Biotransformation of cholesterol using Lactobacillus bulgaricus in a glucose-controlled bioreactor

A novel single-step microbial transformation process for the production of testosterone from cholestrol by Lactobacillus bulgaricus in an aerated fermenter was investigated. The metabolism of glucose possibly supplying reducing power as NADH was necessary for the reduction of androst-4-en-3,17-dione (AD) to testosterone (TS). The growth period for the accumulation of testosterone in maximal amount and the residual glucose increased in parallel with the amount of glucose supplement in growth cultures. After the glucose in the fermentation culture was completely exhausted, most of the testosterone was oxidized to AD. Addition of a larger amount of glucose could prevent oxidation of testosterone to AD. The biotransformation of cholestrol was significantly increased in the presence of cyclodextrin (CD) in the fermenting medium. The addition of 0.1% CD to the growth medium facilitated the transport of the steroid substrate through the microbial cell wall.     

Biotransformation of glucose to gluconic acid by Aspergillus niger—study of mass transfer in an airlift bioreactor

A glucose–gluconic acid biotransformation system was suggested for the experimental study of oxygen transfer in bioreactors. This biosystem was used for the investigation of the effect of the flow rate and biomass concentration on the volumetric oxygen transfer coefficient k_La in a 10 dm³ internal-loop airlift bioreactor. For this purpose, the fermentation broth of the mycelial strain Aspergillus niger was employed, representing a three-phase system, where bubbles come into contact with dense rigid pellets. The results showed that the presented biotransformation system can be successfully utilised for the determination of the oxygen transfer rate in airlift bioreactors. The experiments showed a strong positive influence of the air flow rate on the rate (r_Glu), specific rate of gluconic acid production (k_Glu/X) as well as on the volumetric oxygen transfer coefficient (k_La). This confirmed an expected limitation of production rate by the oxygen transport from the gas to the liquid phase in the whole range of air flow rates applied. Moreover, consistent curves of the production rate r_Glu and k_La values vs. biomass concentration c_X (amount of enzymes) were observed. These exhibited a local maximum for c_X equal to 6.68 g dm⁻³. On the other hand, the specific production rate monotonously decreased with increasing biomass concentration. A decline of k_La values at higher c_X values was attributed to a bubble coalescence promoting effect of mycelial pellets.     

Bioconversion of citronellol by Botrytis cinerea

Summary Bioconversion of citronellol 1 was studied with four strains of Botrytis cinerea. Using grape must predominant transformation of 1 to 2,6-dimethyl-1,8-octandiol 2 and (E)-2,6-dimethyl-2-octen-1,8-diol 3 was observed. In minor amounts 2,6-dimethyl-2,8-octandiol 4, two p-menthan-3,8-diol isomers 5a, 5b, (Z)-2,6-dimethyl-2-octen-1,8-diol 6, isopulegol 7, 2-methyl-2-hepten-6-one-1-ol 8 and 2-methyl--butyrolactone 9 were found. Using a small amount of grape must in a synthetic medium (1:700) the bioconversion products 2, 4, 5a and 5b were absent, but additionally 2-methyl-2-hepten-6-one 10, 2-methyl-2-hepten-6-ol 11 and citronellic acid 12 were detected. The results obtained were strongly dependent on the strains used; one strain did not show any metabolic activity against 1. The bioconversion products were identified by capillary gas chromatography (HRGC) and coupled HRGC techniques, i.e. on-line — mass spectrometry (HRGC-MS) and — Fourier transform infrared spectroscopy (HRGC-FTIR).     

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.     

Laccase production in bioreactor scale under saline condition by the marine-derived basidiomycete Peniophora sp. CBMAI 1063

Laccase production in saline conditions is still poorly studied. The aim of the present study was to investigate the production of laccase in two different types of bioreactors by the marine-derived basidiomycete Peniophora sp. CBMAI 1063. The highest laccase activity and productivity were obtained in the Stirred Tank (ST) bioreactor, while the highest biomass concentration in Air-lift (AL) bioreactor. The main laccase produced was purified by ion exchange and size exclusion chromatography and appeared to be monomeric with molecular weight of approximately 55 kDa. The optimum oxidation activity was obtained at pH 5.0. The thermal stability of the enzyme ranged from 30 to 50 °C (120 min). The Far-UV Circular Dichroism revealed the presence of high β-sheet and low α-helical conformation in the protein structure. Additional experiments carried out in flask scale showed that the marine-derived fungus was able to produce laccase only in the presence of artificial seawater and copper sulfate. Results from the present study confirmed the fungal adaptation to marine conditions and its potential for being used in saline environments and/or processes.     

Chemoenzymatic epoxidation of citronellol catalyzed by lipases

The chemoenzymatic epoxidation of a terpene alcohol, citronellol, is reported. Some experimental conditions, such as the use of lipases from different sources, oxidizing agents (H₂O₂ or urea–hydrogen peroxide, UHP), reaction time, acyl donor type (C6–C16), temperature (15–40 °C) and the influence of organic media, were evaluated. In most cases, citronellol oxide 2 or the ester citronellol oxide 3 were obtained. Depending on the reaction conditions, high yields of products 2 or 3 were obtained (>99%). CAL-B was the most effective catalyst in this reaction. For epoxide 2, the highest yields of 80% and 77% were obtained at 20 °C and 25 °C, respectively, using UHP as an oxidizing agent and octanoic acid as an acyl donor. The organic medium appears to be one of the most important parameters in the reaction. Using chloroform or dichloromethane, product 2 was obtained at a >99% yield after 24 h. When different mixtures consisting of varied organic solvents and an imidazolium-based ionic liquid (IL) were used, the results were dependent on both the solvent and IL counter-ion (18–75%).     

Transformations of acetates of citronellol,geraniol, and linalool by Aspergillus niger: regiospecific hydroxylation of citronellol by a cell-free system

Summary Incubation of acetates of geraniol, citronellol and linalool with Aspergillus niger resulted in their hydrolysis to corresponding alcohols which were further hydroxylated to their respective 8-hydroxy derivatives. In the case of linalyl acetate, besides linalool and 8-hydroxylinalool, small amounts of geraniol and -terpineol were also formed. Microsomes (105 000xg sediment) prepared from induced cells of A. niger were found to convert (1-³H)citronellol to 8-hydroxy citronellol in the presence of NADPH and O₂. The pH optimum for the hydroxylase was found to be 7.6.     

Biotransformation of cephalosporin C to 7-aminocephalosporanic acid with coimmobilized biocatalyst in a batch bioreactor

Biotransformation of cephalosporin C (CPS-C) to 7-aminocephalosporanic acid (7-ACA) was carried out with coimmobilized permeabilized cells of Trigonopsis variabilis and Pseudomonas species entrapped in Ca-pectate gel beads. Good aeration and stirring during the process was assured. The analysis of this complicated biochemical process in a heterogeneous system was based on the identification of individual effects (internal diffusion, reaction) running simultaneously. A spectrophotometric method was proposed for the determination of 7-(-ketoadipyl amido) cephalosporanic acid (CO-GL-7-ACA) and 7-ACA. The reaction-diffusion model containing dimensionless partial differential equations was solved by using the orthogonal collocation method. A good agreement between experimental values and values predicted by the mathematical model was obtained. Numerical simulations were performed on the basis of following the two assumptions:- several times higher activity of both cells,- hydrogen peroxide was continuously supplied in the bioreactor.List of Symbols A m² surface of the bead - c _i mol/dm³ concentration of component in the bead and/or in the solution - c _i0 mol/dm³ initial concentration of component in the solution - c _l0 mol/dm³ initial concentration of CPS-C in the solution - C _jl orthogonal collocation weights of the first derivation - D _ei m²/s effective diffusion coefficient of the components - D _jl orthogonal collocation weights of the second derivation - k ₅ dm³/(mol · s) kinetic parameter of non-enzyme reaction - K _inh mol/dm³ inhibition parameter for the first enzyme reaction - K _i dimensionless Michaelis constant for the first and second enzyme reaction, defined in Eq. (7) - K _l dimensionless inhibition parameter for the first enzyme reaction, defined in Eq. (7) - K _mi mol/dm³ Michaelis constant for the first and second enzyme reaction - n number of beads - P( _i ) symbol of dimensionless reaction rate, defined in Eq. (13) - r m radial coordinate inside the bead - R m radius of the bead - R(c _i ) mol/(dm³ · s) symbol for reaction rate, defined in Eq. (6) - t s time - V _max mol/(dm³ · s) max. reaction rate for the first and second enzyme reaction - V _L dm³ volume of solution excluding the space occupied by beads - voidage in batch bioreactor - _P porosity of the bead - i dimensionless effective diffusion coefficient of the components, defined in Eq. (7) - dimensionless time, defined in Eq. (7) - _mi Thiele modulus, defined in Eq. (7) - _i dimensionless concentration, defined in Eq. (7) - dimensionless radial position inside the bead, defined in Eq. (7) - _l0 initial dimension concentration of CPS-C, defined in Eq. (9), (10) - _i0 initial dimension concentration of component, defined in Eq. (9), (10) The authors wish to thank Dr. P. Gemeiner of Slovak Academy of Sciences for rendering of pectate gel. This work is supported by Ministry of Education (Grant No. 1/990 935/93).     

Biotransformation of cephalosporin C to 7-aminocephalosporanic acid with coimmobilized biocatalyst in a batch bioreactor

The permeabilized cells of Trigonopsis variabilis CCY 15-1-3 having D-amino acid oxidase (DAAO) activity were used to convert cephalosporin C (CPS-C) into 7-(-ketoadipyl amido) cephalosporanic acid (CO-GL-7-ACA) in a batch bioreactor with good aeration and stirring during the process. The deacylation of 7--(4-carboxybutanamido)-cephalosporanic acid (GL-7-ACA) to 7-cephalosporanic acid (7-ACA) by permeabilized cells of Pseudomonas species 3635 having 4--(4-carboxybutamido)-cephalosporanic acid acylase (GL-7-ACA acylase) activity was performed in a batch bioreactor. A spectrophotometric method for the determination of CO-GL-7-ACA and 7-ACA was proposed. Experimental data were fitted by non-linear regression with parameters optimization. The sorption method (without reaction) was applied for the determination of cephalosporin effective diffusion coefficients in Ca-pectate gel beads. These beads were prepared by dropping a potassium pectate gel suspension of inactive permeabilized cells of Trigonopsis variabilis and Pseudomonas species, crosslinked with glutaraldehyde, into a stirred 0.2 M calcium chloride solution. Concentrations of appropriate cephem components were measured by the refractive method. Values of effective diffusion coefficients were calculated by the Fibbonacci optimization method.List of Symbols c _L mol/dm³ concentration on the surface of a bead - c _L0 mol/dm³ initial cephalosporin concentration - c _L mol/dm³ equilibrium cephalosporin concentration in the solution - c _s1 mol/dm³ concentration of CPS-C - c _s2 mol/dm³ concentration of GL-7-ACA - D _ei m²/s effective diffusion coefficient of the components - K _i mol/dm³ inhibition parameter in Eq. (2) - K _{m i} mol/dm³ Michaelis constant in Eq. (1) - K _{m 2} mol/dm³ Michaelis constant in Eq. (2) - n number of beads - q _n nonzero positive roots in Eq. (7) - r ₁ mol/(dm³·s) rate of the conversion of CPS-S to CO-GL-7-ACA - r ₂ mol/(dm³·s) rate of the conversion of GL-7-ACA to 7-ACA - R m radius of the bead - S( ) symbol for total residual sum of squares in Eq. (1) - t s time - V _{m 1} mol/(dm³·s) max. reaction rate in Eq. (1) - V _{m 2} mol/(dm³·s) max. reaction rate in Eq. (2) - V _L dm³ volume of the solution excluding the space occupied by beads - V _s dm³ volume of beads - y _i mol/(dm³ · s) symbol for experimental data in Eq. (1) - _i mol/(dm³· s) symbol for calculated data in Eq. (1) - _P porosity, defined by Eq. (5) - dimensionless parameter, defined by Eq. (6) The authors wish to thank Dr. P. Gemeiner of Slovak Academy of Sciences for rendering of pectate gel. This work is supported by Ministry of Education (Grant No. 1/990 935/93)     

Bioconversion of waste cellulose by using an attrition bioreactor

A new type of reactor, an attrition bioreactor, was tested to achieve a higher rate and extent of enzymatic saccharification of cellulose than is possible with conventional methods. The reactor consisted of a jacketted stainless-steel vessel with shaft, stirrer, and milling media, which combined the effect of the mechanical action of wet milling with cellulose hydrolysis. The substrates tested were newsprint and white-pine heartwood. The performance of the reactor was excellent. The extent and rate of enzymatic hydrolysis could be markedly improved over other methods. The power consumption of the attrition bioreactor was also measured. The cellulase enzyme deactivation during attrition milling was not significant.     

Catalytic removal of sulfide by an immobilized sulfide-oxidase bioreactor

A bioreactor packed with chitosan immobilized sulfide-oxidase from Streptomyces species LD048 was developed to treat a liquid stream of sulfide. The inoculation system was composed of glass with a 0.7 L working volume and enzyme activity of 2 mmol S g^?1 carrier. The sulfide removal efficiency was almost 100% when the volumetric loading was increased up to 3.9 mmol S L^?1 h^?1 at a space velocity of 18 h^?1. The maximal elimination capacity was 22.1 mmol S L^?1 h^?1 with a space velocity of 72 h^?1. When the aeration was increased from 0.05 to 0.1 L min^?1, the average removal efficiency improved from 81% to 94%. A removal efficiency of 90% was obtained after 15 days of operation with a load rate of 8.9 mmol S L^?1 h^?1 and a space velocity of 14.28 h^?1. An operational equation based on the ideal plug flow bioreactor and the Michaelis–Menten model predicted the performance of this bioreactor.     

Dye decolorization by manganese peroxidase in an enzymatic membrane bioreactor

In the present work an enzymatic membrane reactor (EMR) for the oxidation of azo dyes by manganese peroxidase (MnP) has been developed. The configuration consisted of a stirred tank reactor coupled with an ultrafiltration membrane. The membrane allowed for most of the enzymatic activity to be recovered while both the parent dye and the degradation products could pass through. Different operational strategies (batch, fed-batch, and continuous) and parameters such as enzyme activity, H(2)O(2) feeding rate, hydraulic retention time (in continuous operation), and dye loading rate were studied. At best conditions, a continuous operation with a dye decolorization higher than 85% and minimal enzymatic deactivation was feasible for 18 days, attaining an efficiency of 42.5 mg Orange II oxidized/MnP unit consumed.     

Enzymatic synthesis of geraniol and citronellol esters by direct esterification in n-hexane

Summary Short chain fatty acid esters of geraniol and citronellol were synthesized by lipase-catalyzed direct esterification with yields ranging from 90 to 100% molar conversion. The effect of the order of addition of substrates, solvent and enzyme was investigated. Yields were highest when the solvent was added first followed by the substrates and then the lipase.     

Investigation of the bacitracin biosynthesis in an airlift bioreactor

Results of pilot plant studies using an external-loop airlift bioreactor (170 l fermentation volume, liquid height-to-riser diameter: 27, loop-to-tower cross-section-area: 0.1225) have proven the relative merits of such a system in the bacitracin biosynthesis produced by the Bacillus licheniformis submerged aerobic cultivation. The results were compared to those obtained in a pilot-scale stirred-tank bioreactor with the same values of k_La. Excepting the aeration rate of 0.2 vvm, the fermentation process performed at 0.5 vvm and 1/0 vvm, respectively, unfolded similarly in the two fermentation devices with respect to the cell mass production, substrate utilization and bacitracin production during the fermentation process. In the riser section of the airlift bioreactor, the dissolved oxygen levels were higher, while in the downcomer section they were lower than those realized in the stirred tank bioreactor. Power requirements of the airlift fermenter were by 17–64% lower than those for a mechanically agitated system, depending on the aeration rates, which led to an important energy saving. Moreover, the lack of mechanical devices in the airlift system provides safety and a more gentle environment for the cultivation of microorganisms.     

Copper biosorption in an aerobic bioreactor

Abstract

This study evaluates the biosorption of copper by aerobic biomass that was selected from surface waters of the San Pedro River in Sonora, Mexico. Using a batch system, 73% biosorption of copper was obtained in 75 minutes. Continuous biosorption assays were carried out for 133 days in an ascending flow aerobic reactor packed with zeolite (AFAR-PZ) that was inoculated with a bacterial consortium. Strains were grown until 1g L^?1 of biomass was obtained. Tests using continuous biosorption were performed as follows: (i) the addition of 50 mg Cu²⁺ L^?1 without recirculation of biomass; (ii) the addition of 20 mg Cu²⁺ L^?1without recirculation of biomass; and (iii) the biomass were recirculated with the addition of 20 mg Cu²⁺ L^?1 to pH 3 to 4. The fourth and fifth assays varied pH between 4 and 5, with 20 mg Cu²⁺ L^?1and the biomass recirculated. Biosorption capacity of the first and second assays was 96% on the first day of experimentation. During the third trial 97% of biosorption was obtained during 6 days and the process was improved by varying the pH. Copper biosorption equilibrium was investigated under the same operating conditions. Langmuir adsorption isotherms were used to fit experimental data. The biosorption capacity of aerobic biomass was 3.08 mmol g^?1. It was demonstrated that this biomass is capable of biosorbing copper and this method has potential for the treatment of industrial effluents contaminated with heavy metals.     

Continuous antibiotic production by an immobilized cyanobacterium in a seaweed-type bioreactor

The filamentous cyanobacterium,Scytonema sp. TISTR 8208, which produces a cyclic peptide antibiotic, was cultivated for 20 d in a seaweed-type bioreactor containing anchored polyurethan foam strips. Cells immobilized onto the foam strips produced the antibiotic for only several days, and the secreted antibiotic disappeared very rapidly from the medium. Cells accumulated the antibiotic intracellularly in a growth-related manner, and secreted it in the stationary phase. Since the antibiotic has a stable physico-chemical nature, the cells seem to take it up and metabolize it. When continuous cultivation was attempted, stable production of the antibiotic was maintained in the bioreactor for 16 d at a dilution rate of 0.01 h^–1. Three times more antibiotic was produced in the continuous culture than in the batch culture by the 16th day.     

Biotransformation of N-acetylphenothiazine by fungi

Cultures of the fungi Aspergillus niger, Cunninghamella verticillata, and Penicillium simplicissimum, grown in a sucrose/peptone medium, transformed N-acetylphenothiazine to N-acetylphenothiazine sulfoxide (from 13% to 28% of the total) and phenothiazine sulfoxide (from 5% to 27%). Phenothiazin-3-one (4%) and phenothiazine N-glucoside (4%) were also produced by C. verticillata. The probable intermediate, phenothiazine, was detected only in cultures of P. simplicissimum (6%). Received: 15 January 1999 / Received revision: 7 May 1999 / Accepted: 21 May 1999     

Biotransformation of cycloalkanones by microorganisms

Summary Washed-cell suspensions of cyclopentanol-grownPseudomonas sp. NCIB 9872 were shown to preferentially biotransform norbornanone to an equivalent lactone of potential use in the synthesis of prostanoid analogues. Conditions to optimise the yield of product, including incubation at pH 7.1–7.35 and at substrate concentrations <15 mM, have been established.     

Heterokaryon formation in the basidiomycete Schizophyllum commune by electrofusion of protoplasts

Summary Conditions for high frequency electrofusion of protoplasts from the basidiomycete Schizophyllum commune are described. Visual inspection revealed up to 30% of the protoplasts engaged in fusion. Using complementing nutritional mutations, nearly 7% of the regenerated protoplasts could be recovered as heterokaryotic mycelia. The method is probably equally applicable to other basidiomycetes such as Agaricus bisporus, permitting the recovery of fusion products in the absence of selection markers.     

Biotransformation of nitriles by rhodococci

Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.