Production of isopropyl cis-6-hexadecenoate by regiospecific desaturation of isopropyl palmitate by a double mutant of a Rhodococcus strain

Resting cells of a double mutant noted as KSM-MT66, derived from Rhodococcus sp. strain KSM-B-3 by UV irradiation, were found to cis-desaturate isopropyl hexadecanoate, yielding isopropyl cis-6-hexadecenoate. Addition of sodium glutamate (1.0%), Mg SO4 (2 mM), and thiamine (2 mM) increased the productivity of the unsaturated product in phosphate buffer. Optimal temperature and pH for the reaction were around 26 degrees C and 7, respectively. Under the optimized conditions, more than 50 g/l of isopropyl cis-6-hexadecenoate was produced after a 3-day incubation by resting cells of the mutant. Thus, cis-6-hexadecenoic acid, the main component of human sebaceous lipids, can be manufactured economically by the rhodococcal bioconversion.     

Regiospecific internal desaturation of aliphatic compounds by a mutant Rhodococcus strain

A mutant Rhodococcus strain lacking the ability to utilize 1-chlorohexadecane was found to cis-desaturate aliphatic compounds, such as 1-chlorohexadecane, n-hexadecane, and heptadecanonitrile, yielding corresponding products with a double bond mainly at the ninth carbon from the terminal methyl groups. A new oxidative pathway involving the cis-desaturation step was suggested for alkane utilization by Rhodococcus spp.     

Substrate specificity of regiospecific desaturation of aliphatic compounds by a mutant Rhodococcus strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.     

Forced-flow bioreactor for sucrose inversion using ceramic membrane activated by silanization

A forced-flow enzyme membrane reactor system for sucrose inversion was investigated using three ceramic membranes having different pore sizes. Invertase was immobilized chemically to the inner surface of a ceramic membrane activated by a silane-glutaraldehyde technique. With the cross-flow filtration of sucrose solution, the reaction rate was a function of the permeate flux, easily controlled by pressure. Using 0.5 mum support pore size of membrane, the volumetric productivity obtained was 10 times higher than that in a reported immobilized enzyme column reactor, with a short residence time of 5 s and 100% conversion of the sucrose inversion.     

A hollow-fiber membrane bioreactor for the removal of trichloroethylene from the vapor phase

A hollow-fiber membrane bioreactor was used to separate trichloroethylene (TCE) from a gaseous waste stream with subsequent cometabolic biodegradation by a pure culture of Methylosinus trichosporium OB3b PP358. The two-stage bioreactor system was successfully operated for 20 days. PP358 was grown in a continuous-flow chemostat and circulated through the fiber lumen of a hollow-fiber membrane module (HFMM), while TCE contaminated air (141 to 191 microg/L) was pumped through the HFMM shell. Between 54% -84% TCE transfer and 92%-96% TCE cometabolism were obtained in the HFMM reactor loop. Short shell-residence times, 1.6 to 5.0 minutes, demonstrated quick throughput of TCE contaminated air. Best-fit computer modeling of the biological experiments estimated mass transfer coefficients between 2.0 x 10(-3) cm/min and 5.6 x 10(-3) cm/min. The average pseudo-first-order biodegradation rate constant for the biological experiments was 0.46 L/mg TSS/d. These results demonstrate that the hollow-fiber membrane bioreactor represents an attractive technology for the bioremediation of gaseous waste streams.     

Mineralization of 4-fluorocinnamic acid by a Rhodococcus strain

A bacterial strain capable of aerobic degradation of 4-fluorocinnamic acid (4-FCA) as the sole source of carbon and energy was isolated from a biofilm reactor operating for the treatment of 2-fluorophenol. The organism, designated as strain S2, was identified by 16S rRNA gene analysis as a member of the genus Rhodococcus. Strain S2 was able to mineralize 4-FCA as sole carbon and energy source. In the presence of a conventional carbon source (sodium acetate [SA]), growth rate of strain S2 was enhanced from 0.04 to 0.14 h^?1 when the culture medium was fed with 0.5 mM of 4-FCA, and the time for complete removal of 4-FCA decreased from 216 to 50 h. When grown in SA-supplemented medium, 4-FCA concentrations up to 1 mM did not affect the length of the lag phase, and for 4-FCA concentrations up to 3 mM, strain S2 was able to completely remove the target fluorinated compound. 4-Fluorobenzoate (4-FBA) was transiently formed in the culture medium, reaching concentrations up to 1.7 mM when the cultures were supplemented with 3.5 mM of 4-FCA. Trans,trans-muconate was also transiently formed as a metabolic intermediate. Compounds with molecular mass compatible with 3-carboxymuconate and 3-oxoadipate were also detected in the culture medium. Strain S2 was able to mineralize a range of other haloorganic compounds, including 2-fluorophenol, to which the biofilm reactor had been exposed. To our knowledge, this is the first time that mineralization of 4-FCA as the sole carbon source by a single bacterial culture is reported.     

A membrane bioreactor for biotransformations of hydrophobic molecules

The Membrane Bioreactor for Biotransformations (MBB) is based on the aqueous/organic two-phase system, and uses a tubular silicone rubber membrane to separate the two liquid phases. This avoids the key problem associated with direct contact two-phase processes, specifically, product emulsification. The baker's yeast mediated reduction of geraniol to citronellol was used as a model biotransformation to demonstrate MBB operation. Values for the overall mass transfer coefficient were determined for geraniol, (2.0 x 10(-5) ms-1), and for citronellol, (2.1 x 10(-5) ms-1) diffusion across the silicone rubber membrane. Using these values, and the specific activity of the biocatalyst (5 nmols-1g biomass-1), a suitable membrane surface area: biomass ratio was determined as 2.4 x 10(-3) m2g biomass-1. The bioreactor was operated at this surface area: biomass ratio and achieved a product accumulation rate 90-95% that of a conventional direct contact two-phase system. The slight reduction in product accumulation rate was shown not to be due to mass transfer limitations with respect to reactant delivery or product extraction. Copyright 1998 John Wiley & Sons, Inc.     

Cometabolic turnover of aniline,phenol and some of their monochlorinated derivatives by the Rhodococcus mutant strain AM 144

One-step conversion of aniline, phenol and some of their monochlorinated derivatives into the corresponding catechols by resting pre-adapted cells of the Rhodococcus mutant strain AM 144 (defective in synthesis of catechol 1,2-dioxygenase) was shown to depend on the availability of an additional metabolizable carbon substrate, e.g. glucose or acetate. A stoichiometric relation existed between the amount of the latter compounds added and the amount of aniline (or phenol, respectively) converted into catechol suggesting that the primary function of the cosubstrates was to provide reducing power to the oxygenative transformation reaction. The observed cosubstrate-dependence generally parallels that seen in previous studies on turnover of different monochloroaromatic non-growth substrates by aromatics-utilizing Rhodococcus wildtype-strains. Cell cultures of strain AM 144 growing at the expense of acetate also proved able to convert aniline into catechol. Typically, growth of the cells was retarded during the phase of aniline transformation as compared to the respective control cultures. Based on the results of these model experiments, it was concluded that (i) in natural microbial communities cometabolically active bacteria would hardly enrich under cometabolic conditions over fast-growing non-cometabolizing bacteria if the latter organisms will tolerate the particular non-growth substrate, and (ii) cometabolizing bacteria would have a selective advantage only if the non-growth substrate to be transformed is a toxic one or if it can serve as a potential nutrient source (e.g., of nitrogen or sulfur).Abbreviations MCA monochloroaniline - MCP monochlorophenol - MCC monochlorocatechol - TLC thin-layer chromatography - MS mass spectrometry - GLC gas-liquid chromatography - UV ultraviolet (range of the spectrum)     

Characterization of microemulsion structures in the pseudoternary phase diagram of isopropyl palmitate/water/Brij 97:1-butanol

This research was aimed to characterize microemulsion systems of isopropyl palmitate (IPP), water, and 2∶1 Brij 97 and 1-butanol by different experimental techniques. A pseudoternary phase diagram was constructed using water titration method. At 45% wt/wt surfactant system, microemulsions containing various ratios of water and IPP were prepared and identified by electrical conductivity, viscosity, differential scanning calorimetry (DSC), cryo-field emission scanning electron microscopy (cryo-FESEM) and nuclear magnetic resonance (NMR). The results from conductivity and viscosity suggested a percolation transition from water-in-oil (water/oil) to oil-in-water (oil/water) microemulsions at 30% wt/wt water. From DSC results, the exothermic peak of water and the endothermic peak of IPP indicated that the transition of water/oil to oil/water microemulsions occurred at 30% wt/wt water. Cryo-FESEM photomicrographs revealed globular structures of microemulsions at higher than 15% wt/wt water. In addition, self-diffusion coefficients determined by NMR reflected that the diffusability of water increased at higher than 35% wt/wt water, while that of IPP was in reverse. Therefore, the results from all techniques are in good agreement and indicate that the water/oil and oil/water transition point occurred in the range of 30% to 35% wt/wt water.     

Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain

Rhodococcus sp. KT462, which can grow on either benzothiophene (BT) or dibenzothiophene (DBT) as the sole source of sulfur, was newly isolated and characterized. GC and GC-MS analyses revealed that strain KT462 has the same BT desulfurization pathway as that reported for Paenibacillus sp. A11-2 and Sinorhizobium sp. KT55. The desulfurized product of DBT produced by this strain, as well as other DBT-desulfurizing bacteria such as R. erythropolis KA2-5-1 and R. erythropolis IGTS8, was 2-hydroxybiphenyl. A resting cells study indicated that this strain was also able to degrade various alkyl derivatives of BT and DBT.     

Teichoic acids and lipids associated with the membrane of a Bacillus licheniformis mutant and the membrane lipids of the parental strain.

Bacillus licheniformis 6346 MH-1 and a phosphoglucomutase-deficient poorly lytic mutant, B. licheniformis 6346 MH-5, both contain cardiolipin, phosphatidyl ethanolamine, and phosphatidyl glycerol but are devoid of phosphoglycolipids. Gentiobiosyl diglyceride is present in the parent organism but glycolipids are absent from the mutant. Lipoteichoic acid was extracted from the whole cells of MH-5 with hot aqueous phenol and contained fatty acids, glucosamine, and 1,3-polyglycerol phosphate. The fatty acids were predominantly of the branched-chain type and were esterified to hydroxyl groups of a terminal glycerol residue. The polyglycerol phosphate chains contained, on average, 32 to 40 glycerol residues, some of which were substituted at the secondary hydroxyl group with alpha-N-acylglucosaminyl residues. Phenol extraction of the supernatant fluid that remained when walls were removed from preparations of disrupted cells of MH-5 yielded membrane teichoic acid, which consisted of substituted polyglycerol phosphate but was devoid of fatty acids.     

A novel membrane bioreactor enhanced by effective microorganisms for the treatment of domestic wastewater

The activated sludge membrane bioreactor (MBR) has been shown to have some advantages for the processing and reclamation of domestic wastewater. We hypothesized that certain microorganisms, chosen for their abilities to decompose the chemical components of raw sewage, would, when coupled with the MBR, significantly improve the stability and efficiency of this system. We selected environmental bacterial strains which oxidize ammonia and nitrites and produce protease, amylase, and cellulase for the development and testing of a novel biologically enhanced MBR (eMBR). We compared the eMBR with the activated sludge MBR. With the eMBR, the average values of effluent quality were: chemical oxygen demand (COD), 40 mg/l(average efficiency of removal 90.0%); and NH₄ ⁺–N, 0.66 mg/l(average efficiency of removal 99.4%). Effluent qualities met the standard and were stable during the entire 90 days of this study. For the activated sludge MBR, the COD removal rate was 91.7%, and the NH₄ ⁺–N removal (94.8%) was less than that of the eMBR. Start-up time for the eMBR was only 24–48 h, much shorter than the 7–8 days required to initiate function of the standard MBR. The biomass concentrations of total heterotrophic bacteria and autotrophic bacteria in the eMBR did not fluctuate significantly during the course of the study. Various kinds of microorganisms will establish an ecological balance in the reactor. Compared with the activated sludge MBR, the eMBR not only produced an excellent and stable quality of effluent but also resulted in a shorter time to start-up and significantly improved the efficiency of NH₄ ⁺–N removal.     

Evidence for diverse oxidations in the catabolism of toluene by Rhodococcus rhodochrous strain OFS

Rhodococcus rhodochrous strain OFS grew on toluene as a sole source of carbon and energy with a maximum growth rate of 0.011 h⁻¹. Initial reaction products were extracted, derivatized and identified by GC-MS. Oxygen consumption studies indicated that OFS grown on an aliphatic substrate required an induction period before oxidizing toluene. OFS grown on toluene transformed an array of aromatic ground water pollutants including styrene, ethylbenzene and chlorobenzene. Products of these transformations were identified. The sole product of chlorobenzene biotransformation was 4-chlorophenol. Products from toluene oxidation included 3- and 4-methylcatechol as well as benzyl alcohol, p-cresol and cis-toluene dihydrodiol. The identification of these and the products of other aromatic substrate conversions affirm that oxidation occurred on the functional group as well as directly on the aromatic nucleus. Received: 23 July 1999 / Received revision: 4 October 1999 / Accepted: 16 October 1999     

Performance of a membrane bioreactor used for the treatment of wastewater contaminated with heavy metals

In this work the performance of a Membrane bioreactor (MBR) was assessed for the removal of 3-15 mg/l of copper, lead, nickel and zinc from wastewater. The average removal efficiencies accomplished by the MBR system were 80% for Cu(II), 98% for Pb(II), 50% for Ni(II) and 77% for Zn(II). The addition of 5 g/l vermiculite into the biological reactor enhanced metal removal to 88% for copper, 85% for zinc and 60% for nickel due to adsorption of metal ions on the mineral, while it reduced biomass inhibition and increased biomass growth. The metal ions remaining in soluble form penetrated into the permeate, while those attached to sludge flocs were effectively retained by the ultrafiltration membranes. The average heterotrophic biomass inhibition was 50%, while it reduced to 29% when lower metal concentrations were fed into the reactor in the presence of vermiculite. The respective autotrophic biomass inhibition was 70% and 36%. The presence of heavy metals and vermiculite in the mixed liquor adversely impacted on membrane fouling.     

Start-up of the Anammox process in a membrane bioreactor

The start-up of an Anammox process was studied in a membrane sequencing batch reactor (MSBR) in which a submerged hollow fibre membrane module was used to retain the biomass. The reactor was seed with Anammox biomass and fed using the Van de Graaf medium. During a first operating stage, salt precipitation was observed and interfered with microbial activity and caused a decrease of the nitrogen removal rate of the reactor from 100 to only 10 mgl(-1) per day. Salt precipitation was avoided by diminishing adequately the Ca and P concentrations of the Van de Graaf medium during the last operating stage. This action increased quickly the activity of the system, and nitrogen removal rate reached up to 710 mgl(-1) per day with almost full nitrite removal. Sporadic flotation of the sludge was observed in the MSBR. The use of the membrane avoided biomass wash-out from the system. Moreover, a surprising fact was that Anammox biomass did not grow in flocs in the MSBR, but in granules. This fact showed that this kind of microorganisms have a trend to grow in aggregates. Results indicated that the use of the MSBR could be a suitable system for nitrogen removal by using the Anammox reaction.     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Preparation of vanillin by bioconversion in a silicon rubber membrane bioreactor

In this study, the bioconversion of clove oil into vanillin using soybean lipoxygenase (SBLOX) as biocatalyst was investigated in a silicon rubber membrane bioreactor (SRMBR) and shaking flasks. High performance liquid chromatography (HPLC) analysis indicated that the vanillin concentration was 8.14 mg/L after 36 h of conversion in a shaking flask. It reached up to 121.53 mg/L in the receiving solution after 36 h of conversion in the SRMBR. The conversion rate of clove oil was 0.033% in the shaking flask. It was 1.01% in the SRMBR. The peak area ratio of vanillin in the receiving solution of the SRMBR was 70.08%. By adding activated carbon into the conversion broth of the SRMBR, the vanillin concentration in the receiving solution reached 140.27 mg/L, the conversion rate of clove oil increased to 1.14%, and the peak area ratio of vanillin in the receiving solution reached 93.53%.