Optimization of culture media and conditions for cultivating Erwinia sp., a producer of fumarase and aspartase

Mathematical methods of experimental design were used to determine the optimal concentrations of nutrient medium components, aeration conditions, and pH providing for the maximum biomass yields, as well as fumarase and aspartase activities, during submerged cultivation of Erwinia sp. The data showed that different concentrations of carbon source (molasses) and pH of the nutrient medium were required to reach the maximum yields of fumarase and aspartase. Calculations suggested that the combination of these optimized factors would result in 3.2-, 3.4-, and 3.8-fold increases in the Erwinia sp. biomass, aspartase activity, and fumarase activity yields, respectively. The experimental data were consistent with these estimates to a 80% accuracy.     

Biodegradation of diphenyl ethers by a copper-resistant mutant ofErwinia sp.

Summary A bacterium tentatively identified as anErwinia sp. was isolated from sewage by enrichment on methanol and lignin. Several mutants developed from this strain were studied for their ability to degrade aromatic ethers. Different concentrations of the chemicals were incubated with the organisms and the degradation was estimated by high-performance liquid chromatography (HPLC). Among these mutants, one isolate,Erwinia sp. strain CU3614, showed resistance to copper ions (>20 mM CuSO₄) and the ability to degrade 4-hydroxydiphenyl ether (4-HDPE), 4-chlorodiphenyl ether (4-CDPE), 4-nitrodiphenyl ether (4-NDPE) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) in the presence of copper ions. Increased concentrations of copper in the medium resulted in higher degradation of 4-HDPE. Further studies with copper-sensitive mutants obtained fromErwinia sp. CU3614 by Tn5 transposon-induced mutagenesis showed a corresponding decrease in the ability to degrade 4-HDPE. These results suggest the presence of a copper-associated activity in the biotransformation of aromatic ethers.     

Preliminary study on the operating variables of SCP production from grape must

Summary The fermentation of grape must by Candida utilis ISS 28 was studied at different substrate concentrations, pH values, and nutrient supplementation in a shaken-flask fermenter, by using a composite design experiment.The experimental biomass yields were fitted to the only statistically significant factors with a mean standard error less than 8%, by using multiple regression analysis.Optimal conditions for maximum cell yield were established by plotting a series of loci at constant biomass yield and then verified experimentally, thus confirming the remarkable accuracy of the model     

Improvement of production of l-aspartic acid using immobilized microbial cells

Summary In our laboratory, EAPc-7 a strain having higher aspartase activity was derived from Escherichia coli ATCC 11303. For the improvement of l-aspartic acid productivity using EAPc-7 cells immobilized in -carrageenan, it was necessary to eliminate the fumarase activity which converts fumaric acid to l-malic acid. Several treatments for specifically eliminating fumarase activity from EAPc-7 cells were tested and it was found that when EAPc-7 cells were treated in a culture broth (pH 4.9) containing 50 mM l-aspartic acid at 45° C for 1 h, fumarase activity was almost completely eliminated without inactivation of the aspartase.The treated cells, immobilized in -carrageenan, were used for continuous production of l-aspartic acid from ammonium fumarate. The formation of l-malic acid was negligible and the half-life of the immobilized preparation was 126 days.Productivity of immobilized preparation of treated EAPc-7 cells in l-aspartic acid production was six times of that of the parent cell preparation.     

Aspartase-hyperproducing mutants of Escherichia coli B

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aspartase-hyperproducing mutants of Escherichia coli B.

When a wild-type strain of Escherichia coli B was cultured on a medium containing L-aspartic acid as the sole carbon source (Asp-C medium), aspartase formation was higher than that observed in minimal medium. Addition of glucose to Asp-C medium decreased aspartase formation. When also cultured in a medium containing L-aspartic acid as the sole nitrogen source (Asp-N medium), E. coli B showed a low level of aspartase formation and an elongated doubling time. To obtain aspartase-hyperproducing strains, we enriched cells growing faster than cells of the wild-type strain in Asp-N medium by continuous cultivation of mutagenized cells. After plate selection, the doubling times of these mutants were measured. Thereafter, fast-growing mutants were tested for aspartase formation. One of these mutants, strain EAPc7, had a higher level of aspartase formation than did the wild-type strain in medium containing L-aspartic acid as the carbon source, however; addition of glucose to this medium decreased aspartase formation. The other mutant, strain EAPc244, had a higher level of aspartase activity than did the wild-type strain in both media. Therefore, aspartase formation in mutant EAPc244 was released from catabolite repression. In strain EAPc244 the other catabolite-repressible enzymes, beta-galactosidase, tryptophanase, and the three tricarboxylic acid cycle enzymes, were also released from catabolite repression. Both mutants had sevenfold the aspartase formation of the wild-type strain in a medium which contained fumaric acid as the main carbon source and which has been used for industrial production of E. coli B aspartase. However, strain EAPc244 had 2.5-fold the fumarase activity of strain EAPc7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modeling of polygalacturonase enzyme activity and biomass production by <Emphasis Type="Italic">Aspergillus sojae</Emphasis> ATCC 20235

Aspergillus sojae, which is used in the making of koji, a characteristic Japanese food, is a potential candidate for the production of polygalacturonase (PG) enzyme, which of a major industrial significance. In this study, fermentation data of an A. sojae system were modeled by multiple linear regression (MLR) and artificial neural network (ANN) approaches to estimate PG activity and biomass. Nutrient concentrations, agitation speed, inoculum ratio and final pH of the fermentation medium were used as the inputs of the system. In addition to nutrient conditions, the final pH of the fermentation medium was also shown to be an effective parameter in the estimation of biomass concentration. The ANN parameters, such as number of hidden neurons, epochs and learning rate, were determined using a statistical approach. In the determination of network architecture, a cross-validation technique was used to test the ANN models. Goodness-of-fit of the regression and ANN models was measured by the R ² of cross-validated data and squared error of prediction. The PG activity and biomass were modeled with a 5-2-1 and 5-9-1 network topology, respectively. The models predicted enzyme activity with an R ² of 0.84 and biomass with an R ² value of 0.83, whereas the regression models predicted enzyme activity with an R ² of 0.84 and biomass with an R ² of 0.69.     

Crystal structure of thermostable aspartase from Bacillus sp. YM55-1: structure-based exploration of functional sites in the aspartase family

The crystal structure of the thermostable aspartase from Bacillus sp. YM55-1 has been solved and refined for 2.5A resolution data with an R-factor of 22.1%. The present enzyme is a homotetramer with subunits composed of three domains. It exhibits no allosteric effects, in contrast to the Escherichia coli aspartase, which is activated by divalent metal cation and L-aspartate, but is four-times more active than the E.coli enzyme. The overall folding of the present enzyme subunit is similar to those of the E.coli aspartase and the E.coli fumarase C, both of which belong to the same superfamily as the present enzyme. A local structural comparison of these three enzymes revealed seven structurally different regions. Five of the regions were located around putative functional sites, suggesting the involvement of these regions into the functions characteristic of the enzymes. Of these regions, the region of Gln96-Gly100 is proposed as a part of the recognition site of the alpha-amino group in L-aspartate for aspartase and the hydroxyl group in L-malate for fumarase. The region of Gln315-Gly323 is a flexible loop with a well-conserved sequence that is suggested to be involved in the catalytic reaction. The region of Lys123-Lys128 corresponds to a part of the putative activator-binding site in the E.coli fumarase C. The region in the Bacillus aspartase, however, adopts a main-chain conformation that prevents the activator binding. The regions of Gly228-Glu241 and Val265-Asp272, which form a part of the active-site wall, are suggested to be involved in the allosteric activation of the E.coli aspartase by the binding of the metal ion and the activator. Moreover, an increase in the numbers of intersubunit hydrogen bonds and salt-bridges is observed in the Bacillus aspartase relative to those of the E.coli enzyme, implying a contribution to the thermostability of the present aspartase.     

Polygalacturonase enzyme production from bacterial isolated from raw milk and green and black olives

Forty microbial strains isolated from raw milk samples and black and green olives were grown in MP5 (mineral pectin 5) medium containing 0.5% lemon pectin. All strains synthesized an extracellular polygalacturonase. Rhodotorula sp. ONRh9 (0.44 U x mL(-1)) and Leuconostoc sp. LLn1 (0.16 U x mL(-1)), which had a more active polygalacturonase in MP5 medium, were studied in MAPG5 medium containing polygalacturonic acid. Highest biomass and polygalacturonase production by these two strains were observed for polygalacturonic acid concentrations of 10 g x L(-1) (Rhodotorula sp. ONRh9) and 5 g x L(-1) (Leuconostoc sp. LLn1) and for initial pH values of 6 (Rhodotorula sp. ONRh9) and 5.5 (Leuconostoc sp. LLn1). The two strains grown in fermenters in MAPG5 medium generated the following results: with controlled initial pH, Rhodotorula sp. produced maximum biomass (DO) and polygalacturonase (PG) after 20 h (DO, 3.86; PG, 0.24 U x mL(-1)) of growth, and this level was sustained until the end of the culture; Leuconostoc sp. LLn1 synthesized more cells and polygalacturonase between 4 h (DO, 1.80; PG, 0.17 U x mL(-1)) and 24 h (DO, 3.90; PG, 0.27 U x mL(-1)) of culture. With uncontrolled initial pH, the cultures produced maximum biomass and polygalacturonase after 20 h (DO, 3.30; PG, 0.26 U x mL(-1)) for Rhodotorula sp. ONRh9 and 10 h (DO, 2.84; PG, 0.17 U x mL(-1)) for Leuconostoc sp. LLn1.     

Production of l-alanine from ammonium fumarate using two immobilized microorganisms

Summary For the efficient production of l-alanine from ammonium fumarate using the aspartase activity of immobilized Escherichia coli cells and l-aspartate -decarboxylase activity of immobilized Pseudomonas dacunhae cells, alanine racemase and fumarase activities should be eliminated. We investigated various procedures to eliminate these side reactions, and found that both activities of intact E. coli cells could be eliminated by treating the culture broth at pH 5.0 and 45° C for 1 h, and those of intact P. dacunhae cells could be eliminated by treating the culture broth at pH 4.75 and 30° C for 1 h. Further, it was confirmed that l-alanine was efficiently produced using these two immobilized pH-treated microorganisms.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Production and composition of phenylpyrrole metabolites prodcued by Pseudomonas cepacia

Summary In shaken cultures, a strain of Pseudomonas cepacia isolated from apple leaves produced pyrrolnitrin and four other phenylpyrrole antibiotics. The concentrations of these metabolites were determined at intervals for 7 days in three different media at two initial pH levels. Optical density measurements revealed maximum cell concentrations after 24 h in nutrient broth, after 48 h in King's B medium, and after 96 h in minimum salts solution. The effects caused by initiating fermentations at pH 5.8 rather than 7.0 were in most cases not dramatic, although in some instances, especially in minimum salts broth, higher concentrations of metabolites were produced with the lower initial pH. Concentrations of the phenylpyrrole antibiotics were greatly affected by choice of culture medium and incubation time. Concentrations of the two nitrophenyl metabolites, pyrrolnitrin and 2-chloropyrrolnitrin, rose throughout the 7-day incubation and were more than 20 times greater in minimum salts medium than in either King's B medium or nutrient broth. The maximum concentrations of each of the three aminophenyl metabolites (dichloroamino, trichloroamino and monochloroamino) occurred in different media, the monochloro compound in nutrient broth, the dichloro compound in Kings B medium and the trichloro compound in minimum salts medium. The time dependence of the concentrations of the five metabolites supports the proposed biosynthesis of these pyrroles from tryptophan by successive chlorinations followed by oxidation of the amino group at the end of the pathway.     

Biomass and lipid enhancement in Ankistrodesmus sp. cultured with reused and minimal nutrients media

Microalgae are a promising feedstock for biofuel production. Lipid content in microalgae could be enhanced under nutrient depletion. This work investigated the effect of the nutrient on lipid accumulation in Ankistrodesmus sp. culture. Batch cultures were carried out using fresh BG11 medium, and after the harvest, the medium was reused for the next culture; this method was repeated two times. The maximum lipid productivity of 29.75 mg L^?1 day^?1 was obtained from the culture with the second reuse medium. In continuous cultures, Ankistrodesmus sp. was cultured in both fresh and modified BG11 mediums. The modified BG11 medium was adjusted to resemble the content of the first reuse medium. As a comparison between batch and continuous cultures, it was proven that the productivity in the continuous culture was better than in the batch, where the achievable maximum biomass and lipid were 188.30 and 38.32 mg L^?1 day^?1. The maximum lipid content of 34.22% was obtained from the continuous culture at a dilution rate of 0.08 day^?1, whereas the maximum saturated and unsaturated fatty acid productivities of 79.96 and 104.54 mg L^?1 day^?1 were obtained at a dilution rate of 0.16 day^?1.     

Development of Chemically Defined Media Supporting High-Cell-Density Growth of Lactococci, Enterococci, and Streptococci

Lactococcus lactis IL1403 was used as an experimental strain to develop a chemically defined medium for study of the physiology and metabolic pathways of lactococci. An experimental leave-one-out technique was employed to determine the necessity of each of the 57 chemical components used in medium development. A statistical experimental design approach including three fractional factorial designs and a central composite design was used to optimize the fermentation process with 21 variables composed of 19 nutritional factors grouped from the 57 components and two environmental factors (initial pH and temperature). For L. lactis IL1403, the maximum biomass concentrations obtained with the two optimal chemically defined media developed in this study (ZMB1 and ZMB2) were generally 3.5- to 4-fold higher than the maximum biomass concentrations obtained with the previously described best synthetic media (SA) and 50% to 68% higher than the maximum biomass concentrations obtained with M17, a complex medium commonly used for lactococci. The new chemically defined media support high-cell-density growth of numerous strains of L. lactis, Enterococcus faecalis, and Streptococcus thermophilus.     

Biomass production and nutrient uptake by <Emphasis Type="Italic">Neochloris oleoabundans</Emphasis> in an open trough system

The purpose of this paper is to present biomass and nutrient uptake data from Neochloris oleoabundans production in an open trough system. The growth medium used was BG11, temperature ranged from 16.7 °C to 25.3 °C, and pH ranged from 5.52 to 9.94 because the customary pH increase during algal biomass production was moderated by incoming CO₂ gas streams (atmospheric, 2%, 4%, and 6% CO₂). Peak concentrations of algal biomass ranged from 643 to 970 mg L⁻¹, specific growth rates ranged from 0.15 to 0.37 day⁻¹, and doubling times ranged from 4.8 to 1.9 days. Carbon, nitrogen, and phosphorus were incorporated into the biomass at 0.05%, 8.3%, and 54% of supplied amounts. Open growth systems supplemented with CO₂ should be designed to regulate medium pH within the range of 6.3 to 7.1. Future research should examine various media and alternative carbon sources to decrease doubling times, increase peak concentrations, and optimize nutrient uptake.     

Erwinia and yellow-pigmentedEnterobacter isolates from human sources

Sixty-five strains of gram-negative, yellow-pigmented bacilli, including four chromogenicEnterobacter strains and 55 anaerogenic and six aerogenicErwinia strains, were isolated from human sources. The genusErwinia contained two groups; an anaerogenic group which produced aggregates of bacteria (symplasmata) in the syneresis water of slant cultures and biconvex bodies in colonies on agar medium, and an aerogenic group which lacked these characteristics.Erwinia was differentiated fromEnterobacter, since the latter possessed dihydrolase and decarboxylase activity and demonstrated resistance to cephalothin.     

Characteristic Decrease of Nuclear Protein Kinase Activity in G1-Arrested Cells of Saccharomyces cerevisiae cdc28

An alkalopsychrotrophic strain, Micrococcus sp. 207, inducibly and extracellularly produced amylase and pullulanase. The main hydrolysis product from amylose, with a crude enzyme preparation, was maltotetraose. The optimum temperature for activity of the amylase was 60°C and that for pullulanase 55°C. The activities at 0 to 30°C exhibited similar activation energy values. In an optimized production medium at pH 9.7, the highest yields of these enzymes were obtained after cell growth at 18°C for 4 days. At pH 8.5, the yields of amylase and pullulanase became maximum after 3 days cultivation. With more prolonged cultivation, the yield of amylase but not that of pullulanase activity decreased. These enzymes were not produced at temperatures above 30°C. Sucrose was not effective as an inducer, but it stimulated cell growth and enhanced the enzyme productivities with soluble starch.     

Interaction of Mercuric Ions with Different Marine Algal Species

The use of different seaweeds such as Sargassum sp., Turbinaria conoides, and Ulva sp. in removing mercury(II) from aqueous solutions were investigated. The initial experimental runs, conducted at different equilibrium pH conditions, demonstrated that brown seaweeds outperformed green seaweed in Hg(II) biosorption at all pH conditions. In particular, at pH 5, maximum biosorption capacities of 170.3 and 145.8 mg/g were recorded for the brown seaweeds T. conoides and Sargassum sp., respectively, compared with 138.4 mg/g for the green seaweed Ulva sp. Isotherm data were modeled and interpreted using the Langmuir, Freundlich, Redlich-Peterson, and Toth models, with the latter described the Hg(II) isotherms with high correlation coefficients and low % error values. The kinetic data indicate the rapidity of the biosorption process, with the equilibrium achieved within 90 min. Several models, including the Elovich, pseudo-first-order, and pseudo-second-order models, were examined for their suitability with the present data; the correlation coefficient and % error values, along with better prediction of equilibrium uptake values, favored the pseudo-first-order model. The desorption experiments were highly successful for T. conoides biomass with 0.05 M HCl, whereas for the other two seaweeds, 0.05 M HCl resulted in high biomass weight loss. Reusing T. conoides biomass in three successive sorption-desorption cycles resulted in only 8.8% reduction in Hg(II) biosorption capacity compared with its original uptake.     

Influence of the fermentation parameters and optimisation of isomaltulose production from free Erwinia sp. D12 cells using response surface methodology

The food industry is constantly seeking novel ingredients to improve existing products or to allow for the introduction of new products. Isomaltulose is a reducing sugar with a sweet taste and very similar physical and organoleptic properties to those of sucrose. The strain Erwinia sp. is able to convert sucrose into isomaltulose. A two level rotatory central composite design and response surface methodology were applied to verify the influence and conditions for the production of isomaltulose by Erwinia sp. D12 free-cells in a batch process. The statistical analysis carried out at a confidence level of 90% gave a coefficient of determination of 0.90, and the polynomial model resulted in a response surface and contour curve that indicated the best parameters for the conversion of sucrose into isomaltulose as follows: temperature 35 °C, pH 6.5, wet cell mass 10% and sucrose 35%. The free-cells of Erwinia sp. D12 were recycled during repeated-batch processes to produce isomaltulose from sucrose obtaining high isomaltulose yields.     

Continuous production of L-aspartic acid by immobilized aspartase

Various methods were tried for the immobilization of aspartase, and the preparation having the highest activity was obtained when partially purified aspartase from Escherichia coli was entrapped into polyacrylamide gel Iattice. Enzymatic properties of the immobilized aspartase were investigated and compared with those of the native aspartase. With regard to optimum pH, temperature, concentration of Mn⁺⁺, kinetic constants and heat stability, no marked difference was observed between the native and immobilized aspartases. By employing an enzyme column packed with the immobilized aspartase, conditions for continuous production of L -aspartic acid from ammonium fumarate were investigated. When a solution of 1M ammonium fumarate (pH 8.5, containing 1mM MnCl₂) was passed through the aspartase column at the flow rate of SV = 0.08 at 37°C, the highest rate of reaction was attained. From the column effluents, L-aspartic acid was obtained in a good yield.