Structural characterization and surface activities of biogenic rhamnolipid surfactants from Pseudomonas aeruginosa isolate MN1 and synergistic effects against methicillin-resistant Staphylococcus aureus

The aim of present work was to study chemical structures and biological activities of rhamnolipid biosurfactants produced by Pseudomonas aeruginosa MN1 isolated from oil-contaminated soil. The results of liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis revealed that total rhamnolipids (RLs) contained 16 rhamnolipid homologues. Di-lipid RLs containing C₁₀-C₁₀ moieties were by far the most predominant congeners among mono-rhamnose (53.29?%) and di-rhamnose (23.52?%) homologues. Mono-rhamnolipids form 68.35?% of the total congeners in the RLs. Two major fractions were revealed in the thin layer chromatogram of produced RLs which were then purified by column chromatography. The retardation factors (R _f) of the two rhamnolipid purple spots were 0.71 for RL1 and 0.46 for RL2. LC-MS/MS analysis proved that RL1 was composed of mono-RLs and RL2 consisted of di-RLs. RL1 was more surface-active with the critical micelle concentration (CMC) value of 15?mg/L and the surface tension of 25 mN/m at CMC. The results of biological assay showed that RL1 is a more potent antibacterial agent than RL2. All methicillin-resistant Staphylococcus aureus (MRSA) strains were inhibited by RLs that were independent of their antibiotic susceptibility patterns. RLs remarkably enhanced the activity of oxacillin against MRSA strains and lowered the minimum inhibitory concentrations of oxacillin to the range of 3.12?C6.25???g/mL.     

A new assay for rhamnolipid detection—important virulence factors of Pseudomonas aeruginosa

Rhamnolipids (RLs) are heterogeneous glycolipid molecules that are composed of one or two l-rhamnose sugars and one or two β-hydroxy fatty acids, which can vary in their length and branch size. They are biosurfactants, predominantly produced by Pseudomonas aeruginosa and are important virulence factors, playing a major role in P. aeruginosa pathogenesis. Therefore, a fast, accurate and high-throughput method of detecting such molecules is of real importance. Here, we illustrate the ability to detect RL-producing P. aeruginosa strains with high sensitivity, based on an assay involving phospholipid vesicles encapsulated with a fluorescent dye. This vesicle-lysis assay is confirmed to be solely sensitive to RLs. We illustrate a half maximum concentration for vesicle lysis (EC₅₀) of 40 μM (23.2 μg/mL) using pure commercial RLs and highlight the ability to semi-quantify RLs directly from the culture supernatant, requiring no extra extraction or processing steps or technical expertise. We show that this method is consistent with results from thin-layer chromatography detection and dry weight analysis of RLs but find that the widely used orcinol colorimetric test significantly underestimated RL quantity. Finally, we apply this methodology to compare RL production among strains isolated from either chronic or acute infections. We confirm a positive association between RL production and acute infection isolates (p?=?0.0008), highlighting the role of RLs in certain infections.     

Volatile organic compound emissions from Microcystis aeruginosa under different phosphorus sources and concentrations

In eutrophicated water, cyanobacteria massively grow and release an abundance of volatile organic compounds (VOCs) that contribute to the water odor. To uncover the effects of different phosphorus (P) nutrients on the formation of cyanobacteria VOCs and water odor, the cell growth and VOC emissions of Microcystis aeruginosa were investigated under different P nutrient conditions. Among K₂HPO₄, Na₄P₂O₇, and (NaPO₃)₆, K₂HPO₄ showed the largest increase in cell density, while a reduction in P concentration decreased the cell density. There were 26, 23 and 22 compounds in M. aeruginosa VOCs with K₂HPO₄, Na₄P₂O₇ and (NaPO₃)₆ as the sole P source, respectively, including sulfocompounds, terpenoids, benzenes, hydrocarbons, alcohols, aldehydes, and esters. Non‐P markedly promoted the VOC emission, and six additional compounds were observed: α‐pinene, 1‐phenyl‐1‐butanone, 1H‐1‐ethylidene‐indene, 2,6,10‐trimethyl‐tetradecane, 2‐ethyl‐hexanal, and acetic acid 2‐ethylhexyl ester. It can be deduced that cyanobacteria release different VOC blends using various P forms in eutrophicated waters, and the reduction of P amount promotes VOC emission and increases the water odor.     

Novel rhamnolipid biosurfactants produced by a polycyclic aromatic hydrocarbon-degrading bacterium Pseudomonas aeruginosa strain NY3

A novel rhamnolipid biosurfactant-producing and Polycyclic Aromatic Hydrocarbon (PAH)-degrading bacterium Pseudomonas aeruginosa strain NY3 was isolated from petroleum-contaminated soil samples. Strain NY3 was characterized by its extraordinary capacity to produce structurally diverse rhamnolipids. A total of 25 rhamnolipid components and 37 different parent molecular ions, representing various metal ion adducts (Na⁺, 2Na⁺ and K⁺), were detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Among these compounds are ten new rhamnolipids. In addition to its biosurfactant production, strain NY3 was shown to be capable of efficient degradation of PAHs as well as synergistic improvement in the degradation of high molecular weight PAHs by its biosurfactant. These findings have added novel members to the rhamnolipid group and expanded current knowledge regarding the diversity and productive capability of rhamnolipid biosurfactants from a single specific strain with variation of only one carbon source. Additionally, this paper lays the foundation for improvement in the yield of NY3BS and study of the degradation pathway(s) of PAHs in P. aeruginosa strain NY3.     

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH₄) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH₄ by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH₄ in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared with the parent strain, solely from CH₄. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH₄.     

A novel identified Pseudomonas aeruginosa,which exhibited nitrate- and nitrite-dependent methane oxidation abilities,could alleviate the disadvantages caused by nitrate supplementation in rumen fluid fermentation

After the occurrence of nitrate-dependent anaerobic methane oxidation (AMO) in rumen fluid culture was proved, the organisms that perform the denitrifying anaerobic methane oxidizing (DAMO) process in the rumen of dairy goat were investigated by establishing two enrichment culture systems, which were supplied with methane as the sole carbon source and NaNO₃ or NaNO₂ as the electron acceptor. Several Operational Taxonomic Units (OTU) belonging to Proteobacteria became dominant in the two enrichment systems. The identified Pseudomonas aeruginosa, which was isolated from the NaNO₂ enrichment system, could individually perform a whole denitrifying anaerobic methane oxidizing process. Further in vitro rumen fermentation showed that supplementation with the isolated P. aeruginosa could reduce methane emissions, alleviate the nitrite accumulation and prevent the decrease in propionic acid product caused by nitrate supplementation.     

High level phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 563 in solid state culture: response surface optimization,up-scaling,and its partial characterization

Phytase production by Aspergillus niger NCIM 563 was optimized by using wheat bran in solid state fermentation (SSF). An integrated statistical optimization approach involving the combination of Placket–Burman design (PBD) and Box–Behnken design (BBD) was employed. PBD was used to evaluate the effect of 11 variables related to phytase production, and five statistically significant variables, namely, glucose, dextrin, NaNO₃, distilled water, and MgSO₄·7H₂O, were selected for further optimization studies. The levels of five variables for maximum phytase production were determined by a BBD. Phytase production improved from 50 IU/g dry moldy bran (DMB) to 154 IU/g DMB indicating 3.08-fold increase after optimization. A simultaneous reduction in fermentation time from 7 to 4 days shows a high productivity of 38,500 IU/kg/day. Scaling up the process in trays gave reproducible phytase production overcoming industrial constraints of practicability and economics. The culture extract also had 133.2, 41.58, and 310.34 IU/g DMB of xylanase, cellulase, and amylase activities, respectively. The partially purified phytase was optimally active at 55°C and pH 6.0. The enzyme retained ca. 75% activity over a wide pH range 2.0–9.5. It also released more inorganic phosphorus from soybean meal in a broad pH range from 2.5 to 6.5 under emulated gastric conditions. Molecular weight of phytase on Sephacryl S-200 was approximately 87 kDa. The K _m and V _max observed were 0.156 mM and 220 μm/min/mg. The SSF phytase from A. niger NCIM 563 offers an economical production capability and its wide pH stability shows its suitability for use in poultry feed.     

Enhanced curdlan production in <Emphasis Type="Italic">Agrobacterium</Emphasis> sp. ATCC 31749 by addition of low-polyphosphates

A large amount of adenosine triphosphate with high energy phosphate bonds is required for uridine triphosphate regeneration during curdlan biosynthesis by Agrobacterium sp. ATCC 31749. To supply high energy for curdlan synthesis, three low-polyphosphates (Na₄P₂O₇, Na₅P₃O₁₀, and (NaPO₃)₆) with higher energy phosphate bonds were employed to substitute for KH₂PO₄-K₂HPO₄ in fermentation medium. Two genes encoding the polyphosphate metabolizing enzymes, polyphosphate kinase and exopolyphosphatase, were amplified and showed 95% homology to those in Agrobacterium sp. C58 by sequence analysis. The curdlan yields were enhanced by 23 and 134% when phosphate concentrations 0.024 mol/L of Na₅P₃O₁₀ and 0.048 mol/L of (NaPO₃)₆ respectively, were added in the medium. The maximum curdlan yield of 30 ± 1.02 g/L was obtained with the addition of 0.048 mol/L of (NaPO₃)₆ with 5 g/L CaCO₃ in the medium. When CaCO₃ was removed from the culture and the three lowpolyphosphates were added, the pH and biomass yield dropped remarkably and little or no curdlan was produced. The culture containing 0.048 mol/L of (NaPO₃)₆ was mixed with KH₂PO₄-K₂HPO₄ and CaCO₃ in the medium, but showed no effect on curdlan production. However, curdlan yield was improved by 49 ∼ 60% when CaCO₃ was removed from the medium and KH₂PO₄-K₂HPO₄ acted as a buffer. It appears that the positive effect of (NaPO₃)₆ on curdlan production required the buffering capacity of CaCO₃ and the absence of KH₂PO₄-K₂HPO₄ competing as a phosphate supplier.     

Salt tolerance of two differently drought-tolerant wheat genotypes during germination and early seedling growth

Summary Osmotic and specific ion effect are the most frequently mentioned mechanisms by which saline substrates reduce plant growth. However, the relative importance of osmotic and specific ion effect on plant growth seems to vary depending on the drought and/or salt tolerance of the plant under study. We studied the effects of several single salts of Na⁺ and Ca²⁺−NaCl, NaNO₃, Na₂SO₄, NaHCO₃, Na₂CO₃, and Ca(NO₃)₂—on the germination and root and coleoptile growth of two wheat (Triticum aestivum L.) cultivars, TAM W-101 and Sturdy, the former being more drought tolerant than the latter. The concentrations used were: 0, 0.02, 0.04, 0.08, 0.16, and 0.32 mol L⁻¹. Significant two- and three-way interactions were observed between cultivar, kind of salt, and salt concentration for germination, growth of coleoptile and root, and root/coleoptile ratio. Salts differed significantly (P<0.001) in their effect on seed germination, coleoptile and root growth of both cultivars. Germination of TAM W-101 seeds was consistently more tolerant than that of Sturdy to NaCl, CaCl₂, Ca(NO₃)₂, and NaHCO₃ salts at concentrations of 0.02, 0.04, 0.08, 0.16 mol L⁻¹. The osmotic potential, at which the germination of wheat seeds was reduced to 50% of that of the control, was different depending on the kind of salt used in the germination medium. NaCl at low concentrations (0.02 and 0.04 mol L⁻¹) stimulated the germination of both wheat cultivars. At concentrations of 0.02 to 0.16 mol L⁻¹, Ca²⁺ salts (CaCl₂ and Ca(NO₃)₂) were consistently more inhibitory than the respective Na⁺ salts (NaCl and NaNO₃) for germination of Sturdy. This did not consistently hold true for TAM W-101. Among the Na⁺ salts, NaCl was the least toxic and NaHCO₃ and Na₂CO₃ were the most toxic for seed germination. Root and coleoptile (in both wheat cultivars) differed in their response to salts. This differential response of coleoptile and root to each salt resulted in seedlings with a wide range of root/coleoptile ratios. For example, the root/coleoptile ratio of cultivar TAM W-101 changed from 2.09 (in the control) to 3.77, 3.19, 2.8, 2.44, 1.31, 0.32, and 0.0 when subjected to 0.08 mol L⁻¹ of Na₂SO₄, NaCl, CaCl₂, NaNO₃, Ca(NO₃)₂, NaHCO₃, and Na₂CO₃, respectively. Na₂CO₃ at 0.08 mol L⁻¹ inhibited root growth to such an extent that germinated wheat seeds contained coleoptile but no roots. The data indicate that, apart from the clear and more toxic effects of NaHCO₃ and Na₂CO₃ and lesser toxic effect of NaCl on germination and seedling growth, any toxicity-ranking of other salts done at a given concentration and for a given tissue growth may not hold true for other salt concentrations, other tissues and/or other cultivars. The more drought-tolerant TAM W-101, when compared to the less drought tolerant Sturdy, showed higher tolerance (at most concentrations) to NaCl, CaCl₂, Ca(NO₃)₂ and NaHCO₃ during its seed germination and to Na₂SO₄ and CaCl₂ for its root growth. This supports other reports that some drought-tolerant wheat cultivars are more tolerant to NaCl. In contrast, the coleoptile growth of drought-sensitive Sturdy was noticeably more tolerant to NaNO₃, Ca(NO₃)₂ and NaHCO₃ than that of drought-tolerant TAM W-101. Based on the above and the different root/coleoptile ratios observed in the presence of various salts, it is concluded that in these wheat cultivars: a) coleoptile and root tissues are differently sensitive to various salts, and b) at the germination stage, tolerance to certain salts is higher in the more drought-tolerant cultivar.     

INORGANIC NITROGEN NUTRITION OF THE SEEDLINGS OF THE ORCHID,CATTLEYA

When grown in vitro in a medium containing NH₄NO₃ as the sole source of nitrogen, seeds ro the orchid, Cattleya (C. labiata ‘Wonder’ X C. labiata ‘Treasure'), germinated readily and proceeded to form small plantlets. Development of the embryos was accompanied by an increase in their total nitrogen and a decline in the percent dry weight. Growth responses of the seedlings in other ammonium salts like (NH₄)₂SO₄, (NH₄)₂HPO₄, NH₄Cl, ammonium acetate and ammonium oxalate were similar to that in NH₄NO₃. However, when grown in a medium containing NaNO₃, development of the seedlings was drastically inhibited; KNO₃, Ca(NO₃)₂, KNO₂ and NaNO₂ also were poor nitrogen sources. Attempts to grow the seedlings in NaNO₃ by changing the pH or by addition of kinetin, molybdenum or ascorbic acid as supplements were completely unsuccessful. When seedlings growing in NH₄NO₃ for varying periods were transferred to NaNO₃, it was found that those plants allowed to grow for 60 or more days in NH₄NO₃ could resume normal growth thereafter in NaNO₃. Determination of the nitrate reductase activity in seedlings of different ages grown in NaNO₃, after NH₄NO₃, showed that the ability of the seedlings to assimilate inorganic nitrogen was paralleled by the appearance of the enzyme.     

Induced change in <Emphasis Type="Italic">Arthrospira</Emphasis> sp. (<Emphasis Type="Italic">Spirulina</Emphasis>) intracellular and extracellular metabolites using multifactor stress combination approach

The interactive effects of light intensity, NaCl, nitrogen, and phosphorus on intracellular biomass content and extracellular polymeric substance production were assessed for Arthrospira sp. (Spirulina) in a two-phase culture process using principal component analysis and central composite face design. Under high light intensity (120 μmol photons m^?2?s^?1) and low NaCl (1 gL^?1), NaNO₃, and K₂HPO₄ (0.5 g L^?1), the carbohydrate content was maximized to 26.61%. Interaction of both K₂HPO₄ (1.6 gL^?1) and NaCl (1.19 gL^?1) with low NaNO₃ (0.5 gL^?1) achieved the maximum content of lipids (15.62%), while high NaCl (40 gL^?1), K₂HPO₄, and NaNO₃ (4.5 gL^?1) enhanced mainly total carotenoids (0.85%). Conversely, under low light intensity of 10 μmol photons m^?2?s^?1 combined with 11.76 gL^?1 of NaCl, 0.5 gL^?1 of NaNO₃, and 2.68 gL^?1 of K₂HPO₄, the phycobiliprotein content reached its highest level (16.09%). The maximum extracellular polymeric substance (EPS) production (0.902 gg^?1?DW) was triggered under moderate light of 57.25 μmol photons m^?2?s^?1 and interaction of high NaCl (40 gL^?1) and K₂HPO₄ (4.5 gL^?1) with low NaNO₃ (0.5 gL^?1). The maximization ratios of intracellular biomass content in terms of carbohydrate, lipid, total carotenoid, phycobiliprotein, and EPS production were 3.55-, 1.73-, 9.55-, 2.92-, and 1.46-fold, respectively, greater than those obtained at optimal growth conditions. This study demonstrated that the multiple stress factors applied to the adopted two-phase culture process could be a promising strategy to produce biomass enriched in various high-value compound.     

Optimization of novel halophilic lipase production by Fusarium solani strain NFCCL 4084 using palm oil mill effluent

Among different sources of lipases, fungal lipases have continued to attract a wide range of applications. Further, halophilic lipases are highly desirable for biodiesel production due to the need to mitigate environmental pollution caused as result of extensive use of fossil fuels. However, currently, the high production cost limits the industrial application of lipases. In order to address this issue, we have attempted to optimize lipase production by Fusarium solani NFCCL 4084 and using palm oil mill effluent (POME) based medium. The production was optimized using a combinatory approach of Plackett-Burman (PB) design, one factor at a time (OFAT) design and face centred central composite design (FCCCD). The variables (malt extract, (NH₄)₂SO₄, CaCl₂, MgSO₄, olive oil, peptone, K₂HPO₄, NaNO₃, Tween-80, POME and pH) were analyzed using PB design and the variables with positive contrast coefficient were found to be K₂HPO₄, NaNO₃, Tween-80, POME and pH. The significant variables selected were further analyzed for possible optimum range by using OFAT approach and the findings revealed that K₂HPO₄, NaNO₃, and Tween-80 as the most significant medium components, and thus were further optimized by using FCCCD. The optimum medium yielded a lipase with an activity of 7.8 U/ml, a significant 3.2-fold increase compared to un-optimized medium. The present findings revealed that POME is an alternative and suitable substrate for halophilic lipase production at low cost. Also, it is clearly evident that the combinatory approach employed here proved to be very effective in producing high activity halophilic lipases, in general.     

Rhamnolipid biosurfactants: evolutionary implications,applications and future prospects from untapped marine resource

Rhamnolipid-biosurfactants are known to be produced by the genus Pseudomonas, however recent literature reported that rhamnolipids (RLs) are distributed among diverse microbial genera. To integrate the evolutionary implications of rhamnosyl transferase among various groups of microorganisms, a comprehensive comparative motif analysis was performed amongst bacterial producers. Findings on new RL-producing microorganism is helpful from a biotechnological perspective and to replace infective P. aeruginosa strains which ultimately ensure industrially safe production of RLs. Halotolerant biosurfactants are required for efficient bioremediation of marine oil spills. An insight on the exploitation of marine microbes as the potential source of RL biosurfactants is highlighted in the present review. An economic production process, solid-state fermentation using agro-industrial and industrial waste would increase the scope of biosurfactants commercialization. Potential and prospective applications of RL-biosurfactants including hydrocarbon bioremediation, heavy metal removal, antibiofilm activity/biofilm disruption and greener synthesis of nanoparticles are highlighted in this review.     

Preparation of zymosan from yeast cell walls

Cell walls of the yeastSaccharomyces cerevisiae after disintegration and protoplasm removal by centrifugation and repeated washing were suspended in 0.5m Na₂HPO₄, pH 7.8–8.0, as a 5% or 10% suspension, depending on the mode of heating. The suspension was boiled for 3h, purified by repeated washing with water and ethanol and dried. The yield was approximately 1.8% of the starting amount of pressed commercial baker’s yeast.     

Optimization and production of curdlan gum using Bacillus cereus PR3 isolated from rhizosphere of leguminous plant

Curdlan gum is a neutral water-insoluble bacterial exopolysaccharide composed primarily of linear β-(1,3) glycosidic linkages. Recently, there has been increasing interest in the applications of curdlan and its derivatives. Curdlan is found to inhibit tumors and its sulfated derivative possess anti-HIV activity. Curdlan is biodegradable, non-toxic towards human, environment and edible which makes it suitable as drug-delivery vehicles for sustained drug release. The increasing demand for the growing applications of curdlan requires an efficient high yield fermentation production process so as to satisfy the industrial needs. In this perspective, the present work is aimed to screen and isolate an efficient curdlan gum producing bacteria from rhizosphere of ground nut plant using aniline-blue agar. High yielding isolate was selected based on curdlan yield and identified as Bacillus cereus using gas-chromatography fatty acid methyl ester analysis. B. cereus PR3 curdlan gum was characterized using FT-IR spectroscopy, SEM, XRD and TGA. Fermentation time for curdlan production using B. cereus PR3 was optimized. Media constituents like carbon, nitrogen and mineral sources were screened using Plackett–Burman design. Subsequent statistical analysis revealed that Starch, NH₄NO₃, K₂HPO₄, Na₂SO₄, KH₂SO₄ and CaCl₂ were significant media constituents and these concentrations were optimized for enhancement of curdlan production up to 20.88?g/l.     

Growth characteristics and salt requirement ofDeleya halophila in a defined medium

Growth characteristics ofDeleya halophila (CCM 3662^T), were determined using a defined medium.Deleya halophila presented its optimal growth at 7.5% (wt/vol) total salts when it was grwon at incubation temperatures of 32° and 42°C; when the temperature was lowered to 22°C, it had optimal growth at 5% (wt/vol) total salts. This bacterium had an absolute requirement for the Na⁺ cation; it could not be replaced by other cations. NaBr, Na₂SO₄, or Na₂S₂O₃ could be substituted for NaCl in the growth medium, but, when MgCl₂, KCl, LiCl, NaI, NaF, or NaNO₃ was substituted for NaCl, the medium did not support growth. Growth rates of the strain were diverse when NaCl was partially replaced by other sodium salts. Finally,D. halophila suffered loss of viability when the culture was diluted into different low NaCl concentrations (0, 0.5%, and 1%, wt/vol) at various incubation temperatures.     

Effects of air pollutants on proton and sucrose transport at the plasma membrane of Ricinus communis

The effects of the air pollutants O₃, SO₂ and NO₂ on aspects of sucrose/proton cotransport across the plasma membrane of Ricinus communis plants have been investigated. The H⁺-ATPase hydrolytic activity in cotyledon plasma membrane vesicles purified by phase partitioning showed small stimulations by Na₂SO₃ or NaNO₃ added separately or together to the assay medium. ATPase activity from plants pretreated by fumigation with SO₂ or O₃ also showed an increase, the effect of O₃ being quite marked. Plasma membrane H⁺-pumping in KI-treated microsomal fractions and medium acidification by intact cotyledons both showed small decreases in the presence of Na₂SO₃ or NaNO₂. Both Na₂SO₃ and NaNO₂ at high concentrations (2 mol m^–3) had significant effects on sucrose uptake by intact cotyledons, although sucrose efflux was unaffected. No significant effects on sucrose uptake or efflux by intact cotyledons were observed in plants pretreated by fumigation with SO₂ or O₃. Proton-coupled sucrose transport in isolated plasma membrane vesicles was inhibited in the presence of Na₂SO₃ or NaNO₂. However, both pollutants also significantly inhibited the uptake of acetate by the vesicles, indicating a dissipation of the pH gradient across the membrane. It was concluded that no specific aspect of the sucrose/proton cotransport mechanism was damaged by these air pollutants, and that the effects of these pollutants on carbohydrate partitioning are more likely to be due to general effects on membrane integrity or on other aspects such as leaf carbohydrate metabolism.     

Statistical optimization of culture media for production of phycobiliprotein by Synechocystis sp. PCC 6701

Synechocystis sp. PCC 6701 has a brilliantly colored pigment, phycobiliprotein containing phycoerythrin. Culture medium was optimized by sequential designs in order to maximize phycobiliprotein production. The observed fresh weights after 6 days were 0.58 g/L in BG-11, 0.83 g/L in medium for Scenedesmus sp. and 0.03∼0.52 g/L in the other tested media. Medium for Scenedesmus sp. was selected to be optimized by fractional factorial design and central composite design since the medium maintained a more stable pH within a desirable range due to higher contents of phosphate. The fractional factorial design had seven factors with two levels: KNO₃, NaNO₃, NaH₂PO₄, Na₂HPO₄, Ca(NO₃)₂, FeEDTA, and MgSO₄. From the result of fractional factorial design, nitrate and phosphate were identified as significant factors. A central composite design was then applied with four variables at five levels each: nitrate, phosphate, pH, and light intensity. Parameters such as fresh weight and phycobiliprotein contents were used to determine the optimum value of the four variables. The proposed optimum media contains 0.88 g/L of nitrate, 0.32 g/L of phosphate under 25 μE·m⁻²·s⁻¹ of light intensity. The maximum phycobiliprotein contents have been increased over 400%, from 4.9 to 25.9 mg/L after optimization.     

Application of response-surface methodology to evaluate the optimum medium components for the enhanced production of lichenysin by Bacillus licheniformis R2

Biosurfactants have gained attention because they exhibit some advantages such as biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates. They are widely used for environmental applications for bioremediation and also in biomedical field. However, the high cost of production is the limiting factor for widespread industrial applications. Thus, optimization of the growth medium for biosurfactant-lichenysin production by Bacillus licheniformis R2 was carried out using response-surface methodology. A preliminary screening phase based on a two-level fractional factorial design led to the identification of NH₄NO₃, glucose, Na₂HPO₄ and MnSO₄·4H₂O concentrations as the most significant variables affecting the fermentation process. The 2⁴ full-factorial central composite design was then applied to further optimize the biosurfactant production. The optimal levels of the aforementioned variables were (g/l): NH₄NO₃, 1.0; glucose, 34.0; KH₂PO₄, 6.0; Na₂HPO₄, 2.7; MgSO₄·7H₂O, 0.1; CaCl₂, 1.2 × 10⁻³; FeSO₄·7H₂O, 1.65 × 10⁻³; MnSO₄·4H₂O, 1.5 × 10⁻³ and Na–EDTA, 2.2 × 10⁻³. With the optimization procedure, the relative lichenysin yield expressed as the critical micelle dilution (CMD) was fourfold higher than that obtained in the non-optimized reference medium.     

Interference of some aqueous two-phase system phase-forming components in protein determination by the Bradford method

The interference of some specific aqueous two-phase system (ATPS) phase-forming components in bovine serum albumin (BSA) determination by the Bradford method was investigated. For this purpose, calibration curves were obtained for BSA in the presence of different concentrations of salts and polymers. A total of 19 salts [Na₂SO₄, (NH₄)₂SO₄, MgSO₄, LiSO₄, Na₂HPO₄, sodium phosphate buffer (pH 7.0), NaH₂PO₄, K₂HPO₄, potassium phosphate buffer (pH 7.0), KH₂PO₄, C₆H₈O₇, Na₃C₆H₅O₇, KCHO₂, NaCHO₂, NaCO₃, NaHCO₃, C₂H₄O₂, sodium acetate buffer (pH 4.5), and NaC₂H₃O₂] and 7 polymers [PEG 4000, PEG 8000, PEG 20000, UCON 3900, Ficoll 70000, PES 100000, and PVP 40000] were tested, and each calibration curve was compared with the one obtained for BSA in water. Some concentrations of salts and polymers had considerable effect in the BSA calibration curve. Carbonate salts were responsible for the highest salt interference, whereas citric and acetic acids did not produce interference even in the maximum concentration level tested (5 wt%). Among the polymers, UCON gave the highest interference, whereas Ficoll did not produce interference when used in concentrations up to 10 wt%. It was concluded that a convenient dilution of the samples prior to the protein quantification is needed to ensure no significant interference from ATPS phase-forming constituents.