Search

Abstract: The gas vesicles isolated from the cells of filamentous cyanobacterium Anabaena flos-aquae were treated and sterilized with glutaraldehyde and then evaluated for their effectiveness as gas carriers in cell culture. Anchorage-dependent Vero cells were grown in a packed bed of microcarrier beads under the perfusion of Dulbecco’s Modified Eagle’s Medium with 1% serum. The culture medium supplemented with 1.8% (v/v) gas vesicles was found to support a 30% higher maximum glucose utilization rate than the same medium without gas vesicles. The gas vesicle suspension was confirmed to have no apparent effects on cell metabolism in T-flask cultures. The study results indicated that the gas vesicles, with high oxygen carrying capacity, can be used to increase the oxygen supply in cell culture systems.

Cellulase could not be selectively collected from fermentation broth by simple foam fractionation, because of the presence of other more surface-active compounds. A new approach of affinity foam fractionation was investigated for improvement. A hardwood hydrolysate (containing cellulose oligomers, substrates to cellulase) and two substrate analogs, i.e., carboxymethyl cellulose (CMC) and xylan hydrolysate, were added before the foaming process. The substrates and substrate analogs were indeed found to bind the cellulase selectively and form more hydrophobic complexes that partition more readily onto bubble surfaces. In this study, the effects of the type and concentration of substrate/analog as well as the presence of cells at different growth stages were examined. The foam fractionation properties evaluated included foaming speed, foam stability, foamate volume, and enrichment of filter paper unit (FPU) and individual cellulase components (i.e., endoglucanases, exoglucanases, and β-glucosidases). Depending on the broth and substrate/analog employed, the foamate FPU could be more than fourfold higher than the starting broth FPU. Addition of substrate/analog also deterred the enrichment of other extracellular proteins, resulting in the desired cellulase purification in the foamate. The value of E/P (enzyme activity-FPU/g/L of proteins) in the foamate reached as high as 18, from a lactose-based fermentation broth with original E/P of 5.6. Among cellulase components, exoglucanases were enriched the most and β-glucosidases the least. The study with CMC of different molecular weights (MW) and degrees of substitution (DS) indicated that the CMC with low DS and high MW performed better in cellulase foam fractionation.

Sawdust hydrolysates were investigated for their ability to support cell growth and cellulase production, and for potential inhibition of Trichoderma reeseiRut C30. Simultaneous fermentations were conducted to compare the hydrolysate-based media with the controls having equivalent concentrations of glucose and Avicel cellulose. Six hydrolysates differing in the boiling durations in the hydrolysis procedure were evaluated. The hydrolysates were found to support cell growth and induce active cellulase synthesis. The maximum specific cellulase production rate was 0.046 filter paper units (FPU)/(g of cells · h) in the hydrolysate-based systems, much higher than that (0.017 FPU/[g of cells · h]) in the controls.

For the development of pH-sensitive surfactants to be used in water-in-oil fermentation, the free-radical terpolymerization of methacrylic acid (MAA), methoxy poly(ethylene glycol) methacrylate (MPEGMA), and lauryl methacrylate (LMA), at a molar ratio of 1.0:0.04:0.76, was studied with two initiators, azobisisobutyronitrile (AIBN) and hydrogen peroxide, at different concentrations. The polymer synthesized with 0.45% AIBN as the initiator was the most promising, giving similar conversions of all three monomers throughout the 10-h polymerization. The subsequent study on AIBN-initiated systems indicated that MPEGMA caused an increase-then-decrease profile of the MAA conversion with a plateau around an ethylene glycol/MAA ratio of 1–2. This observation was fairly consistent with the well-known type II template polymerization of poly(ethylene glycol) (PEG)–MAA systems. The reactivity ratios obtained in this study suggested that the polymer synthesized with AIBN as the initiator had a structure of alternating blocks of MAA and LMA, with isolated PEG grafts. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 2950–2959, 2004

The direct enzymatic polymerization of lactonic sophorolipids (SLs) was investigated with four lipases, including porcine pancreatic lipase (PPL), immobilized Mucor mieheilipase (MML), lyophilized Candidaantarcticalipase (Fraction B, CAL‐B), and lyophilized Pseudomonassp. lipase (PSL). Several organic solvents, covering a wide range of polarity, were compared for suitability as the reaction medium. Isopropyl ether and toluene were found most effective. According to the quantification and structure identification by HPLC and LC‐MS, the reaction proceeded with the formation of monoacetylated lactonic SLs and the subsequent conversion of the intermediates to oligomers and polymers, presumably through ring‐opening polymerization. Temperature was found to have significant effects on the reaction. Both the conversion of reactant SLs and the subsequent formation of oligomers and polymers from the intermediates were faster at 60 °C than at 50 °C. The substrate selectivity among the three dominant reactant SLs also differed with the temperature. The conversion rate increased with the ring size of the lactones at 60 °C, but it decreased with the size at 50 °C.

Both heterotrophs and autotrophic nitrifiers are present in aerobic sludge digestion systems. The endogenous heterotrophic metabolism releases ammonium that, via oxidation to nitrite and nitrate, serves as the energy substrate for growth and survival of nitrifiers. The population interaction was used to explain the two-phase decay behavior observed in this study for the biosolids collected from rotating biological contactors (RBC) as well as the mixed primary and secondary sludge. Separated by an intermediate transition, the two phases followed different first-order decay kinetics, with the first phase being much faster. The two-phase phenomenon was supported by the on-line fluorescence profile monitored by an NAD(P)H fluorometer. The fluorescence showed an initial rise to a high plateau, a sharp decline after staying at the plateau for 30-50 h, and a trailing very slow decrease. The initial plateau corresponded to the first-phase of rapid sludge digestion; the trailing fluorescence decline corresponded to the second phase of slow digestion. The transition between the two phases occurred during the sharp decline from the plateau. The fluorescence profile was consistent with our earlier observation of negligible NAD(P)H fluorescence from nitrifiers compared to that from heterotrophs. The VSS reduction was, therefore, modeled by considering the decay and interaction of heterotrophs and nitrifiers. The model described the experimental results very well. The best-fit average decay constants were 0.067 d-1for nitrifers and 0.95 d-1for heterotrophs in the RBC biosolids. The apparent heterotrophic decay constants (in the presence of nonbiomass VSS) were found to be 0.48-0.55 d-1, consistent among different runs of mixed sludge digestion.

Being biosurfactants, rhamnolipids create severe foaming when produced in aerobic Pseudomonas aeruginosa fermentation. The necessary reduction of aeration causes oxygen limitation and restricts cell and product concentrations. In this study, we evaluate the new strategy of rhamnolipid production under denitrification conditions. Because hydrocarbons used in earlier aerobic fermentations were not metabolizable in the absence of oxygen, other potential C substrates were examined, including palmitic acid, stearic acid, oleic acid, linoleic acid, glycerol, vegetable oil, and glucose. All were found able to support cell growth under anaerobic denitrification. The growth on the two solid substrates (palmitic acid and stearic acid) was slower but could be enhanced substantially by initial addition of rhamnolipids (0.06 g/L). The effects of different limiting nutrients (N, P, S, Mg, Ca, and Fe) were also investigated. The commonly used N limitation could not be adopted in the denitrifying fermentation because the nitrate added for anaerobic respiration would also be assimilated for growth. P limitation was most effective, giving four- to fivefold higher specific productivity than the conventional N limitation. S limitation was comparable to N limitation; Mg limitation was much poorer. Ca and Fe were ineffective in limiting cell growth. The new strategy was further evaluated in a P-limited fermentation with palmitic acid as the substrate. The fermentation was first carried out under denitrification and later switched to aerobic condition. The specific productivity under denitrification was found to be about one-third that of the aerobic condition. The denitrification process was, however, free of foaming or respiratory limitation. Much higher cell concentrations may be employed to attain higher volumetric productivity and product concentrations, for more economical product recovery and/or purification. © 2001 John Wiley & Sons, Inc. Biotechnol Bioeng 72: 25–33, 2001.

Rhamnolipids and poly(β‐hydroxyalkanoic acids) (PHAs) are important fermentation products of Pseudomonasaeruginosa. Both contain β‐hydroxyalkanoic acids as main constituents. To investigate the possible relationship between their syntheses, we studied the n‐hexadecane fermentation by P.aeruginosa(ATCC 10145). PHA synthesis was found to occur only during active cell growth, while substantial rhamnolipid production began at the onset of the stationary phase. The specific synthesis rate of β‐hydroxyalkanoic acids was estimated as 12.6 mg HA/(g dry cells·h) from the PHA formation during the exponential‐growth phase. A similar rate was obtained from the β‐hydroxyalkanoic acid incorporation in the rhamnolipids produced during the early stationary phase. A regulatory switch of the flow of β‐hydroxyalkanoic acids from PHA polymerization to rhamnolipid synthesis is clearly indicated to occur when the culture reaches the stationary phase. Five rhamnolipid structures were identified using HPLC‐MS. Three are monorhamnolipids, two dirhamnolipids. All have a chain of two β‐hydroxyalkanoic acids. The two major components contain only β‐hydroxydecanoic acids; the three minors also have a β‐hydroxydecanoic acid linked to the sugar but a β‐hydroxydodecanoic acid or β‐hydroxydodecenoic acid as the second acid. The PHA accumulation reached about 7.5% of the cell dry weight. The monomer composition was relatively constant at different stages of production: in weight fractions, β‐hydroxyoctanoic acid, 0.25 (±0.05); β‐hydroxydecanoic acid, 0.41 (±0.06); β‐hydroxydodecanoic acid, 0.11 (±0.05), β‐hydroxytetradecanoic acid, 0.11 (±0.06), and β‐hydroxyhexadecanoic acid, 0.12 (±0.06). β‐Hydroxydecanoic acid was clearly the primary monomer.

As potential gas microcarriers, gas vesicles (GVs) were isolated from cultures of the filamentous cyanobacterium Anabaena flos‐aquaeand treated with glutaraldehyde. The effects of glutaraldehyde treatment on the stability of GVs, against elevated temperatures (40−121 °C) and protein‐stripping agents such as urea and sodium dodecyl sulfate (SDS), were then examined with the pressure collapse curves generated using pressure nephelometry. The treatment was very beneficial to GVs against the exposure to SDS and urea; however, it did not make the evolution‐optimized vesicle structure stronger or more temperature‐resistant. In the presence of these protein‐stripping agents, the treated vesicles had higher median (50%) collapse pressures (by ≥1 atm) than the untreated ones, at both room temperature and 40 °C. This increase has been presumably attributed to the cross‐linking of the large GvpC protein to the ribbed GvpA shell, thereby resisting the stripping of GvpC that provides the primary mechanical strength to the vesicle wall. The glutaraldehyde treatment also restored the strength of GVs weakened by a 5‐week storage in a refrigerator and, therefore, is expected to improve the stability of GVs for long‐term storage. GVs could not be autoclaved. If necessary for the intended applications, glutaraldehyde treatment may also serve to chemically sterilize the vesicles, with the glutaraldehyde subsequently removed by dialysis.

The enzymatic hydrolysate of wastepaper was evaluated for its cellulase‐inducing capability and production characteristics in continuous culture of Trichoderma reeseiRUT C30. Under the study conditions, i.e., pH 5.0, temperature 25 °C, and typical medium C:N ratio, the apparent cell yield constant was found to be 0.76 (g of dry cell weight/g of reducing sugar), and the maximum specific cell growth rate was 0.26 h−1. The study on the effects of medium C:N ratio confirmed an important role of N sources in the cellulase synthesis. The cellulase production decreased significantly when the feed concentrations of N sources were reduced. An experiment at pH 7.5 with 4‐fold N source concentrations also led to poorer cellulase production. When compared with cellulose, the wastepaper hydrolysate was found to have similar cellulase‐inducing strength and to induce an apparently complete set of cellulase components. The hydrolysate was also concluded to be a better soluble inducer than sophorose. While comparable at a low dilution rate (0.012 h−1), the specific cellulase productivities of the hydrolysate‐supported and the sophorose‐induced systems exhibited opposite trends with increasing dilution rates. The specific productivity in sophorose‐induced systems decreased with an increase in the dilution rate. On the other hand, with increasing dilution rate the specific productivity in the hydrolysate‐supported systems increased from 2.2 FPU/g·h at D= 0.012 h−1to 12.2 FPU/g·h at D= 0.122 h−1before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.

An online fluorometer designed for following intracellular NAD(P) H was used to monitor aerobic sludge digestion experiments. The fluorescence showed an initial rise to a high plateau, a sharp decline after staying at the plateau for 20–60 h, and a trailing very slow decrease. The characteristic fluorescence profile was shown to result mainly from the solids‐associated fluorescence, after ruling out other factors such as pH, temperature, and supernatant fluorescence. The fluorescence profile was, however, not a mere result of the decreasing solids concentration. The varying sludge viability and population composition (e.g., the decay of heterotrophs and the increasing fraction of nitrifiers) played important roles. The fluorescence profile correlated well with the profile of the viable heterotrophic cell numberconcentration evaluated with TSB‐agar plates. The initial increase of the number concentration was attributed to the growth of multiple small bacteria from the lysate of each large microorganism, which was demonstrated in the experiments with baker's yeast as the starting culture for digestion. The fluorescence profiles observed in the yeast experiments were similar to those in the sludge experiments. Responding to glucose additions and the switch from aerobic to anaerobic conditions, the yeast systems showed typical step increases of fluorescence as expected from the change of NAD(P) H level associated with heterotrophic metabolism. However, no such fluorescence responses were detectable in the sludge digestion systems. NAD(P) H were thus uncertain to be responsible for the online fluorescence observed. Nonetheless, the initial fluorescence plateau corresponded to the period of rapid digestion and, for the plant studied, the EPA regulation criteria of VSS reduction >38% and/or SOUR <1.5 mg of O2(g of TS) −1h−1were satisfied at the end of the plateau. The online fluorescence provides an effective means of monitoring the aerobic sludge digestion process.

Transport of lactic acid in supported liquid membranes containing tertiary amines (Alamine 336, Henkel Corp.) as the carrier was investigated. Both equilibrium extraction constants (Kt) and effective diffusion coefficients (D) of the acid-amine complex were measured for systems with various diluents. Larger Kt values and, thus, more efficient extraction were found when diluents were used, especially with oleyl alcohol which improved the polarity of the oil membrane and led to an approximately sixtyfold increase in the Kt value. Experimental results of D for different supported liquid membranes were found in the range of 2 to 7 × 10-7 cm2/s. Although much lower than those predicted by the Wilke-Chang equation, the values are consistent in the orders of magnitude with the literature results for other permeates in similar supported liquid membranes.

The W/O xanthan fermentation is simulated by integrating the microbial kinetic behaviors and the multiple-phase process characteristics. Model 1 assumes uniform redistribution of cells, substrates and product by frequent droplet breakup and coalescence. Model 2 simulates the system of viscous aqueous phase with minimal droplet breakup and component redistribution. The real fermentation should proceed within the bounds set by the two models. Effects of various parameters are evaluated. The aqueous-phase xanthan concentration (Xn) and volumetric productivity (QP) achieved at 200 h are used as the indicators. Xn and QP increase with nitrogen-source concentration (SNO) initially but plateau (Model 1) or decrease slightly (Model 2) at high SNO. Xn (at 200 h) decreases with increasing aqueous-phase volume fraction (f). QP, however, increases with f reflecting its basing on the total dispersion volume. Increasing agitation and aeration result in higher Xn and QP. The higher agitation enhances the G/O interfacial oxygen transfer and reduces the droplet size. Increasing aeration improves the G/O interfacial transfer but increases the droplet size. Its net positive effect implies a rate-limiting step at G/O interface. The W/O fermentation can produce far higher Xn (> 200 kg/m3) and QP( > 0.8 kg/m3-h) than the conventional fermentation (Xn ∼ 50 kg/m3, QP ∼ 0.5 kg/m3-h).

Cyanobacterium Anabaena flos-aquae was cultivated in photobioreactors for production of intracellular gas vesicles (GVs), as potential oxygen microcarriers. Natural flotation of the buoyant culture was investigated as a potential means of cell harvesting, because filtration and centrifugation tended to destroy the vesicles. Best flotation was found with actively growing culture and when conducted in the dark. The flotation-related cell properties, including the specific GV content, vesicle-collapsed filament density, and intracellular carbohydrate content, were measured to understand the phenomena. During the batch culture, the specific GV content remained relatively constant at 370 μL/(g dry cells) but the filament density (ranging 1.02 to 1.08 g/cm³) showed a decrease-then-increase profile. The increase began when the growth slowed down because of the reduced light availability at high cell concentrations. The dark flotation was studied with both actively growing (μ ≈ 0.2 day⁻¹) and stationary-phase cultures. The specific GV content of the stationary-phase culture remained relatively constant while that of the growing culture increased slightly. The intracellular carbohydrate content of the growing culture decreased much faster and more significantly, from 57 to 10 mg/(g dry cells) in  8 h. The filament density also decreased, apparently parallel to the profiles of carbohydrate content. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 60: 636–641, 1998.

The occurrence of biological oxygen transfer enhancement was investigated with samples of influent wastewater and wastewater solids collected from a nearby wastewater treatment plant. The potential mechanisms for the enhancement, namely, cell respiration, physical presence of solids, and wastewater modification, were studied in isolation by careful experimental design. The physical presence of nonrespiring solids was found to retard oxygen transfer, presumably because of the lower oxygen permeability in the solids layer accumulated near the bubble surfaces. On the other hand, cell respiration and wastewater modification enhanced the oxygen transfer rates. Overall oxygen transfer enhancements of approximately 20% were observed.

ABSTRACTPolyvinylpyridine (PVP) and activated carbon were examined for the adsorption characteristics pertinent to their application in lactic acid fermentation. For PVP the linear adsorption constant, Kad, was between 0.7 and 1.0 for an equilibrium pH range of 3 to 9. The pH was adjusted by acid/base addition, similar to pH control in fermentation. The values of Kad>in the pH-adjusted systems were much lower than that reported for pure lactic acid solutions, i.e., about 9.7. Furthermore, no clear effect of pH was observed. These are attributed mainly to the competition of anions (Cl∼ and lactate) for the adsorption sites of protonated pyridinal N. Its adsorption capacity was also found to decrease with the base regeneration (by about 14% each time) after being contacted with the culture broth. These limit its potential application in lactic acid fermentation. Activated carbon was much more effective in lactic acid/lactate adsorption than PVP. At pH 5.5 (optimal for fermentation), the value of Kadof activated carbon was about 7. The adsorption further favored lower pH under acid (HC1) addition. Activated carbon has been reported to adsorb glucose. However, the presence of glucose in 0–70 g/L was found in this study to have an insignificant effect on lactate adsorption. Cells of Lactobacillus delbrueckiialso adsorbed rapidly on activated carbon. This cell adsorption had a negative effect on lactate adsorption.

The sulfur found in coal is generally divided into three forms: pyritic, sulfate, and organic sulfur. Originating from irt-situ oxidation of metal sulfides, sulfates axe present in coal at low concentrations. Being soluble in water, they are relatively easy to be leached from the coal. Similarly, microbial depyritization promotes the oxidative conversion of inorganic sulfur compounds to water-soluble products. The pyrite removal results from the combined effects of direct bacterial attack and indirect chemical solubilization. In the former, pyrite (FeS2) is oxidized by bacteria into Fe2(SO4) 3; in the latter, ferric iron is the actual oxidizing agent and microorganisms serve to regenerate the ferric iron from ferrous iron.Organic sulfur is chemically bound to the carbon skeleton of coal. It is believed to be present mainly in the forms of organic sulfides, disulfides, thiols and thiophenes. Two pathways have been proposed for microbial desulfurization of heterocycles. One emphasized the oxidation of carbon ring structures to polar teachable derivatives. The other stressed the sulfur-specific metabolism without degradation of the carbon skeleton. Both pathways have been demonstrated with model organosulfur compounds, especially dibenzothiophene (DBT). Reports on organic sulfur removal from coal and water-soluble coal-derived products by microorganisms are also available. Unfortunately, due to the difficulty in accurate analysis.of organic sulfur content, the technical feasibility of microbial organic sulfur removal from coal is still in dispute.The costs of independent microbial depyritization and organic:sulfur removal were estimated to be $10-14 and $14 per ton of coal, respectively. A cost analysis for a tow-step process, which was designed to achieve complete removal of inorganic sulfur and a 40% reduction of organic sulfur, yieded $11 per ton of coal. These are lower than the costs of other techniques, e.g. $20-40 per ton of coal for flue-gas desulfurization.

When nitrate was added to anaerobic resting cultures of Escherichia coli, two different profiles of NAD(P)H fluorescence were observed. E. coli is known to reduce nitrate to ammonia via nitrite as an anaerobic respiration mechanism. The profile showing single-stage response corresponded to situations where the nitrite formed from nitrate reduction was immediately converted to ammonia. The other profile showing two-stage response resulted from a much slower reduction of nitrite than nitrate. Nitrite thus accumulated during the first stage and was gradually reduced to ammonia when nitrate was depleted, i.e. in the second stage. An undamped oscillation of NAD(P)H fluorescence was also observed in the cultures showing the two-stage response. The oscillation was always detected during the second stage and seldom during either the first stage or the recovered anaerobic stage (after complete nitrite reduction). It never occurred in the cultures showing the single-stage response. The period of oscillation ranged from 1 to 5min. The possibility of the common glycolytic oscillation being responsible is low, as judged from the current knowledge of the nitrate/nitrite reductases of E. coli and the observations in this study. This is the first report on the occurrence of oscillatory NAD(P)H fluorescence in E. coli.

Several nonionic surfactants, Pluronic F68 and F88 (BASF) as well as Tween 20 and 80, were found in this study to enhance the enzymatic hydrolysis of pretreated newsprint. Pluronic F68 was the most effective among the surfactants studied. With 2% (w/v) F68 the cellulose (10 g/L) conversion achieved with 2 g/L cellulase reached 52%, about the same as the 48% conversion achieved with 10 g/L cellulase in a surfactant‐free system. An empirical model, x= Kt0.5‐(t/τ)1/2, has been developed to describe the extended hydrolysis, where xis conversion, tis reaction time, and Kand τ correspond to the initial saccharification rate and the characteristic time for its tendency to deviate from the initial power‐law (exponent = 0.5) behavior. The best‐fit Kand τ in the extended hydrolysis experiments confirmed that F68 significantly enhanced both the initial rate and long‐term hydrolysis. A study was also made to evaluate the compatibility of these surfactants, at concentrations up to 2% (w/v), with cultures of Saccharomyces cerevisiaeand Dekkera clausenii. While Tween 20 was found strongly inhibitory to D.clausenii, the culture growth in all other surfactant‐added systems was comparable to that in the surfactant‐free controls.

The objective of this paper is to determine oxygen diffusion coefficients, permeabilities, and solubilities in a commonly used o/w topical ointment (similar to USP XXI hydrophilic ointment) and four commercial 1% anthralin creams. A new method developed by Ju and Ho in 1986 by applying a coupled steady-state and unsteady-state analysis for oxygen diffusion through a thin layer of test material on the surface of a membrane-covered polarographic oxygen electrode was used in the study. The results indicate that oxygen diffusion coefficients decrease with an increasing petrolatum concentration in o/w topical creams. The oxygen diffusion coefficients in four commercial creams containing 1% of extremely oxygen-sensitive anthralin were also determined using the same method. The information generated in this study can lead to a better understanding of the diffusion transport mechanism of oxygen in the topical creams and, thus, be useful to the formulation design of oxygen-sensitive drugs in semisolid dosage forms intended for topical, ophthalmic, and parenteral use.

Xanthan fermentations in W/O dispersions performed better than the control in both small flasks and a 6.6-L fermentor. The better bulk mixing and oxygen transfer achieved in the dispersion resulted in a still rising xanthan concentration of 65 g/L, compared with 26 g/L in the control. A phase inversion phenomenon was observed when n-hexadecane recovered from previous runs was used as the oil.

The NAD(P)H fluorescence ofPseudomonas aeruginosadropped sharply upon addition of nitrate to an anaerobic culture, indicating that denitrification is not limited by mass transfer of nitrate through cell membrane to reach nitrate reductase. The effect of added nitrate concentration on fluorescence drop followed a typical saturation kinetics. The maximum specific denitrification rate under the studied condition was found to be 0.26±0.05 g NO3−-N/g cells-hr.