms using a medium with limited amounts of yeast extract and peptone and n-butyrate as a co-substrate. Under these conditions the phenol-degrading bacterium was lost and phenol accumulated in the medium. No growth and no dehalogenation of 2-chlorophenol was obtained when yeast extract and peptone were omitted completely. Besides serving as a source of supplementary components, yeast extract and peptone were apparently required as the main source of carbon, wereas reducing equivalents for reductive dehalogenation were obtained by oxidation of n-butyrate. A spirochaete-like organism was presumably the dechlorinating bacterium. The mixed culture lost its dehalogenation capability if this organism was lost. n-Butyrate could be replaced by n-valerate, hexanoate, heptanoate, octanoate, pelargonic acid, n-decanoic acid or palmitate as co-substrates for dehalogenation of either 2-chlorophenol, 2-bromophenol or complete dechlorination of 2,6-dichlorophenol, whereas from 2,4-dichlorophenol only the substituent in the ortho-position could be eliminated.Dedicated to Professor O. Kandler on the occassion of his 70th birthdayOffprint requests to: J. Winter     

Unraveling the potential and constraints associated with corn steep liquor as a nutrient source for industrial fermentations

Costly complex media components such as yeast extract and peptone are still widely used in industrial bioprocesses, despite their ill-defined composition. Side stream products such as corn steep liquor (CSL) present a compelling economical alternative that contains valuable nutrients required for microbial growth, that is, nitrogen and amino acids, but also vitamins, trace elements, and other minerals. However, as a side stream product, CSL may be subject to batch-to-batch variations and compositional heterogeneity. In this study, the Respiration Activity MOnitoring System designed for shake flasks (RAMOS) and 96-well microtiter plates (μTOM) were applied to investigate the potential and constraints of CSL utilization for two model microorganisms: E. coli and B. subtilis. Considering the dry substance content of complex nutrients involved, CSL-based media are more efficient in biomass production than the common lysogeny broth (LB) medium, containing 5 g/L yeast extract, 10 g/L peptone, and 5 g/L NaCl. At a glucose to CSL (glucose/CSL, g/g) ratio of 1/1 (g/g) and 2/1 (g/g), a secondary substrate limitation occurred in E. coli and B. subtilis cultivations, respectively. The study sheds light on differences in the metabolic activity of the two applied model organisms between varying CSL batches, which relate to CSL origin and production process, as well as the effect of targeted nutrient supplementation. Through a targeted nutrient supplementation, the most limiting component of the CSL-glucose medium used for these applied model microorganisms was identified to be ammonium nitrogen. This study proves the suitability of CSL as an alternative nutrient source for E. coli and B. subtilis. The RAMOS and μTOM technique detected differences between CSL batches, allowing easy and early identification of varying batches. A consistent performance of the CSL batches in E. coli and B. subtilis cultivations was demonstrated.