Utilization of hydrophobic bacterium <Emphasis Type="Italic">Rhodococcus opacus</Emphasis> B-4 as whole-cell catalyst in anhydrous organic solvents

Rhodococcus opacus strain B-4, which has recently been isolated as an organic solvent-tolerant bacterium, has a high hydrophobicity and exhibits a high affinity for hydrocarbons. This bacterium was able to survive for at least 5 days in organic solvents, including n-tetradecane, oleyl alcohol, and bis(2-ethylhexyl) phthalate (BEHP), which contained water less than 1% (w/v). The biocatalytic ability of R. opacus B-4 was demonstrated in the essentially nonaqueous BEHP using indigo production from indole as a model conversion. By the catabolism of oleic acid for NADH regeneration, indigo production increased up to 71.6 μg ml⁻¹ by 24 h.     

Effects of organic solvents on membrane of<Emphasis Type="Italic">Taxus cuspidata</Emphasis> cells in

The effects of organic solvents (oleic acid and dibutyl phthalate) on viability and membrane integrity of Taxus cuspidata cells were investigated in two-liquid-phase suspension cultures. It has been found that the cell viability, electrical conductivity and concentration of malonyl dialdehyde did not change obviously when the content of oleic acid or dibutyl phthalate was 2% (v/v), but varied markedly when the contents of oleic acid or dibutyl phthalate were raised to 6% (v/v) or more, indicating that the organic solvents at higher concentrations severely affected the cell membrane permeability.     

Isolation and characterization of a thermotolerant ene reductase from Geobacillus sp. 30 and its heterologous expression in Rhodococcus opacus

Rhodococcus opacus B-4 cells are adhesive to and even dispersible in water-immiscible hydrocarbons owing to their highly lipophilic nature. In this study, we focused on the high operational stability of thermophilic enzymes and applied them to a biocatalytic conversion in an organic reaction medium using R. opacus B-4 as a lipophilic capsule of enzymes to deliver them into the organic medium. A novel thermo- and organic-solvent-tolerant ene reductase, which can catalyze the enantioselective reduction of ketoisophorone to (6R)-levodione, was isolated from Geobacillus sp. 30, and the gene encoding the enzyme was heterologously expressed in R. opacus B-4. Another thermophilic enzyme which catalyzes NAD⁺-dependent dehydrogenation of cyclohexanol was identified from the gene-expression library of Thermus thermophilus and the gene was coexpressed in R. opacus B-4 for cofactor regeneration. While the recombinant cells were not viable in the mixture due to high reaction temperature, 634 mM of (6R)-levodione could be produced with an enantiopurity of 89.2 % ee by directly mixing the wet cells of the recombinant R. opacus with a mixture of ketoisophorone and cyclohexanol at 50 °C. The conversion rate observed with the heat-killed recombinant cells was considerably higher than that obtained with a cell-free enzyme solution, demonstrating that the accessibility between the substrates and enzymes could be improved by employing R. opacus cells as a lipophilic enzyme capsule. These results imply that a combination of thermophilic enzymes and lipophilic cells can be a promising approach for the biocatalytic production of water-insoluble chemicals.     

Single cell oil production by <Emphasis Type="Italic">Gordonia</Emphasis> sp. DG using agro-industrial wastes

Lipid accumulation by Gordonia sp. DG using sodium gluconate as carbon source in comparison with Rhodococcus opacus PD630 was studied. Maximum lipid content 80% was observed at the beginning of the stationary phase for R. opacus and 72% at the end of stationary phase for Gordonia sp. Different agro-industrial wastes were used as carbon source. The cells of the two organism accumulated lipid more than 50% of the biomass with most tested agro-industrial wastes. The maximum value was in presence of sugar cane molasses (93 and 96%) for R. opacus and Gordonia sp. respectively. Maximum triacyglycerols (TAGs), 88.9 and 57.8 mg/l, was obtained using carob and orange waste by R. opacus and Gordonia sp. respectively. The use of orange waste as carbon source by R. opacus, increased lipid unsaturation with C18:3 as the major unsaturated fatty acid. On the other hand, C22:0 and C6:0 were the dominant fatty acids (54.5% of the total identified fatty acids) produced by Gordonia sp. in presence of orange waste as carbon source. Statistical optimization of the medium revealed that maximum lipid content was achieved with 60% orange waste, 0.05 g/l ammonium chloride and 0.2 g/l magnesium sulphate.     

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.

     

Paraffin oil as a “methane vector” for rapid and high cell density cultivation of <Emphasis Type="Italic">Methylosinus trichosporium</Emphasis> OB3b

Slow growth and relatively low cell densities of methanotrophs have limited their uses in industrial applications. In this study, a novel method for rapid cultivation of Methylosinus trichosporium OB3b was studied by adding a water-immiscible organic solvent in the medium. Paraffin oil was the most effective at enhancing cell growth and final cell density. This is at least partially due to the increase of methane gas transfer between gas and medium phases since methane solubility is higher in paraffin than in water/nitrate minimal salt medium. During cultivation with paraffin oil at 5% (v/v) in the medium, M. trichosporium OB3b cells also showed higher concentrations of the intermediary metabolites, such as formic acid and pyruvic acid, and consumed more methane compared with the control. Paraffin as methane vector to improve methanotroph growth was further studied in a 5-L fermentor at three concentrations (i.e., 2.5%, 5%, and 10%). Cell density reached about 14 g dry weight per liter with 5% paraffin, around seven times higher than that of the control (without paraffin). Cells cultivated with paraffin tended to accumulate around the interface between oil droplets and the water phase and could exist in oil phase in the case of 10% (v/v) paraffin. These results indicated that paraffin could enhance methanotroph growth, which is potentially useful in cultivation of methanotrophs in large scale in industry. Bing Han and Tao Su contributed equally to this work.     

Effect of Soy Soluble Polysaccharide on the Stability of Soy-Stabilised Emulsions During In Vitro Protein Digestion

This study investigated physicochemical properties of soy soluble polysaccharide (SSP) and pectinase-hydrolysed soy soluble polysaccharide (PH-SSP) from okara, the residue from soy milk production, and their influences when used as a fibre source in oil-in-water (o/w) emulsions. Although pectinase hydrolysed only the carbohydrate fraction in SSP, it resulted in the self-association of PH-SSP to the large-size aggregates. When PH-SSP was added to liquid emulsion containing 3.33% (w/v) rice bran oil and 3.75% (w/v) heated soy protein, it regulated the contents of protein in serum phase, sediment phase and at oil–water interface. The types and contents of soy proteins in the serum phase and sediment phase could be manipulated by pre-heating of soy proteins at 80 °C for 30 min and the addition of PH-SSP. The presence of PH-SSP (0–6% w/v) induced different distribution of proteins to the sediment phase and subsequent in vitro protein digestion in the emulsion. Overall, this study proposed the means to design the distributions of proteins in different phases of o/w emulsion for different degrees of oil release, emulsion stability and protein-polysaccharide coacervation during the course of in vitro peptic and tryptic digestion.     

Phototrophic bacterial production of oleic acid in an illuminated upflow anaerobic sludge blanket reactor

Phototrophic bacterial cells in the effluent from a lighted upflow anaerobic sludge blanket reactor supplied with a medium containing 142 mg S (as SO₄ ^2–) l^–1 accumulated a 6.8% w/w oleic acid content in cells and 19 mg cell-bound oleic acid l^–1 in the effluent. Pure cultures of Rhodopseudomonas palustris and Blastochloris sulfoviridis isolated from the effluent also accumulated 5.1 and 6.4% w/w oleic acid contents in cells, respectively. The oleic acid content in the cells recovered from the LUASB reactor effluent was related to the phototrophic bacterial population in the LUASB reactor. The inverse relationship was observed in the LUASB reactor between phototrophic bacterial growth and sulfate concentration in the influent.     

Permeabilization of<Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> SY509 cells with organic solvents for whole cell biocatalyst

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.     

Identification and characterization of <Emphasis Type="Italic">o</Emphasis>-xylene-degrading <Emphasis Type="Italic">Rhodococcus</Emphasis> spp. which were dominant species in the remediation of <Emphasis Type="Italic">o</Emphasis>-xylene-contaminated soils

Soils contaminated with o-xylene were more difficult to bioremediate than those contaminated with other BTEX hydrocarbons (benzene, toluene, ethylbenzene, m-xylene and p-xylene). In order to identify microorganisms responsible for o-xylene degradation in soil, microbial community structure analyses were carried out with two soil samples in the presence of o-xylene and mineral nutrients. In two different soil samples, Rhodococcus opacus became abundant. We were also able to isolate o-xylene degrading Rhodococcus species from these soil samples. A primer set was developed to specifically detect a cluster of this Rhodococcus group including isolated Rhodococcus strains, Rhodococcus opacus and Rhodococcus koreensis. The growth of this bacterial group in an o-xylene-contaminated soil was followed by competitive PCR (cPCR). The decrease in o-xylene clearly paralleled the growth of the Rhodococcus group.     

Evaluation of the biocompatibile ionic liquid 1-methyl-3-methylimidazolium dimethylphosphite pretreatment of corn cob for improved saccharification

Ionic liquid (IL) pretreatment of lignocellulose materials is a promising process in biomass conversion to renewable biofuel. More in-depth research involving environment-friendly IL is much needed to explore pretreatment green route. In our case, IL 1-methyl-3-methylimidazolium dimethylphosphite ([Mmim]DMP) was chosen as an environment-friendly solvent to pretreat corn cob in view of its biocompatibility with both lignocellulose solubility and cellulase activity. The pretreatment/saccharification process and in situ saccharification process involving [Mmim]DMP were efficiently performed in bioconversion of corn cob to sugars, and more than 70% saccharification rates were obtained. Furthermore, the fermentability of reducing sugars obtained from the hydrolyzates was evaluated using Rhodococcus opacus strain ACCC41043 (R. opacus). High lipid production 41–43% of cell dry matter was obtained after 30 h of cultivation. GC/MS analysis indicated that lipids from R. opacus contained mainly long-chain fatty acids with four major constituent/oleic acid, stearic acid, palmitic acid, palmitoleic acid which are good candidates for biodiesel. These elucidated that corn cob pretreated by IL [Mmim]DMP did not bring negative effects on saccharification, cell growth, and accumulation of lipid of R. opacus. In conclusion, the IL [Mmim]DMP shows promise as green pretreatment solvent for cellulosic materials.     

10(R)-Hydroxystearic acid production by a novel microbe,NRRL B-14797, isolated from compost

Summary A bacterium, NRRL B-14797, isolated from composted manure, converted oleic acid exclusively to 10(R)-hydroxystearic acid in 3-day batch cultures. 9(Z)-Unsaturated fatty acids in a lipid extract from soybean soapstock were also hydrated effectively. Aerobic bioconversions by isolate B-14797 were compared with those byPseudomonas B-2994 andNocardia 5767, which produce mixtures of 10-hydroxy- and 10-ketostearic acids. The results of studies with resting cells and cell-free extracts were consistent with action of a hydratase and absence of secondary alcohol dehydrogenase in strain B-14797.The mention of firm names or trade products does not imply that they are endorsed or recommended by the US Department of Agriculture over other firms or similar products not mentioned.     

Investigation of the synthesis of xylose/glucose isomerases in sixArthrobacter sp. strains

The substrate specificity of isomerases produced by six strains ofArthrobacter sp. was studied. The role of utilizable carbon sources in controlling enzyme biosynthesis was established. All of the strains studied were found to produce xylose isomerases efficiently, converting D-xylose into D-xylulose and D-glucose into D-fructose. All but A.ureafaciens B-6 strains showed low activity toward D-ribose,Arthrobacter sp. B-5 was slightly active toward L-arabinose, andA. ureafaciens B-6 andArthrobacter sp. B-2239, toward L-rhamnose. InArthrobacter sp. B-5, the synthesis of xylose/glucose isomerase was constitutive (i.e., it was not suppressed by readily metabolizable carbon sources. The synthesis of xylose/glucose isomerase induced by D-xylose inArthrobacter sp. strains B-2239, B-2240, B-2241, and B-2242 and by D-xylose and xylitol inA. ureafaciens B-6 was suppressed by readily metabolizable carbon sources in a concentration-dependent manner. The data obtained suggest that D-xylose and/or its metabolites are involved in the regulation of xylose/glucose isomerase synthesis in theArthrobacter sp. strains B-5, B-2239, B-2240, and B-2241.     

Bioproduction of vanillin using an organic solvent-tolerant <Emphasis Type="Italic">Brevibacillus agri</Emphasis> 13

Nowadays, majority of vanillin supplied to the world market is chemically synthesized from a petroleum-based raw material, raising a concern among the consumers regarding the product safety. In this study, an organic solvent-tolerant Brevibacillus agri 13 previously reported for a strong predilectic property was utilized as a whole-cell biocatalyst for bioproduction of vanillin from isoeugenol (IG). B. agri 13 is the first biocatalyst reported for bioproduction of vanillin at a temperature as high as 45°C. Both pH and temperature were found to affect vanillin production significantly. An extreme level of organic solvent tolerance of B. agri 13 allowed us to utilize it in a biphasic system using organic solvents generally considered as highly toxic to most bacteria. With an addition of butyl acetate at 30% (v/v) as an organic second phase, toxicity of IG exerted onto the biocatalyst was reduced dramatically while faster and more efficient vanillin production was obtained (1.7 g/L after 48 h with 27.8% molar conversion).     

TADH,the thermostable alcohol dehydrogenase from <Emphasis Type="Italic">Thermus</Emphasis> sp. ATN1: a versatile new biocatalyst for organic synthesis

The alcohol dehydrogenase from Thermus sp. ATN1 (TADH) was characterized biochemically with respect to its potential as a biocatalyst for organic synthesis. TADH is a NAD(H)-dependent enzyme and shows a very broad substrate spectrum producing exclusively the (S)-enantiomer in high enantiomeric excess (>99%) during asymmetric reduction of ketones. TADH is active in the presence of 10% (v/v) water-miscible solvents like 2-propanol or acetone, which permits the use of these solvents as sacrificial substrates in substrate-coupled cofactor regeneration approaches. Furthermore, the presence of a second phase of a water-insoluble solvent like hexane or octane had only minor effects on the enzyme, which retained 80% of its activity, allowing the use of these solvents in aqueous/organic mixtures to increase the availability of low-water soluble substrates. A further activity of TADH, the production of carboxylic acids by dismutation of aldehydes, was investigated. This reaction usually proceeds without net change of the NAD⁺/NADH concentration, leading to equimolar amounts of alcohol and carboxylic acid. When applying cofactor regeneration at high pH, however, the ratio of acid to alcohol could be changed, and full conversion to the carboxylic acid was achieved.     

Squalene-based oil-in-water emulsion adjuvants perturb metabolism of neutral lipids and enhance lipid droplet formation

Oil-in-water emulsions are used as vaccine adjuvants, but the mechanism of action remains unknown. In this paper we used phagocytes (monocytes, macrophages, dendritic cells) and non-phagocytic cells (fibroblasts, skeletal muscle cells) to study internalization of emulsions in vitro, and to characterize the influence of emulsion uptake on cellular metabolism of neutral lipids. We found that all tested cell types endocytose the emulsion droplets, and that the uptake leads to an acute accumulation of neutral lipids in the form of cytoplasmic lipid droplets. The accumulated lipids comprise not only the delivered squalene, but also cholesteryl esters, triacylglycerols, fatty acids, and diacylglycerols. Lipid metabolism and innate immunity are closely linked, and accumulation of lipids in non-adipose tissues is known to induce inflammatory conditions. We propose that one aspect of o/w emulsion adjuvanticity could depend on their ability to rapidly change lipid metabolism of the target cells.     

Anaerobically grown <Emphasis Type="Italic">Thauera aromatica</Emphasis>, <Emphasis Type="Italic">Desulfococcus multivorans</Emphasis>, <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> are more sensitive towards organic solvents than aerobic bacteria

The effect of seven important pollutants and three representative organic solvents on growth of Thauera aromatica K172, as reference strain for nitrate-reducing anaerobic bacteria, was investigated. Toxicity in form of the effective concentrations (EC50) that led to 50% growth inhibition of potential organic pollutants such as BTEX (benzene, toluene, ethylbenzene, and xylene), chlorinated phenols and aliphatic alcohols on cells was tested under various anaerobic conditions. Similar results were obtained for Geobacter sulfurreducens and Desulfococcus multivorans as representative for Fe³⁺-reducing and sulphate-reducing bacteria, respectively, leading to a conclusion that anaerobic bacteria are far more sensitive to organic pollutants than aerobic ones. Like for previous studies for aerobic bacteria, yeast and animal cell cultures, a correlation between toxicity and hydrophobicity (log P values) of organic compounds for different anaerobic bacteria was ascertained. However, compared to aerobic bacteria, all three tested anaerobic bacteria were shown to be about three times more sensitive to the tested substances.     

Lipase activity of resting cells of Aspergillus flavus after solvent washing; memory effects from endogenous substrate arising from the original growth medium

Aspergillus flavusresting cells were washed with solvents of different polarity for 2, 6, and 24 h and then suspended in isooctane containing either oleic acid and 1-propanol or 1-propanol alone. Propyl oleate and propyl linoleate were produced in all experiments after 24 h due to the presence of residual fatty acids originating from the sunflower oil used for growing the mycelium. After 24 h washing, most solvents produced a 70 to 90% decrease in lipase activity and a 0 to 99% decrease in the amount of residual acids. 0.7 M 1-propanol in hexane was the best washing solvent among all those assayed (93% remaining activity, 0.3% residual oleic acid).     

Attachment and Biofilm Formation of <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> on a Hydrophobic Surface at the Air Interface

Properties of attachment of Mycobacterium marinum to hydrophobic surfaces and subsequent␣biofilm formation were investigated. Binding of M. marinum to polypropylene occured under aerobic and anaerobic/microaerophilic conditions. However, aerobic conditions were necessary for biofilms to persist. Highly non-polar organic solvents were found to efficiently remove attached bacteria from the polypropylene surface, indicating strong hydrophobic interactions between the M. marinum cell wall and the surface. Increased capsular material, occurring during stationary phase, correlated with a decrease in attachment of cells to polypropylene. A protein of approximately 40 kDa appears to be present in increased amounts during the stationary phase. The protein has been identified by LC MS/MS analysis as alanine dehydrogenase.     

Microwave-assisted extraction of embelin from <Emphasis Type="Italic">Embelia ribes</Emphasis>

A rapid and efficient microwave-assisted extraction (MAE) process for the selective extraction of embelin from Embelia ribes was developed. Solvent selection, microwave energy input and solid loading were optimized. The rate of extraction and purity of embelin depended upon the solvent used and exposure time to microwaves. Maximum MAE was achieved in acetone with total yield of 92% (w/w) embelin with 90% (w/w) purity with 1% (w/v) raw material loading at 150 W power level in 80 s. Non-polar solvents, such as hexane and dichloromethane, were not effective for the selective extraction of embelin.