Alginate polycation microcapsules. II. Some functional properties.

The main cause of alginate polycation capsule breakage under physiological conditions is probably the osmotic swelling of the alginate core owing to the Donnan equilibrium set up by the negative charges of the carboxyl groups not involved in cooperative binding of counterions in the junction zones of the network. In the present paper we show how capsules can be stabilized extensively by reducing their swelling capacity in various ways. Alginate polycation capsules with good chemical and mechanical stability have been made by controlling their swelling behaviour through selection of capsule material according to chemical structure and molecular weight, as well as by controlling the kinetics of the capsule formation. Stable capsules have been made either by increasing the strength of the polyanion-polycation membrane, or by keeping a low-swelling gel network in the core. The latter capsules are made from an alginate rich in guluronic acid both in the core and in an outer coating, and with anisotropic distribution of the polymer material in the core where the concentration at the surface is higher than that in the centre of the capsule. Some functional properties of these capsules, such as porosity, have also been studied.

Alginate polycation microcapsules. I. Interaction between alginate and polycation.

The interactions between alginate and polycations have been studied by using different labelling techniques. Binding of poly-L-lysine (PLL) to alginate in the gel state is mainly governed by the amount of dissociable negative charges on the bead surface. PLL was found to bind more rapidly to gel beads made from alginate with a high content of mannuronic acid. The binding was enhanced by increasing the alginate concentration on the surface by making inhomogeneous beads. When the capsules were stored in the presence of cations with high affinity for alginate (Ca2+, Sr2+), PLL was washed off. Less PLL is bound to strontium alginate than to calcium alginate beads. Two mechanisms appear to be responsible for the binding of sodium alginate to alginate PLL capsules (coating): (i) an electrostatic interaction between the soluble coating material and excess positive charges on PLL on the surface; (ii) the formation of a calcium alginate gel on the surface owing to leaching of calcium ions from the core. The stability and efficiency of the coating as a function of molecular size and sequential structure of the coating polymer have also been investigated.

Effects of non-ionic surfactants on the uptake and hydrolysis of fluoresceindiacetate by alkane-oxidizing bacteria.

Biological effects of non-ionic surfactants on alkane-oxidizing bacteria were studied by assessing their influence on the uptake of prefluorochrome fluoresceindiacetate (FDA) and its intracellular hydrolysis to fluorescein. Both decreasing and increasing rates of hydrolysis as a consequence of the presence of surfactants were observed. The surfactants influenced the uptake of FDA, but not its intracellular hydrolysis. The effects of the surfactants on the uptake rate depended strongly on the structure and physico-chemical properties of the surfactants. There was no qualitative or significant quantitative difference in surfactant susceptibility between induced (alkane grown) and non-induced bacteria (acetate grown), even though the induced cells possess greater cell surface hydrophobicity.

Chemically emulsified crude oil as substrate for bacterial oxidation: differences in species response.

Four bacterial species were tested for their abilities to oxidize alkanes in crude oil in water emulsions. The emulsions were prepared by nonionic sorbitan ester and polyoxyethylene ether surfactants. The oxidation rates were measured as initial attack on the emulsions by resting cells pregrown in crude oil media. The bacteria responded differently and both positive and negative effects of surfactant amendment were observed. The same surfactant affected various bacteria differently and the response to the surfactant amendment depended on the physiological state of the bacteria, i.e., exponential versus stationary growth phase. The surfactants caused a marked decrease in cell adhesion to the oil phase for all the bacteria, irrespective of the growth phase and any positive effect on the oil oxidation rates. The response of Deleya salina 128 to polyoxyethylene surfactant emulsified crude oil depended on the length and structure of the hydrophobic tail, the number of hydrophilic ethoxy groups, and the relative proportion of the hydrophobic and hydrophilic constituents in the individual surfactant.

Bacterial degradation of emulsified crude oil and the effect of various surfactants.

A Rhodococcus sp. 094 bacterium was tested for its ability to oxidize alkanes in crude oil emulsified by nonionic chemical and biological surfactants. Oxidation rates were measured in a 3-h period by Warburg respirometry. 14CO2 recovery was measured from the [1-14C]hexadecane spiked crude oil. Response to emulsified oil depended on the physiological state of the bacteria (i.e., cells harvested in the exponential and stationary growth phases) were tested. Oxidation rates by cells in the exponential growth phase were negatively affected by surfactant amendment. Oxidation rates by cells in the stationary growth phase were in some cases stimulated by surfactants. The stimulatory effect depended on both the chemical structure and the physicochemical properties (i.e., hydrophilic-lipophilic balance (HLB)) of the surfactants. Surfactants with intermediate HLB values (8-12) gave the best results. Neither the biosurfactants nor the commercial oil-spill dispersants tested had any significant stimulatory effect.

Effects of surfactant mixtures, including Corexit 9527, on bacterial oxidation of acetate and alkanes in crude oil.

Mixtures of nonionic and anionic surfactants, including Corexit 9527, were tested to determine their effects on bacterial oxidation of acetate and alkanes in crude oil by cells pregrown on these substrates. Corexit 9527 inhibited oxidation of the alkanes in crude oil by Acinetobacter calcoaceticus ATCC 31012, while Span 80, a Corexit 9527 constituent, markedly increased the oil oxidation rate. Another Corexit 9527 constituent, the negatively charged dioctyl sulfosuccinate (AOT), strongly reduced the oxidation rate. The combination of Span 80 and AOT increased the rate, but not as much as Span 80 alone increased it, which tentatively explained the negative effect of Corexit 9527. The results of acetate uptake and oxidation experiments indicated that the nonionic surfactants interacted with the acetate uptake system while the anionic surfactant interacted with the oxidation system of the bacteria. The overall effect of Corexit 9527 on alkane oxidation by A. calcoaceticus ATCC 31012 thus seems to be the sum of the independent effects of the individual surfactants in the surfactant mixture. When Rhodococcus sp. strain 094 was used, the alkane oxidation rate decreased to almost zero in the presence of a mixture of Tergitol 15-S-7 and AOT even though the Tergitol 15-S-7 surfactant increased the alkane oxidation rate and AOT did not affect it. This indicated that there was synergism between the two surfactants rather than an additive effect like that observed for A. calcoaceticus ATCC 31012.

A theoretical analysis of the biosynthesis of actinorhodin in a hyper-producing Streptomyces lividansstrain cultivated on various carbon sources.

A stoichiometric equation for the biosynthesis of actinorhodin (ACT) was derived taking into consideration both the requirements of the carbon precursors (acetyl-CoA) and reducing power (NADPH). The estimate for reducing power was derived from a detailed molecular analysis of each step in the ACT biosynthetic pathway. Even though ACT is slightly more oxidized than most carbon substrates, e.g. glucose, reducing power (NADPH and NADH) is necessary due to reducing steps and to monooxygenase steps. The equation was used to evaluate, in a metabolic network context, the experimental results from batch fermentations with eight different carbon sources using a Streptomyces lividans 1326 derived strain containing the pathway-specific activator gene ( actII-ORF4) on a multicopy plasmid (pIJ68). The yield of ACT on the various carbon sources ranged from 0.04 to 0.18 Cmol ACT/Cmol carbon source in the stationary phase. Glucose was the best carbon source and supported a yield of 25% of the maximum theoretical yield. There are no obvious constraints in the primary metabolic pathways that can explain why the various carbon sources allowed different levels of ACT production, because their potential for supplying acetyl-CoA and NADPH are far from fully utilized. For the observed ACT yields, there is an excess production of NADPH that has to be reoxidized either by a transhydrogenase or a NADPH oxidase. This study discusses the central metabolic pathways, focusing on providing precursors for ACT synthesis.

High-yield actinorhodin production in fed-batch culture by a Streptomyces lividans strain overexpressing the pathway-specific activator gene actll-ORF4.

Streptomyces lividans 1,326 usually does not produce the red/blue colored polyketide actinorhodin in liquid culture even though it carries the entire actinorhodin biosynthesis gene cluster. The bacterium can be forced to produce this secondary metabolite by introducing actII-ORF4, the actinorhodin pathway-specific activator gene from Streptomyces coelicolor, on a multicopy plasmid. The production of actinorhodin by such a strain has been optimized by medium and process manipulations in fed-batch cultures. With high-yield cultivation conditions, 5 g actinorhodin/l are produced during 7 days of cultivation; or approximately 0.1 g actinorhodin/g dry weight (DW)/day in the production phase. The yield in this phase is 0.15 Cmol actinorhodin/Cmol glucose, which is in the range of 25% to 40% of the maximum theoretical yield. This high-level production mineral medium is phosphate limited. In contrast, nitrogen limitation resulted in low-level production of actinorhodin and high production of a-ketoglutaric acid. Ammonium as nitrogen source was superior to nitrate supporting an almost three times higher actinorhodin yield as well as a two times higher specific production rate. The wild-type strain lacking the multicopy plasmid did not produce actinorhodin when cultivated under any of these conditions. This work examines the actinorhodin-producing potential of the strain, as well as the necessity to improve the culture conditions to fully utilize this potential. The overexpression of biosynthetic pathway-specific activator genes seems to be a rational first step in the design of secondary metabolite overproducing strains prior to alteration of primary metabolic pathways for redirection of metabolic fluxes.

Continuous cultivations of a Penicillium chrysogenum strain expressing the expandase gene from Streptomyces clavuligerus: Kinetics of adipoyl-7-aminodeacetoxycephalosporanic acid and byproduct formations.

The production kinetics of a transformed strain of Penicillium chrysogenum expressing the expandase gene from Streptomyces clavuligerus was investigated in chemostat cultivations. The recombinant strain produces adipoyl-7-aminodeacetoxycephalosporanic acid (ad-7-ADCA) as the major product; however, during the cultivations, the appearance of a major unknown and poorly secreted product was observed. Investigations using high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS) showed that this byproduct has a six-membered dihydrothiazine ring, which is characteristic for cephalosporins. The byproduct may be formed via isopenicillin N by as-yet unknown mechanisms, but involving expandase. It is likely that the unknown compound (UC) is deacetoxycephalosporin C (DAOC). Investigation of the instability of the various beta-lactams produced showed higher instability for compounds with a five-membered thiazolidine ring than those with a six-membered dihydrothiazine ring. Furthermore, secretion of products and byproducts was shown to be quite different. The productivity was studied as a function of the dilution rate in the range 0.015 to 0.090 h(-1). The specific productivity of total beta-lactams was compared with that of the penicillin-G-producing host strain, and it was found to be lower at dilution rates of <0.06 h(-1). Quantification of the fluxes through the pathway leading to ad-7-ADCA showed a decrease in flux toward ad-7-ADCA, and an increase in flux toward UC as the dilution rate increased. Northern analysis of the biosynthetic genes showed that expression of the enzymes involved in the ad-7-ADCA pathway decreased as the dilution rate increased.

Deletion of scbA enhances antibiotic production in Streptomyces lividans.

Antibiotic production in many streptomycetes is influenced by extracellular gamma-butyrolactone signalling molecules. In this study, the gene scbA, which had been shown previously to be involved in the synthesis of the gamma-butyrolactone SCB1 in Streptomyces coelicolor A3(2), was deleted from the chromosome of Streptomyces lividans 66. Deletion of scbA eliminated the production of the antibiotic stimulatory activity previously associated with SCB1 in S. coelicolor. When the S. lividans scbA mutant was transformed with a multi-copy plasmid carrying the gene encoding the pathway-specific activator for either actinorhodin or undecylprodigiosin biosynthesis, production of the corresponding antibiotic was elevated significantly compared to the corresponding scbA(+) strain carrying the same plasmid. Consequently, deletion of scbA may be useful in combination with other strategies to construct host strains capable of improved bioactive metabolite production.