Lu-Kwang Ju

Estimation of the polar parameters of the surface tension of liquids by contact angle measurements on gels

Abstract In a previous paper it was shown that negative interfacial tensions between predominantly monopolar surfaces (i.e., surfaces with mainly H-acceptor properties) and polar liquids are real phenomena. Such negative interfacial tensions do however decay rapidly. For miscible liquids, the decay of the interface is, in general, so rapid that it practically excludes measurement of interfacial tension. However, if one liquid is present in the form of a gel, and if the other liquid is placed as a drop upon the gel, there is often enough time to measure contact angles. This may be done at various concentrations of the liquid encased in the gel, and an extrapolation made to zero concentration of the gelling agent. With this method we found the existence of negative interfacial tensions at liquid/liquid interfaces.

Rhamnolipid production by Pseudomonas aeruginosa under denitrification: Effects of limiting nutrients and carbon substrates

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.

Degradation of n-Hexadecane and Its Metabolites by Pseudomonas aeruginosa under Microaerobic and Anaerobic Denitrifying Conditions

ABSTRACT A strategy for sequential hydrocarbon bioremediation is proposed. The initial O2-requiring transformation is effected by aerobic resting cells, thus avoiding a high oxygen demand. The oxygenated metabolites can then be degraded even under anaerobic conditions when supplemented with a highly water-soluble alternative electron acceptor, such as nitrate. To develop the new strategy, some phenomena were studied by examining Pseudomonas aeruginosafermentation. The effects of dissolved oxygen (DO) concentration onn-hexadecane biodegradation were investigated first. Under microaerobic conditions, the denitrification rate decreased as the DO concentration decreased, implying that the O2-requiring reactions were rate limiting. The effects of different nitrate and nitrite concentrations were examined next. When cultivated aerobically in tryptic soy broth supplemented with 0 to 0.35 g of NO2−-N per liter, cells grew in all systems, but the lag phase was longer in the presence of higher nitrite concentrations. However, under anaerobic denitrifying conditions, even 0.1 g of NO2−-N per liter totally inhibited cell growth. Growth was also inhibited by high nitrate concentrations (>1 g of NO3−-N per liter). Cells were found to be more sensitive to nitrate or nitrite inhibition under denitrifying conditions than under aerobic conditions. Sequential hexadecane biodegradation by P. aeruginosa was then investigated. The initial fermentation was aerobic for cell growth and hydrocarbon oxidation to oxygenated metabolites, as confirmed by increasing dissolved total organic carbon (TOC) concentrations. The culture was then supplemented with nitrate and purged with nitrogen (N2). Nitrate was consumed rapidly initially. The live cell concentration, however, also decreased. The aqueous-phase TOC level decreased by about 40% during the initial active period but remained high after this period. Additional experiments confirmed that only about one-half of the derived TOC was readily consumable under anaerobic denitrifying conditions.

Enhancing Enzymatic Saccharification of Waste Newsprint by Surfactant Addition

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 x is conversion, t is reaction time, and K and 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 K and 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 cerevisiae and 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.

Aerobic Denitrification of Pseudomonas aeruginosa Monitored by Online NAD(P)H Fluorescence

ABSTRACT Continuous cultures of Pseudomonas aeruginosa (ATCC 9027) maintained at different dissolved oxygen concentrations (DO) were studied for the effects of DO on various culture properties, especially aerobic respiration and denitrification. The DO was varied from 0 mg/liter (completely anoxic conditions) to 1.3 mg/liter and measured with optical sensors that could accurately determine very low DO based on oxygen-quenched luminescence. The strain was found to perform aerobic denitrification; while the specific rate decreased with increasing DO, denitrification persisted at approximately 1/8 of the maximum rate (1.7 mmol/g of cells/h) even at relatively high DO (1 to 1.3 mg/liter). In the presence of nitrate, the cultures Monod half-rate saturation constant for O2 was very small, <0.1 mg/liter. Aerobic denitrification appeared to function as an electron-accepting mechanism supplementary to or competitive with aerobic respiration. The shift of the cultures respiratory mechanism was also clearly detected with a fluorometer targeting intracellular NAD(P)H, i.e., the reduced forms of the NAD(P) coenzymes. Comparatively, the NAD(P)H fluorescence under the anoxic, denitrifying conditions (NFUDN) was highest, that under fully aerobic conditions (NFUOX) was lowest, and that under conditions in which both denitrification and aerobic respiration occurred (NFU) was intermediate. Representing a quantitative measure of the cultures “fractional approach” to the fully denitrifying state, the normalized fraction (NFU − NFUOX)/(NFUDN − NFUOX) was correlated with DO and the calculated fraction of electrons accepted by denitrification. The NFU fraction decreased with increasing DO, following an empirical exponential relationship. The fraction of denitrification-accepted electrons increased with the NFU fraction: the increase was gradual and approximately linear at DO of ≥0.1 mg/liter but much sharper at lower DO. Online NAD(P)H fluorescence was demonstrated as a feasible technique for effective monitoring and quantitative description of the microaerobic state of microorganisms.

Sophorolipid production from different lipid precursors observed with LC-MS

Abstract An HPLC-MSn system was used to quantify and identify the structures of individual sophorolipid components produced in Torulopsis bombicola fermentation on glucose with or without hexadecane or soybean oil. With glucose alone, the SL production was minimal and the products were complex mixtures with mainly acidic SLs. The SLs produced with glucose plus soybean oil were also complex, containing both lactonic and acidic SLs with saturated and unsaturated C16 and C18 fatty acid moieties. The glucose plus hexadecane system gave the highest production rate and product selectivity, forming primarily two diacetylated lactonic isomers with palmitate as the fatty acid moiety. A close structure correspondence between the SL’s lipid moiety and the lipid precursor used was observed. The change of the composition of SL mixtures along batch fermentation was further examined. The concentrations of acidic SLs increased very gradually throughout the process. The production of lactonic SLs became appreciable following the addition of hexadecane or soybean oil at 24 h, and increased much more rapidly after the culture reached the stationary phase. The combined percentage of the main lactonic SLs leveled off at ∼ 80% for the hexadecane system and ∼ 50% for the soybean oil system. The yields of crude SLs were 0.84, 0.20, and 0.03 g per gram of hexadecane, soybean oil, and glucose consumed during the SL production phase. Hexadecane is thus a more efficient second C-source for sophorolipid production.

Improved scale-up strategies of bioreactors

Effective scale-up is essential for successful bioprocessing. While it is desirable to keep as many operating parameters constant as possible during the scale-up, the number of constant parameters realizable is limited by the degrees of freedom in designing the large-scale operation. Scale-up of aerobic fermentations is often carried out on the basis of a constant oxygen transfer coefficient, kLa, to ensure the same oxygen supply rate to support normal growth and metabolism of the desired high cell populations. In this paper, it is proposed to replace the scale-up criterion of constant kL by a more direct and meaningful criterion of equal oxygen transfer rate at a predetermined value of dissolved oxygen concentration. This can be achieved by using different oxygen partial pressures in the influent gas streams for different scales of operation. One more degree of freedom, i.e., gas-phase oxygen partial pressure, is thus added to the process of scale-up. Accordingly, one more operating factor can be maintained constant during scale-up. It can be used to regulate the power consumption in large-scale fermentors for economical considerations or to describe the fluid mixing more precisely. Examples are given to show that the results of optimization achieved in the bench-scale study can be translated to the production-scale fermentor more successfully with only a small change in the gas-phase oxygen partial pressure employed in the bench-scale operation.

Purification of lactonic sophorolipids by crystallization

Various experimental methods for purifying lactonic sophorolipids (SLs) via crystallization were studied. The commonly used solvent, ethanol, was found to have much higher solubility of the lactonic SLs than the acid SLs. Consequently, for purification of lactonic SLs, ethanol not only lacked the selectivity in removing acidic SLs but also resulted in significant loss of desired products. Aqueous buffers were subsequently studied as the solvent, based on the rationale that acidic SLs, having the free carboxylic acid group(s), are more hydrophilic than the lactonic SLs, especially at higher pH. Both phthalate and phosphate buffers were found more suitable for purifying lactonic SLs than ethanol. A practical and effective method for purifying lactonic SLs to about 99% purity using phosphate buffers was developed. The FTIR spectra also showed significantly less SL components with free acid groups in the purified SLs than in the crude SLs, confirming the removal of acidic SLs in the purification.

Pretreatment of guayule biomass using supercritical carbon dioxide-based method

Guayule, a desert shrub harvested for commercial production of hypoallergenic latex and resins constitutes <20% of the biomass. Converting the remaining bagasse to biorefinery feedstock for value-added products is an optimal economic option. A supercritical CO(2)-based process had been developed previously for resin extraction. In this study, the feasibility of including a supercritical CO(2)-based bagasse pretreatment method was evaluated. The pretreatment involved: adding water to the bagasse, raising system temperature, pressurizing using supercritical CO(2), holding the system for a period of time, and exploding the bagasse. The pretreated biomass was subjected to enzyme hydrolysis. The yields of released sugars were used as pretreatment effectiveness indicators. Supercritical method outperformed other methods and gave much higher overall sugar yields for guayule (as high as 77% for glucose and 86% for total reducing sugars through both pretreatment and hydrolysis, as compared to 50% for glucose and 52% for total sugars with the dilute-acid pretreatment and 36% for glucose and 52% for total sugars with the delignification pretreatment). The enzymatic hydrolyzates were tested on the cellulase-producing fungus Trichoderma reesei Rut C-30. No inhibitory/toxic effects were apparent in terms of cell growth, sugar consumption, and cellulase and xylanase production. The supercritical CO(2)-based method was found to be very promising for pretreatment of waste biomass as the feedstock for subsequent enzymatic hydrolysis and fermentation to produce value-added bioproducts.

Wastepaper hydrolysate as soluble inducing substrate for cellulase production in continuous culture of Trichoderma reesei

The enzymatic hydrolysate of wastepaper was evaluated for its cellulase‐inducing capability and production characteristics in continuous culture of Trichoderma reesei RUT 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−1 to 12.2 FPU/g·h at D = 0.122 h−1 before beginning to decline. The initial increasing trend was attributed to the higher concentrations of inducing oligomer intermediates at larger dilution rates.