Evaluating the Optimal Oil Concentrations in the Startup Performance of a Membrane Bioreactor Treating Oily Noodle-soup Wastewater.

The discharge of high-strength oily wastewater adversely affects the environment; therefore, the treatment of wastewater containing fats, oils, and grease from the food industry is of importance. In this study, we used a membrane bioreactor (MBR) to treat Ramen noodle-soup wastewater, and we evaluated the optimal oil concentration in the wastewater for the startup of the MBR treatment in winter and summer. The MBR system had a sufficient startup in both seasons when fed with a 20-fold dilution of the original oily wastewater, containing approximately 950 to 1,200 mg/L oil and approximately 3,000 to 4,400 mg/L biological oxygen demand (BOD; BOD-SS load of 0.1 to 0.2 kg/kg/d). The reactor performance in winter were relatively stable during the operation. While, activated sludge microbes in summer were not highly active with a 40-fold dilution of wastewater, because of the decreased mixed liquor suspended solid concentration during the operation period. Population shifts in the sludge microbiome with increasing oil concentrations were analyzed using high-throughput sequencing, and the relative abundance of operational taxonomic units belonging to the phylum Bacteroidetes were highest in both winter and summer when fed with 20-fold dilution of the wastewater. In particular, the family Chitinophagaceae was dominant, with relative abundances of 13.5% in winter and 5.1% in summer, suggesting that this family may play important roles in the startup of a MBR treating the wastewater.

Activated sludge microbiome in a membrane bioreactor for treating Ramen noodle-soup wastewater.

Population shifts in the activated sludge microbiome of a membrane bioreactor (MBR) during the treatment of Ramen noodle-soup wastewater were analyzed by high-throughput sequencing. An MBR underwent stable treatment of wastewater containing increasing oil concentrations (from 135 to 1,350 mg/L) for 26 days; however, after feeding with wastewater containing 2,700 mg/L of oil, the mixed liquor suspended solids and transmembrane pressure exhibited gradual and rapid increases, respectively, leading to clogging of the membrane. Phylogenetic analysis revealed an oil supply-dependent increase in the abundance of Cupriavidus gilardii (relative abundance of 26.2% at Day 30) in the sludge together with Parasegetibacter terrae (9.9%) and Ferruginibacter yonginensis (9.4%). These dominant species may play important roles in noodle-soup wastewater treatment.

Fine-scale monitoring of shifts in microbial community composition after high organic loading in a pilot-scale membrane bioreactor.

In biological wastewater treatment, municipal wastewater sometimes undergoes unexpected changes in physicochemical parameters, such as organic carbon concentration. The aim of this study was to understand how microbial communities in activated sludge in a membrane bioreactor (MBR) adapt to high organic loading and maintain their degradation ability during reactor operation. A pilot-scale MBR was operated for 19 days. On day 8, the concentration of organic matter in the synthetic wastewater increased from 450 to 900 mg chemical oxygen demand (COD)/L. Even under conditions of high organic loading, COD removal rates were high, ranging from 85.3 to 91.4%. High-throughput sequencing of 16S rRNA genes revealed that microbial communities changed drastically with increased organic loading. After day 8, Aquabacterium- and Azospira-related operational taxonomic units (OTUs) belonging to the class ß-proteobacteria became dominant; this potentially enhanced the degradation of organic substances and decreased activated sludge microbial diversity. Due to the use of dissolved oxygen (DO) for degradation of organic substances, DO levels in the reactor decreased. This led to an increase in a subset of OTUs related to not only aerobic but also anaerobic bacteria, e.g., those in the class Clostridia. During this period, anaerobic microorganisms may have contributed to the degradation of organic substances to maintain MBR performance. On the other hand, high-throughput sequencing also made it possible to identify yet-to-be cultured or minor microorganisms affiliated with the candidate phylogenetic division SR1 and ammonia-oxidizing archaea in activated sludge.

Use of a Gluconobacter frateurii mutant to prevent dihydroxyacetone accumulation during glyceric acid production from glycerol.

To prevent dihydroxyacetone (DHA) by-production during glyceric acid (GA) production from glycerol using Gluconobacter frateurii, we used a G. frateurii THD32 mutant, ΔsldA, in which the glycerol dehydrogenase subunit-encoding gene (sldA) was disrupted, but ΔsldA grew much more slowly than the wild type, growth starting after a lag of 3 d under the same culture conditions. The addition of 1% w/v D-sorbitol to the medium improved both the growth and the GA productivity of the mutant, and ΔsldA produced 89.1 g/l GA during 4 d of incubation without DHA accumulation.

Two-stage electrodialytic concentration of glyceric acid from fermentation broth.

The aim of this research was the application of a two-stage electrodialysis (ED) method for glyceric acid (GA) recovery from fermentation broth. First, by desalting ED, glycerate solutions (counterpart is Na+) were concentrated using ion-exchange membranes, and the glycerate recovery and energy consumption became more efficient with increasing the initial glycerate concentration (30 to 130 g/l). Second, by water-splitting ED, the concentrated glycerate was electroconverted to GA using bipolar membranes. Using a culture broth of Acetobacter tropicalis containing 68.6 g/l of D-glycerate, a final D-GA concentration of 116 g/l was obtained following the two-stage ED process. The total energy consumption for the D-glycerate concentration and its electroconversion to D-GA was approximately 0.92 kWh per 1 kg of D-GA.

Microbial production of glyceric acid, an organic acid that can be mass produced from glycerol.

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.

Production of glyceric acid by Gluconobacter sp. NBRC3259 using raw glycerol.

Gluconobacter sp. NBRC3259 converted glycerol to glyceric acid (GA). The enantiomeric composition of the GA produced was a mixture of DL-forms with a 77% enantiomeric excess of D-GA. After culture conditions, such as initial glycerol concentration, types and amounts of nitrogen sources, and initial pH, were optimized, Gluconobacter sp. NBRC3259 produced 54.7 g/l of GA as well as 33.7 g/l of dihydroxyacetone (DHA) from 167 g/l of glycerol during 4 d of incubation in a jar fermentor with pH control. GA production from raw glycerol samples, the main by-product of the transesterification process in the biodiesel production and oleochemical industries, was also evaluated after proper pretreatment of the samples. Using a raw glycerol sample with activated charcoal pretreatment, 45.9 g/l of GA and 28.2 g/l of DHA were produced from 174 g/l of glycerol.

[Optical resolution of 2-alkanol by lipase-catalyzed acetylation with vinyl acetate in packed-bed reactor with recycling system].

The lipase-catalyzed acetylation of 2-alkanol with vinyl acetate was studied using Burkholderia cepacia lipase (BCL), three alcohol and three organic solvents in a packed-bet reactor with a recycling system (flow method). The optical resolution data were found in agreement with those of the batch method in which BCL was suspended in the substrate solution. Repeated reaction results clearly indicated BCL in the packed-bed to be quite stable and to be usable for at least 50 reaction runs or to remain effective for as long as two months in the water-insoluble solvents such as hexane and 1,2-dichloroethane. In the reaction using a water-soluble solvent such as acetonitrile, the catalytic power of BCL showed only a 1% decrease of conversion per run or solvent recycling possibly owing to compression of BCL in the bed although enantioselectivity was independent of the number of reaction repetitions. The present method showed thus be applicable to kinetic resolution by enzyme-catalyzed acylation in hydrophobic organic solvents with no waste of enzyme.

Kinetic studies on lipase-catalyzed acetylation of 2-alkanol with vinyl acetate in organic solvent.

Lipase-catalyzed acetylation of 2-alkanol with vinyl acetate has been studied kinetically using Burkholderia cepacia lipase (BCL), enantiomerically pure (R)- and (S)-2-alkanols and different organic solvents. The rate equation was derived by the steady state method for the simplified mechanism. The second order rate constants (k(R) and k(S)) for (R)- and (S)-2-alkanols were evaluated from the slopes of the double reciprocal plots, v(-1) vs. [2-alkanol](-1), where v is the initial rate of the reaction. The log k(R) value increased with the solvent hydrophobicity log P, where P is a partition coefficient of a given solvent between octanol and water. The log k(S) value also increased with log P except the bulky solvents such as 1,4-dioxane and cyclohexane, in which the rates were faster than those expected from the log k(S) vs. log P plot. The slope of log k(S) vs. log P plot was larger than that for (R)-2-alkanol. Thus, log E (E=k(R)/k(S): enantioselectivity) decreased with log P except the bulky solvents. The rate constants and the enantioselectivity were different depending on the structure (carbon number CN) of 2-alkanol. The log E vs. CN plot was minimized at CN=8 and 10 and the log k(S) vs. CN plot maximized at CN=8 and 10. In contrast the log k(R) vs. CN plot showed a different feature from the log E vs. CN plot. These facts suggest that dependence of E on CN is more strongly affected by the reactivity of (S)-2-alkanol than that of (R) isomer in this acetylation.

Monolayers assembled from a glycolipid biosurfactant from Pseudozyma (Candida) antarctica serve as a high-affinity ligand system for immunoglobulin G and M.

A carbohydrate ligand system has been developed which is composed of self-assembled monolayers (SAMs) of mannosylerythritol lipid-A (MEL-A) from Pseudozyma antarctica, serving for human immunoglobulin G and M (HIgG and HIgM). The estimated binding constants from surface plasmon resonance (SPR) measurement were Ka = 9.4 x 10(6) M(-1) for HIgG and 5.4 x 10(6) M(-1) for HIgM, respectively. The binding site was not in the Fc region of immunoglobulin but in the Fab region. Large amounts of HIgG and HIgM bound to MEL-A SAMs were directly observed by atomic force microscopy.

Naturally engineered glycolipid biosurfactants leading to distinctive self-assembled structures.

Self-assembling properties of "natural" glycolipid biosurfactants, mannosyl-erythritol lipids A and B (MEL-A, MEL-B), which are abundantly produced from yeast strains, were investigated by using the fluorescence-probe method, dynamic light-scattering (DLS) analysis, freeze-fracture transmission electron microscopy (FF-TEM), and synchrotron small/wide-angle X-ray scattering (SAXS/WAXS) analysis, among other methods. Both MEL-A and MEL-B exhibit excellent self-assembly properties at extremely low concentrations; they self-assemble into large unilamellar vesicles (LUV) just above their critical-aggregation concentration (CAC). The CAC(I) value was found to be 4.0x10(-6) M for MEL-A and 6.0x10(-6) M for MEL-B. Moreover, the self-assembled structure of MEL-A above a CAC(II) value of 2.0x10(-5) M was found to drastically change into sponge structures (L3) composed of a network of randomly connected bilayers that are usually obtained from a complicated multicomponent "synthetic" surfactant system. Interestingly, the average water-channel diameter of the sponge structure was 100 nm. This is relatively large compared with those obtained from "synthetic" surfactant systems. In addition, MEL-B, which has a hydroxyl group at the C-4' position on mannose instead of an acetyl group, gives only one CAC; the self-assembled structure of MEL-B seems to gradually move from LUV to multilamellar vesicles (MLV) with lattice constants of 4.4 nm, depending on the concentration. Furthermore, the lyotropic-liquid-crystal-phase observation at high concentrations demonstrates the formation of an inverted hexagonal phase (H2) for MEL-A, together with a lamella phase (L(alpha)) for MEL-B, indicating a difference between MEL-A and MEL-B molecules in the spontaneous curvature of the assemblies. These results clearly show that the difference in spontaneous curvature caused by the single acetyl group on the head group probably decides the direction of self-assembly of glycolipid biosurfactants. The unique and complex molecular structures with several chiral centers that are molecularly engineered by microorganisms must have led to the sophisticated self-assembling properties of the glycolipid biosurfactants.

Thermodynamically stable vesicle formation from glycolipid biosurfactant sponge phase.

Thermodynamically stable vesicle (L(alpha1)) formation from glycolipid biosurfactant sponge phase (L(3)) and its mechanism were investigated using a "natural" biocompatible mannosyl-erythritol lipid-A (MEL-A)/L-alpha-dilauroylphosphatidylcholine (DLPC) mixture by varying the composition. The trapping efficiency for calcein and turbidity measurements clearly indicated the existence of three regions: while the trapping efficiencies of the mixed MEL-A/DLPC assemblies at the compositions with X(DLPC)< or =0.1 or X(DLPC)> or =0.8 were almost zero, the mixed assemblies at the compositions with 0.1 or =0.8 were multilamellar vesicles (L(alpha)) with diameter from 2 to 10 microm. Meanwhile, dynamic light scattering (DLS) measurement revealed that the average size of the vesicles at the composition of X(DLPC)=0.3 was 633.2 nm, which is remarkably small compared to other compositions. Moreover, the mixed vesicle solution at the composition of X(DLPC)=0.3 was slightly bluish and turbid and kept its dispersion stability at 25 degrees C for more than 3 months, indicating the formation of a thermodynamically stable vesicle (L(alpha1)). These results exhibited the formation of a thermodynamically stable vesicle (L(alpha1)) with a high dispersibility from the MEL-A/DLPC mixture. The asymmetric distribution of MEL-A and DLPC in the two vesicle monolayers caused by the difference in geometrical structures is very likely to have changed their self-assembled structure from a sponge phase (L(3)) to a thermodynamically stable vesicle (L(alpha1)).

Mannosylerythritol lipids, yeast glycolipid biosurfactants, are potential affinity ligand materials for human immunoglobulin G.

Three mannosylerythritol lipids (MEL-A, -B, and -C), yeast glycolipid biosurfactants, were independently attached to poly (2-hydroxyethyl methacrylate) beads (PHEMA), and the three obtained MEL-PHEMA composites were examined for their binding affinity to human immunoglobulin G (HIgG). Of the three composites, the composite bearing MEL-A exhibited the highest binding capacity for HIgG. The binding amount of HIgG increased with increased applied concentration, reaching 106 mg HIgG (per g of composite), with a binding yield of 81%. Interestingly, the protein binding to the composite appeared to follow two different modes (Langmuir type and Freundlich type) depending on the applied concentration. The binding amount of human serum albumin to the composite was much smaller than that of HIgG. The bound human serum albumin, however, had minimal effect on the subsequent binding of HIgG, indicating that the two proteins have different binding sites onto the composite. More significantly, the bound HIgG was efficiently recovered under significantly mild elution conditions: Approximately 90% of the protein was eluted from the composite with phosphate buffer at pH 7. These results indicate that the glycolipid biosurfactant may have great potential as an affinity ligand material for HIgG.

TiO2-montmorillonite composites via supercritical intercalation.

The successful preparation of TiO2-montmorillonite mesoporous composites using intercalation of titanium isopropoxide dissolved in supercritical carbon dioxide involved ion exchange of interlayer cations by hydrophobic cations.