Enhanced biotransformations and product recovery in a membrane bioreactor through application of a direct electric current

The simultaneous enhancement of biotransformation coupled to product recovery, purification and concentration is presented. The nitrilase of Rhodococcus rhodochrous LL100-21 catalyses the single-step hydrolytic biotransformation of benzonitrile to benzoic acid and ammonia. When a direct electric current is applied across a bioreactor containing the bacterium and benzonitrile, the charged product (benzoic acid) can be removed in situ across an anion exchange membrane and recovered in a separate compartment. Over the course of a 24-hour biotransformation, benzonitrile was converted to benzoic acid which was completely removed from the bioreactor chamber and concentrated 3-fold in a separate chamber. The rate of production of benzoic acid increased by 42% when the current was applied (0.044 mmol/min/g dry cell weight in the presence of current as compared to 0.03 mmol/min/g dry cell weight in its absence). The enhanced reaction rate was achieved irrespective of product separation and therefore appears to be a direct effect upon the bacterial cells. This process has potential for enhanced productivity from biotransformations through a simultaneous increase in metabolic activity and in situ product recovery.     

Evaluation of an electrochemical bioreactor system in the biotransformation of 6-bromo-2-tetralone to 6-bromo-2-tetralol

Biotransformation of 6-bromo-2-tetralone (Br-beta-tetralone) to 6-bromo-2-tetralol (Br-beta-tetralol) by yeast cells of Trichosporon capitatum (ATCC 74312) and its partially purified Br-beta-tetralone reductase was evaluated in an electrochemical bioreactor. The biotransformation rates and final product formation were significantly affected by substrate concentration, biomass and electric potential. At 2 g/l of substrate, the initial reaction rate and final product were increased by 35% and 15%, respectively, with -1.5 V of electric potential compared to without electric potential. Additional substrate (2 g/l) provided by pulse feeding to the reaction mixture at different intervals resulted in 2.1 g/l Br-beta-tetralol compared to a total of 1.2 g/l without feeding. However, the increased production was not proportionate to the amount of additionally fed substrate. Increased substrate availability by the addition of 5% (v/v) ethanol resulted in the highest reaction rate and product formation, but addition of ethanol at a concentration higher than 5% decreased the reaction rate. At low biomass, the initial reaction rates were enhanced significantly when electric potential was high, but a higher biomass was necessary to obtain a similar reaction rate when electric potential was reduced. The highest initial reaction rate (59.2 mg/l per min) was achieved with a two-fold biomass concentration of 15.6 g of dry cell weight/l, substrate at 4 g/l and electric potential at -6 V. The conversion of Br-beta-tetralone to Br-beta-tetralol with partially purified Br-beta-tetralone reductase was slow in the presence of electric potential.     

Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli

The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 μmol/min/g(dcw)) than on n-octane (21 μmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed.     

Production of toluene cis-diol by immobilized pseudomonas putida UV4 cells in barium alginate beads

In the present study, we have investigated the biotransformation of toluene to its cis-dihydrodiol (cis-diol) with immobilized Pseudomonas putida UV4 cells using different conditions of immobilization with a view to improving its production. The choice of alginate and its concentration for the immobilization of the cells were found to be the most important factors affecting the production of toluene cis-diol. The concentration of minerals and oxygen in the reaction medium and the methodology of substrate addition were investigated and the optimal conditions were defined. Once the optimal conditions for biotransformations and entrapment were determined, a packed-bed and fluidized-bed reactor were evaluated for the biotransformation process. The results using air as the gas supply showed an increase in the total production from 0.15 mol cis-diol · g⁻¹ dry cell weight (dcw) in the packed-bed reactor to 0.28 mol cis-diol · g⁻¹ dcw in the fluidized-bed reactor. When pure oxygen was used in place of air in the fluidized-bed reactor, a dramatic increase in total production up to a maximum of 6.1 mol cis-diol · g⁻¹ dcw using a medium flow rate of 100 ml min⁻¹ was achieved. Under optimal conditions, a maximum rate of production of 86.9 mmol cis-diol g⁻¹ dcw h⁻¹ was achieved for 48 h. This was seven times higher than the rate previously reported in the literature and for a much longer period of time; consequently, the overall production observed was more than 75 times higher than the values reported in the literature.     

An efficient enzymatic Baeyer-Villiger oxidation by engineered Escherichia coli cells under non-growing conditions

Economical methods of supplying NADPH must be developed before biotransformations involving this cofactor can be considered for large-scale applications. We have studied the enzymatic Baeyer-Villiger oxidation of cyclohexanone as a model for this class of reactions and developed a simple approach that uses whole, non-growing Escherichia coli cells to provide high productivity (0.79 g epsilon-caprolactone/L/h = 18 micromol epsilon-caprolactone/min/g dcw) and an 88% yield. Glucose supplied the reducing equivalents for this process, and no exogenous cofactor was required. The volumetric productivity of non-growing cells was an order of magnitude greater than that achieved with growing cells of the same strain. Cells of an engineered E. coli strain that overexpresses Acinetobacter sp. cyclohexanone monooxygenase were grown under inducing conditions in rich medium until the entry to stationary phase; the subsequent cyclohexanone oxidation was carried out in minimal salts medium lacking a nitrogen source. After the biotransformation was complete, the lactone product was adsorbed to a solid support and recovered by washing with an organic solvent.     

Tools for characterizing the whole-cell bio-oxidation of alkanes at microscale

This article describes the first reported microwell whole-cell bioconversion using a water immiscible substrate that matches the specific activity and yield achieved in a 1.2 L stirred tank bioreactor. Maximum yields of 0.6 g/L(total) 1-dodecanol achieved in 24 h compare favorably to 0.28 g/L(total) 1-dodecanol after 48 h obtained in a stirred tank reactor. Using the microwell platform we present a rapid and systematic approach to identify the key bottlenecks in the bio-oxidation of long-chain alkanes using Escherichia coli expressing the alkane hydroxylase (alkB) complex. The results indicate that mass transfer rates limit productivity in the n-dodecane bio-oxidation system, rather than inherent enzyme activity. Furthermore, substrate solubility, oxygen availability and glucose concentration act cooperatively to affect the amount of by-product, dodecanoic acid. Optimizing these factors using response surface methodology enabled specific yields of 1-dodecanol to increase eightfold and overoxidation to dodecanoic acid to be reduced from 95% to 55%. This resulted in specific activities of 10.4 μmol/min/g(dcw) on n-dodecane; approximately 50% of the 21 μmol/min/g(dcw) obtained with n-octane. For the first time, this in vivo rate difference is within the range reported for the purified enzyme. Finally, the results obtained also provide strong evidence that the mechanism of E. coli interaction with alkanes is mainly via uptake of alkanes dissolved in the aqueous phase rather than by direct cell-droplet contact.     

Accumulation of sesquiterpenes and polysaccharides in cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 10 L bioreactor

We developed a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe), using 100 g fresh weight inoculum in a batch culture. The maximum cell biomass of 68.46 g/L fresh weight was obtained after 14 days of culture in a 10 L bioreactor with a pitch-blade impeller maintained at an agitation speed of 150 rpm and an aeration rate of 2.5 L/min. The accumulation of sesquiterpenes and polysaccharide in zedoary cells from 2 to 18 days was measured by HPLC and a phenol-sulfuric acid assay, respectively. The total polysaccharide concentration increased between 2 to 10 days of culture and reached a maximum value of 6.55%. HPLC revealed several eluted peaks of sesquiterpenes, which increased in amplitude from days 2 to 10. Furthermore, our results indicated that biotransformation occurred in the cell suspension, transforming certain sesquiterpenes into other types during culture.     

生物转化合成苯乳酸工程菌的培养条件优化

本研究旨在优化重组大肠杆菌Escherichia coli BL21 (DE3) harboring pRSF-aad-ldh10-fdh菌株的培养条件,获得高密的供生物转化苯丙氨酸为苯乳酸的细胞。实验考察了摇瓶发酵培养基碳源、氮源种类和浓度,3 L发酵罐中转速和通气量及恒速补料、DO-stat和pH-stat等不同分批补料策略对菌体密度的影响。结果表明,当碳源为4 g/L葡萄糖,氮源为24 g/L安琪酵母浸粉FM802,细胞干重最大可达9.24 g/L;当转速为400 r/min和通气量为1.5 vvm时,细胞干重最大可达10.18 g/L;以4 g/(L·h)恒速流加葡萄糖时,细胞干重最大可达13.71 g/L。本研究还对工程菌酶表达的诱导条件进行了优化,菌体培养2 h后,添加终浓度为0.08 mmol/L IPTG诱导剂,在25℃下诱导培养14 h所得细胞有利于生物转化。底物苯丙氨酸浓度为60 g/L,转化为苯丙酮酸的转化率为50.2%,转化为苯乳酸的转化率为35.2%。     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

The feasibility of growing cells of Saccharomyces cerevisiae for citronellol production in a continuous-closed-gas-loop bioreactor (CCGLB)

The present work aims to address the gas-phase biotransformation of geraniol into citronellol using growing cells of Saccharomyces cerevisiae (baker's yeast) in a continuous-closed-gas-loop bioreactor (CCGLB). This study revealed that the gaseous geraniol had a severe effect on the production of biomass during the growing cell biotransformation resulting in the decrease in the specific growth rate from 0.07 to 0.05 h?1. The rate of reaction of the growing cell biotransformation was strongly affected by agitation and substrate flow rates. The highest citronellol concentration of 1.18 g/L and initial rate of reaction of 7.06 × 10?? g/min g(cell) were obtained at 500 rpm and 8 L/min, respectively.     

Enhanced biotransformation of 1,3-dichloro-2-propanol to epichlorohydrin via resin-based in situ product removal process

Biotransformation of 1,3-dichloro-2-propanol (DCP) to epichlorohydrin (ECH) by the whole cells of recombinant Escherichia coli expressing halohydrin dehalogenase was limited by product inhibition. To solve this problem and improve the ECH yield, a biotransformation strategy using resin-based in situ product removal (ISPR) was investigated. Seven macroporous resins were examined to adsorb ECH: resin HZD-9 was the best. When 10 % (w/v) HZD-9 was added to batch biotransformation, 53.3 mM ECH was obtained with a molar yield of 88.3 %. The supplement of the HZD-9 increased the ECH volumetric productivity from 0.5 to 2.8 mmol/l min compared to without addition of resin. In fed-batch biotransformation, this approach increased ECH from 31 to 87 mM. These results provide a promising basis for the biosynthesis of ECH.     

Beta-adrenergic stimulation of volume-sensitive chloride transport in lamprey erythrocytes

We measured the effects of a beta-adrenergic agonist, isoproterenol, on chloride transport and volume regulation of lamprey (Lampetra fluviatilis) erythrocytes in isotonic (288 mosm L(-1)) and hypotonic (192 mosm L(-1)) medium. Isoproterenol at a high concentration (10(-5) M) did not influence chloride transport in isotonic medium but markedly increased chloride fluxes in hypotonic conditions: unidirectional flux increased from 100 mmol kg dcw(-1) h(-1) in the absence to 350 mmol kg dcw(-1) h(-1) (dcw=dry cell weight) in the presence of isoproterenol. Simultaneously, the half-time for volume recovery decreased from 27 to 9 min. Isoproterenol caused an increase in cellular cyclic AMP (cAMP) concentration. The stimulation of chloride transport in hypotonic conditions could be induced by application of the permeable cAMP analogue, 8-bromo-cyclic AMP, suggesting that the effect of beta-adrenergic stimulation on chloride transport occurs downstream of cAMP production. As isoproterenol did not affect unidirectional rubidium fluxes in hypotonic conditions, the transport pathway influenced by beta-adrenergic stimulation is most likely the swelling-activated chloride channel. Because the beta-adrenergic agonist only influenced the transport in hypotonic conditions despite the fact that cAMP concentration also increased in isotonic conditions, the activation may involve a volume-dependent conformational change in the chloride channel.     

Adsorption of chromium (VI) ion from aqueous solution by succinylated mercerized cellulose functionalized with quaternary ammonium groups

Succinylated mercerized cellulose (cell 1) was used to synthesize an anion exchange resin. Cell 1, containing carboxylic acid groups was reacted with triethylenetetramine to introduce amine functionality to this material to obtain cell 2. Cell 2 was reacted with methyl-iodide to quaternize the amine groups from this material to obtain cell 3. Cells 2 and 3 were characterized by mass percent gain, degree of amination and quaternization, FTIR and CHN. Cells 2 and 3 showed degrees of amination and quaternization of 2.8 and 0.9 mmol/g and nitrogen content of 6.07% and 2.13%, respectively. Cell 3 was used for Cr (VI) adsorption studies. Adsorption equilibrium time and optimum pH for Cr (VI) adsorption were found to be 300 min and 3.1, respectively. The Langmuir isotherm was used to model adsorption equilibrium data. The adsorption capacity of cell 3 was found to be 0.829 mmol/g. Kinetic studies showed that the rate of adsorption of Cr (VI) on cell 3 obeyed a pseudo-second-order kinetic model.     

Alkaline biocatalysis for the direct synthesis of N-acetyl-D-neuraminic acid (Neu5Ac) from N-acetyl-D-glucosamine (GlcNAc)

Integration between the alkaline epimerization of N-acetyl-D-glucosamine (GlcNAc) to N-Acetyl-D-mannosamine (ManNAc) and the N-acetyl-D-neuraminic acid (Neu5Ac) aldolase-catalyzed biotransformation has been assessed experimentally. GlcNAc epimerization took place above pH 9.0, and the initial rate of ManNAc formation increased exponentially to 10.37 mmol/L per hour at pH 12. However, above this pH, severe degradation of pyruvate occurred. A value of 31.3% molar conversion on Pyr was achieved in an integrated biotransformation. The "pseudo"-steady state at the end of the reaction was comparable to the equilibrium achieved with a combination of an epimerase and aldolase enzymes. The integrated reaction proved feasible, but at the expense of pyruvate and Neu5Ac aldolase degradation.     

Optimization of saccharification and ethanol production by simultaneous saccharification and fermentation (SSF) from seaweed, Saccharina japonica

Ethanol was produced using the simultaneous saccharification and fermentation (SSF) method with macroalgae polysaccharide from the seaweed Saccharina japonica (Sea tangle, Dasima) as biomass. The seaweed was dried by hot air, ground with a hammer mill and filtered with a 200-mesh sieve prior to pretreatment. Saccharification was carried out by thermal acid hydrolysis with H(2)SO(4) and the industrial enzyme, Termamyl 120 L. To increase the yield of saccharification, isolated marine bacteria were used; the optimal saccharification conditions were 10% (w/v) seaweed slurry, 40 mM H(2)SO(4) and 1 g dcw/L isolated Bacillus sp. JS-1. Using this saccharification procedure, the reducing sugar concentration and viscosity were 45.6 ± 5.0 g/L and 24.9 cp, respectively, and the total yield of the saccharification with optimal conditions and S. japonica was 69.1%. Simultaneous saccharification and fermentation was carried out for ethanol production. The highest ethanol concentration, 7.7 g/L (9.8 ml/L) with a theoretical yield of 33.3%, was obtained by SSF with 0.39 g dcw/L Bacillus sp. JS-1 and 0.45 g dcw/L of the yeast, Pichia angophorae KCTC 17574.     

The two-step biotransformation of monosodium glutamate to GABA by Lactobacillus brevis growing and resting cells

Gama-aminobutyric acid (GABA) is a natural functional amino acid. In the current study, Lactobacillus brevis TCCC13007, a high GABA-producing strain, was isolated from naturally pickled Chinese vegetables. A two-step cellular bioconversion process was established using L. brevis TCCC13007 for the production of GABA. First, L. brevis cells were grown anaerobically in 7% monosodium glutamate (MSG)-containing medium at an initial pH of 6.0 and a controlled pH of 4.6 for 16 to 66 h; approximately 38 g L(-1) of GABA was obtained after 66 h of fermentation at a conversion rate of 98.6%. In the second stage of the process, about 7.6 g L(-1) of GABA was produced three more times at a conversion rate of 92.2% using the same batch of resting cells in the substrate-containing buffer under optimized conditions. Thus, the total GABA yield reached 61 g L(-1). A model system for the biotransformation of MSG to GABA was established using L. brevis TCCC13007 resting cells. The reaction rates were found to follow the classic Michaelis-Menten equation at low substrate concentrations (<80 mM). Kinetic analysis of the biotransformation revealed that L. brevis TCCC13007 resting cells produced GABA similar to that produced by purified glutamate decarboxylase from L. brevis.     

Rapid characterization of microbial biodegradation pathways by FT-IR spectroscopy

Fourier transform-infrared (FT-IR) spectroscopy has become an important tool for rapid analysis of complex biological samples. The infrared absorbance spectrum could be regarded as a "fingerprint" which is characteristic of biochemical substances. In this study, Pseudomonas putida NCIMB 9869 was grown with either 3,5-xylenol or m-cresol as the sole carbon source, each inducing different metabolic pathways for m-cresol biotransformation. FT-IR spectroscopy was capable of differentiating both induced cultures of P. putida NCIMB 9869 as well as the resulting biotransformation product mixtures. FT-IR spectral analysis indicated that carboxylic acids were key chemicals responsible for distinguishing the products of the two catabolic pathways. Gas chromatography-mass spectrometry (GC-MS) was performed to validate the FT-IR analysis, indicating that two carboxylic acids, 3-hydroxybenzoic acid and 2,5-dihydroxybenzoic acid, were present as m-cresol biotransformation products from 3,5-xylenol-grown cells, but were absent in m-cresol-grown cells. The ability to use FT-IR to rapidly distinguish between biotransformation product mixtures as well as differentially induced bacterial strains suggests this approach might be a valuable tool for screening large biotransformation assays for novel products and metabolic mutants.     

Highly efficient biotransformation of eugenol to ferulic acid and further conversion to vanillin in recombinant strains of Escherichia coli

The vaoA gene from Penicillium simplicissimum CBS 170.90, encoding vanillyl alcohol oxidase, which also catalyzes the conversion of eugenol to coniferyl alcohol, was expressed in Escherichia coli XL1-Blue under the control of the lac promoter, together with the genes calA and calB, encoding coniferyl alcohol dehydrogenase and coniferyl aldehyde dehydrogenase of Pseudomonas sp. strain HR199, respectively. Resting cells of the corresponding recombinant strain E. coli XL1-Blue(pSKvaomPcalAmcalB) converted eugenol to ferulic acid with a molar yield of 91% within 15 h on a 50-ml scale, reaching a ferulic acid concentration of 8.6 g liter(-1). This biotransformation was scaled up to a 30-liter fermentation volume. The maximum production rate for ferulic acid at that scale was 14.4 mmol per h per liter of culture. The maximum concentration of ferulic acid obtained was 14.7 g liter(-1) after a total fermentation time of 30 h, which corresponded to a molar yield of 93.3% with respect to the added amount of eugenol. In a two-step biotransformation, E. coli XL1-Blue(pSKvaomPcalAmcalB) was used to produce ferulic acid from eugenol and, subsequently, E. coli(pSKechE/Hfcs) was used to convert ferulic acid to vanillin (J. Overhage, H. Priefert, and A. Steinbüchel, Appl. Environ. Microbiol. 65:4837-4847, 1999). This process led to 0.3 g of vanillin liter(-1), besides 0.1 g of vanillyl alcohol and 4.6 g of ferulic acid liter(-1). The genes ehyAB, encoding eugenol hydroxylase of Pseudomonas sp. strain HR199, and azu, encoding the potential physiological electron acceptor of this enzyme, were shown to be unsuitable for establishing eugenol bioconversion in E. coli XL1-Blue.     

Pseudomonas resinovorans SPR1, a newly isolated strain with potential of transforming eugenol to vanillin and vanillic acid

In this study a novel strain was isolated with the capability to grow on eugenol as a source of carbon and energy. This strain was identified as Pseudomonas resinovorans (GenBank accession no. HQ198585) based on phenotypic characterization and phylogenetic analysis of 16S rDNA gene. The intermediates coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin and vanillic acid were detected in the culture supernatant during eugenol biotransformation with this strain. The products were confirmed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and spectral data achieved from UV-vis, FTIR and mass spectroscopy. Using eugenol as substrate and resting cells of P. resinovorans SPR1, which were harvested at the end of the exponential growth phase, without further optimization 0.24 g/L vanillin (molar yield of 10%) and 1.1g/L vanillic acid (molar yield of 44%) were produced after 30 h and 60 h biotransformation, respectively. The current work gives the first evidence for the eugenol biotransformation by P. resinovorans.     

静息细胞转化生产3-羟基丙酸的条件优化

本文研究了静息细胞生物转化生产3-羟基丙酸的反应体系。考察了以甘油为底物,利用静息细胞转化生产3一羟基丙酸的相关因素,确定了最佳的转化条件：细胞浓度20g／L,甘油浓度20g／L,辅酶VB12浓度10mg／L,NAD＋浓度0．15mmol／L,温度35℃,反应体系为0．05mol／LpH7．0Tris—HCl缓冲液。在上述条件下反应6h后,3-羟基丙酸的产量达到为3．17g／L,底物转化率为28．33％。由上述结果可知,采用静息细胞转化法为3-HP的生物合成提供了一种可能的方法。