Methods for rapid screening and isolation of bacteria producing acidic lipase: feasibility studies and novel activity assay protocols

Acidic lipase finds its commercial values in medical applications and bioremediation of food wastes. In this work, approaches for rapid screening of lipase-producing bacteria were developed and the feasibility assessment of the screening methods was performed. From food waste samples, the proposed screening procedures allowed isolation of sixteen pure bacterial strains expressing higher lipase activity at acidic pH (pH 6.0) than at alkaline pH (pH 9.0). To enhance the accuracy of lipase activity determination under acidic conditions, a novel assay procedure was also developed by deactivating lipase activity by microwave treatment prior to back titration. This additional step could minimize interferences arising from residual lipase activity during conventional direct back-titration methods in measuring lipase activity at acidic pH. Using the four strategies proposed in this work, the best acidic-lipase-producing isolate was obtained by strategy C (SSC) and was identified as Aeromonas sp. C14, displaying an optimal lipase activity of 0.7 U/ml at an acidic pH of 6.0.     

Plant Anaerobic Stress as a Novel Trend in Ecological Physiology,Biochemistry, and Molecular Biology: 1. Establishment of a New Scientific Discipline

This review attempted to follow the establishment of a novel branch of biology arisen at the interfaces between plant physiology, biochemistry, and molecular biology—plant anaerobic stress. Most attention was given to the early period of these investigations, the activity of the members of International Society for Plant Anaerobiosis in particular, and the contribution of Russian scientists, who played a significant role at that time in the establishment and international recognition of this new trend. In this connection, the following points are considered: (1) Crawford's metabolic theory, which could not withstand experimental verification but induced an active discussion, thus stimulating further investigations in this field; (2) a concept of two main strategies of plant adaptation to anaerobic stress (true and apparent adaptation), which was put forward based on the following experimental data: (a) a discovery of a paradoxical phenomenon of hyper-sensitivity, but not hyper-resistance to anoxia, of the flood-tolerant plant roots (“apparent” tolerance); (b) the elucidation of the physiological role of oxygen transported from aerated organs of flood-tolerant plants to the roots inhabiting anaerobic environment; (c) demonstration of the key role of both energy metabolism, and (d) substrate providing for glycolysis and ethanolic fermentation in plants manifesting “true” tolerance to oxygen deprivation; (3) the discovery of plant stress proteins; and finally (4) pH-stat theory put forward by Davies.     

Extent of intracellular pH changes during H+ extrusion by maize root-tip cells

³¹P-Nuclear-magnetic-resonance spectra of maize (Zea mays L.) root tips, that had been induced to extrude large amounts of H⁺ in response to fusicoccin (FC) in the presence of potassium salts, indicate that the cytoplasmic pH does not become higher than that of controls. In fact, the cytoplasmic pH may become slightly (approx. 0.1 pH unit) lower in cells extruding H⁺. Estimations of the buffer capacity of the cells show that without active intracellular pH regulation, H⁺ extrusion caused by FC would cause the intracellular pH to rise by at least 0.6 pH unit h^-1. Our results indicate that intracellular pH is tightly regulated even during extreme rates of acid extrusion, and that a rise in cytoplasmic pH is not the signal linking H⁺ extrusion with enhanced organic-acid synthesis or other intracellular responses to H⁺ pumping.Abbreviations FC fusicoccin - P_i inorganic phosphate - NMR nuclear magnetic resonance - chemical shift - MDP methylene diphosphonic acid     

Polymeric and Compositional Properties of Novel Extracellular Microbial Polyglucosamine Biopolymer from New Strain of Citrobacter sp. BL-4

A novel polyglucosamine polymer, PGB-2, was produced extracellularly from a new strain Citrobacter sp. BL-4 using pH-stat fed batch cultivation. It was composed of 97.3% glucosamine and 2.7% rhamnose; its average molecular weight, solubility in 2% acetic acid and viscosity were 20 kDa, 5 g l⁻¹ and 2.9 cps, respectively. FT-IR and ¹H NMR spectra of PGB-2 revealed a close identity with chitosan from crab shells. Received 20 September 2005; Revisions requested 6 October 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Fermentative production of P(3HB-co-3HV) from propionic acid by Alcaligenes eutrophus in fed-batch culture with pH-stat continuous substrate feeding method

The feeding of propionic acid for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] by Alcaligenes eutrophus ATCC17697 was optimized using a fed-batch culture system. The concentration of propionic acid was maintained at 3 g l^–1 as growth was inhibited by propionic acid in the broth. A pH-stat substrate feeding system was used in which propionic acid was fed automatically to maintain a pH of the culture broth at 7.0. By feeding a substrate solution containing 20% (w/v) propionic acid, 4.9% (w/v) ammonia water [at a molar ratio of carbon to nitrogen (C/N molar ratio) of 10] in cell growth phase, the concentration of propionic acid in the broth was maintained at 3 g l^–1 giving a specific growth rate of 0.4 h^–1. To promote P(3HB-co-3HV) production, two stage fed-batch culture which consisted of the stage for the cell growth and the stage for the P(3HB-co-3HV) accumulation was carried out. When the substrate solution whose C/N molar ratio was 50 was fed in P(3HB-co-3HV) accumulation phase, the cell concentration and the P(3HB-co-3HV) content in the cells reached 64 g l^–1 and 58% (w/w) in 55.5 h, respectively.     

A sensitive computer-controlled pH-stat system allows the study of net H+ fluxes related to nitrogen uptake of intact plants in situ

Proton fluxes related to the acid–base balance of intact plants were investigated in detail. A multi-channel titration system was developed in order to regulate the pH in two different sets of nutrient solutions. This system also allowed computation of the dynamics of proton fluxes associated with nutrient uptake in situ. The pH-stat system presented here has proved to be very reliable and sensitive. By additions of acid or base to the nutrient solutions, the set pH could typically be maintained within 0·01 pH units. Experiments investigating net proton fluxes correlated with nitrogen uptake are described. The results show a rapid response of proton fluxes to changes in the form of nitrogen supplied, indicating that alterations in net proton fluxes are directly induced by the nature of the nitrogen source. The stoichiometry of proton fluxes connected to nitrogen uptake could be followed online, and the results are discussed in relation to the charge and acid–base balances of the whole plant.     

An effective and simplified pH-stat control strategy for the industrial fermentation of vitamin B12 by Pseudomonas denitrificans

In order to improve the productivity of vitamin B(12) by Pseudomonas denitrificans carried out in a 120-m(3) fermenter, the effect of pH on vitamin B(12) biosynthesis was investigated. Results obtained from shake flask experiments showed that the feeding of carbon source (beet molasses or glucose) and methyl-group donor (betaine or choline chloride) significantly influenced the pH and the biosynthesis of vitamin B(12). In contrast to beet molasses or choline chloride, using glucose as a feed medium and betaine as a methyl-group donor, pH could be maintained at a stable range. As a result, higher vitamin B(12) production was achieved. Accordingly, an effective and simplified pH-stat control strategy was established for the fermentation of vitamin B(12) in a 120-m(3) industrial fermenter. When the new pH control strategy was applied, pH was stably kept in the range of 7.15-7.30 during fermentation. Thus, 214.3 mug/mL of vitamin B(12) was achieved.     

Controlling the sucrose concentration increases Coenzyme Q10 production in fed-batch culture of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

The production yield of Coenzyme Q₁₀ (CoQ₁₀) from the sucrose consumed by Agrobacterium tumefaciens KCCM 10413 decreased, and high levels of exopolysaccharide (EPS) accumulated after switching from batch culture to fed-batch culture. Therefore, we examined the effect of sucrose concentration on the fermentation profile by A. tumefaciens. In the continuous fed-batch culture with the sucrose concentration maintained constantly at 10, 20, 30, and 40 g l⁻¹, the dry cell weight (DCW), specific CoQ₁₀ content, CoQ₁₀ production, and the production yield of CoQ₁₀ from the sucrose consumed increased, whereas EPS production decreased as maintained sucrose concentration decreased. The pH-stat fed-batch culture system was adapted for CoQ₁₀ production to minimize the concentration of the carbon source and osmotic stress from sucrose. Using the pH-stat fed-batch culture system, the DCW, specific CoQ₁₀ content, CoQ₁₀ production, and the product yield of CoQ₁₀ from the sucrose consumed increased by 22.6, 13.7, 39.3, and 39.3%, respectively, whereas EPS production decreased by 30.7% compared to those of fed-batch culture in the previous report (Ha SJ, Kim SY, Seo JH, Oh DK, Lee JK, Appl Microbiol Biotechnol, 74:974–980, 2007). The pH-stat fed-batch culture system was scaled up to a pilot scale (300 l), and the CoQ₁₀ production results obtained (626.5 mg l⁻¹ of CoQ₁₀ and 9.25 mg g DCW⁻¹ of specific CoQ₁₀ content) were similar to those obtained at the laboratory scale. Thus, an efficient and highly competitive process for microbial CoQ₁₀ production is available.     

Repeated pH-stat fed-batch fermentation for rhamnolipid production with indigenous Pseudomonas aeruginosa S2

Rhamnolipid is one of the most commonly used biosurfactants with the ability to reduce the surface tension of water from 72 to 30 mN/m. An indigenous isolate Pseudomonas aeruginosa S2 possessing excellent ability to produce rhamnolipid was used as a model strain to explore fermentation technology for rhamnolipid production. Using optimal medium and operating conditions (37°C, pH 6.8, and 250 rpm agitation) obtained from batch fermentation, P. aeruginosa S2 was able to produce up to 5.31 g/l of rhamnolipid from glucose-based medium. To further improve the rhamnolipid yield, a pH-stat fed-batch culture was performed by maintaining a constant pH of 6.8 through manipulating glucose feeding. The effect of influent glucose concentration on rhamnolipid yield and productivity was investigated. Using the pH-stat culture, a maximum rhamnolipid concentration (6.06 g/l) and production rate (172.5 ml/h/l) was obtained with 6% glucose in the feed. Moreover, combining pH-stat culture with fill-and-draw operation allowed a stable repeated fed-batch operation for approximately 500 h. A marked increase in rhamnolipid production was achieved, leading to the best rhamnolipid yield of approximately 9.4 g/l during the second repeated run.     

Manipulating cytoplasmic pH under anoxia: A critical test of the role of pH in the switch from aerobic to anaerobic metabolism

Ethanol production by maize (Zea mays L.) root tips, measured by an enzymic assay of the suspending medium, was correlated with changes in the cytoplasmic pH, determined by in-vivo ³¹P nuclear magnetic resonance (NMR) spectroscopy, following the onset of anoxia. Strong evidence for the role of the cytoplasmic pH in triggering the switch to ethanol production under anoxia was obtained by: (i) varying the pH of the suspending medium between pH 4 and pH 10; and (ii) using the permeant weak base methylamine to combat the acidification of the cytoplasm induced by the anoxic conditions. Experimentally, it proved to be much easier to manipulate the cytoplasmic pH under anoxia after the pH had stabilised, rather than during the initial rapid acidification that occurred following the onset of anoxia, and in the presence of methylamine, it was possible to impose a normal aerobic cytoplasmic pH value on tissue that was metabolising anaerobically. By this means it was possible to demonstrate the reversibility of the pH effect on ethanol production under anoxia and thus to provide good evidence in support of the biochemical pH-stat model of the anoxic response. The NMR measurement of the cytoplasmic pH in the presence of methylamine was achieved by using a manganese pretreatment technique to eliminate interference between the cytoplasmic and vacuolar P_i signals, and it seems likely that the experimental approach used here will have further applications in studies of the metabolic response to anoxia.Abbreviations Caps 3-(cyclohexylamino)-1-propane sulphonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - NMR nuclear magnetic resonance - Pi inorganic phosphate We acknowledge the financial support of the Agricultural and Food Research Council and G.G.F. acknowledges the receipt of a Research Fellowship from the Royal Commission for the Exhibition of 1851.