Fed-batch biotransformation of β-ionone by Aspergillus niger

Aspergillus niger IFO 8541 was found to be an efficient biocatalyst for the biotransformation of -ionone into hydroxy and oxo derivatives. The reaction had to be carried out with an inoculum made of about 4 × 10⁷ fresh spores/l and with a preliminary growth period giving at least 3 g/l biomass. The fungus developed in the form of pellets when cultivated as free mycelium; entrapment of the microorganism in calcium alginate beads was an efficient way to mimic this feature in an aerated, stirred bioreactor. The biotransformation was carried out using a fed-batch mode of operation involving sequential precursor addition. -Ionone stopped the fungal growth and was converted into metabolites only when the carbon source remained present in the medium; it was fully oxidized after sucrose exhaustion. These conditions allowed recovery of about 2.5 g/l aroma compounds after 230 h cultivation with a molar yield close to 100%.     

Biodegradation of toluene at high initial concentration in an organic–aqueous phase bioprocess with nitrate respiration

An aerobic organic–aqueous system with forced aeration was shown to be inefficient in preventing significant volatile aromatic compounds loss in gassed systems since air sparging in n-hexadecane under abiotic conditions could reduce the toluene concentration from 2.1 g/L to about 0.5 g/L in 3 days with a gassing rate of 1VVM at 20 °C. However, the presence of such an organic phase was found to significantly reduce substrate loss in aerobic conditions in comparison to pure aqueous systems. It was thus decided to develop a new bioprocess based on an anaerobic microbial system operated in an organic–aqueous phase with nitrate respiration. The denitrifying bacterium used, Thauera aromatica K172, was produced by cultivation on sodium benzoate as carbon source under anaerobic conditions. This cultivated biomass (1.5 g/L) was shown to retain its ability to efficiently metabolize toluene in a biphasic medium without any significant loss of organic compound in the gas phase. Toluene biodegradation was thus performed in a biphasic system using a fed-batch technique involving sequential adding of both toluene and nitrate. The reaction rate with an initial concentration of toluene close to 14.5 g/L in hexadecane was found to be close to 0.5 g/L day and the molar stoichiometry of solute metabolization to nitrate reduction was close to 1:6. This work demonstrated that the denitrifying bacteria could efficiently degrade toluene in hexadecane–aqueous phase systems in which toxic compound release in the environment was prevented.     

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- -aspartyl- -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- -aspartic acid (ZA) and -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.     

Microbial conversion of beta-ionone by immobilized Aspergillus niger in the presence of an organic solvent

Aspergillus niger JTS191 was capable of the conversion of beta-ionone to a mixture of its derivatives that is utilized to an essential oil of tobacco. The authors attempted this microbial conversion in the presence of an organic solvent to improve its reaction rate. The addition of isooctane accelerated the microbial conversion of beta-ionone. It took three days to complete the reaction whereas without isooctane it took more than six days. The addition of isooctane also improved the resistance of A. niger to the antifungal property of beta-ionone. A. niger pellets were immobilized in hydrophobic polymer, PU-3, and applied for the microbial conversion of beta-ionone. Further improvement of the resistance to the antifungal property of beta-ionone was achieved by immobilization. PU-3 immobilized A. niger was repeatedly used for microbial conversion of beta-ionone in the presence of isooctane for more than 480 hours.     

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.     

Improved co-oxidation of beta-carotene to beta-ionone using xanthine oxidase-generated reactive oxygen species in a multiphasic system

beta-Ionone, an aroma compound exhibiting flower notes, can be obtained from beta-carotene in a cooxidation system utilizing xanthine oxidase-generated reactive oxygen species (ROS). ROS have to be controlled as, although they can give rise to beta-ionone, they may also degrade it. In this work, the biotransformation of beta-carotene into beta-ionone was investigated in systems containing variable proportions of decane to extract beta-ionone before degradation. The use of 50% or 90% decane resulted in increased production yields. Tween 80, which was added to further improve the production, slightly decreased the reactivity of the medium and the extraction of beta-carotene, but increased the extraction of beta-ionone. In total, the addition of Tween 80 significantly improved the yields of conversion, which reached 34% with 50% decane and 2.5 g/L Tween 80 compared to 10% without decane and Tween 80. These results show that it is possible to control a ROS-mediated reaction by the addition of a solvent phase and by modifing the medium composition.     

Optimization of isonovalal production from α-pinene oxide using permeabilized cells of <Emphasis Type="Italic">Pseudomonas rhodesiae</Emphasis> CIP 107491

Optimization studies on the synthesis of isonovalal from alpha-pinene oxide by Pseudomonas rhodesiae CIP 107491 operated in a biphasic medium are presented. Three key parameters are identified. The first is the need for a permeabilization of cells by freezing them and then treating the thawed material with an organic solvent such as chloroform, toluene or diethyl ether. This operation allows both enzyme release into the aqueous phase outside the cells and an improvement in the transport properties of both substrate and product across the cell membrane, strongly increasing reaction rates. The second is that the enzyme alpha-pinene oxide lyase, which exhibits an irreversible inactivation by isonovalal (or a by-product), presents a constant turn-over, i.e., the total product synthesis is proportional to the biomass loading and is close to 108 mmol (16.4 g) isonovalal l(-1) g(-1) biomass. The third phenomenon is that the biphasic system used is not phase-transfer-limited, a feature attributed to the spontaneous formation of an oil-in-water emulsion. It is thus possible to carry out a very efficient process, allowing the recovery of 2.63 mol isonovalal l(-1) (400 g l(-1)) from 25 g biomass l(-1) in 2.5 h, corresponding to an average reaction rate as high as 0.70 mmol min(-1) g(-1) cells (160 g l(-1) h(-1)).     

Improved production of (R)-2-octanol via asymmetric reduction of 2-octanone with Oenococcus oeni CECT4730 in a biphasic system

Abstract

Oenococcus oeni CECT4730, which catalyses the asymmetric reduction of 2-octanone to (R)-2-octanol with high enantioselectivity, was further studied to exploit its potential for production of (R)-2-octanol in an aqueous/organic solvent biphasic system. Variables such as the volume ratio of aqueous to organic phase (V_a/V_o), buffer pH, reaction temperature, shaking speed, co-substrates and the ratio of biocatalyst to substrate were examined with respect to the molar conversion, the initial reaction rate and the product enantiomeric excess (e.e.). Under the optimized conditions (V_a/V_o=1:1 (v/v), buffer pH=8.0, reaction temperature=30°C, shaking speed=150 rev/min, ratio of glucose to biomass=5.4:l (w/w), ratio of biocatalyst to substrate=0.51:l (g/mol)), the highest space time yield of (R)-2-octanol, 24 mmol L^?1 per h, and >98% product e.e. were obtained at a substrate concentration close to 1.0 mol L^?1 after 24 h reduction.     

Nutrient broth/PEG200/TritonX114/Tween80/Chloroform microemulsion as a reservoir of solubilized sitosterol for biotransformation to androstenedione

Microemulsions (ME) can act as a reservoir of solubilized hydrophobic substrates. The biotransformation of hydrophobic sitosterol to androstenedione (AD) with MEs prepared from nutrient broth and PEG 200 (1:1) as aqueous phase, 40 g/l sitosterol dissolved in chloroform as organic phase, Triton X114 and Tween 80 (1:1) as surfactant phase, was investigated. The phase behavior of this system was studied for ten different ratios(w/w), 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10 of the organic phase and surfactant at 30 °C. A pseudoternary phase diagram was constructed to demarcate the region giving stable MEs. The maximum solubility of sitosterol in ME medium was observed to be 8 g/l, which is 3 orders of magnitude higher than the reported sitosterol solubility of 2–4 mg/l in aqueous medium. The ME medium was used for biotransformation studies and a comparative result has been reported. Transmission electron microscopy of cells grown in ME having oil, surfactant and aqueous phase in the ratio of 6:14:80 showed a weakened cell wall structure that permitted production of 465.86 mg/l AD.     

Characterization of a newly isolated strain Rhodococcus erythropolis ZJB-09149 transforming 2-chloro-3-cyanopyridine to 2-chloronicotinic acid

2-Chloronicotinic acid is receiving much attention for its effective applications as a key precursor in the synthesis of pesticides and medicines. In this study, a strain ZJB-09149 converting 2-chloro-3-cyanopyridine to 2-chloronicotinic acid was newly isolated and identified as Rhodococcus erythropolis, based on its physiological and biological tests, and 16S rDNA sequence analysis. In addition, the effects of inducer, carbon source and nitrogen source were examined. Maximum activity was achieved when the above parameters were set as 8 g/l ?-caprolactam, 7 g/l yeast extract and 5 g/l maltose. Moreover, the biotransformation pathway of 2-chloro-3-cyanopyridine to 2-chloronicotinic acid in strain ZJB-09149 was investigated as well. This study revealed that the nitrile hydratase (NHase) and amidase expressed in R. erythropolis ZJB-09149 are responsible for the conversion of 2-chloro-3-cyanopyridine. This is the first time to report on the biotransformation preparation of 2-chloronicotinic acid.     

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.     

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l(-1) at a flow rate of 0.414 l h(-1) was delivered to the system at a loading capacity of 140 g m(-3) h(-1), and an elimination capacity of 133 g m(-3 )h(-1) was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)(-1) h(-1) was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l(-1). If it assumed that this specific benzene utilization rate (0.57 g g(-1) h(-1)) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.     

Effects of different pretreatment strategies on corn stalk acidogenic fermentation using a microbial consortium

The effects of sulfuric acid, acetic acid, aqueous ammonia, sodium hydroxide, and steam explosion pretreatments of corn stalk on organic acid production by a microbial consortium, MC1, were determined. Steam explosion resulted in a substrate that was most favorable for microbial growth and organic acid productions. The total amounts of organic acids produced by MC1 on steam exploded, sodium hydroxide, sulfuric acid, acetic acid, and aqueous ammonia pretreated corn stalk were 2.99, 2.74, 1.96, 1.45, and 2.21 g/l, respectively after 3 days of fermentation at 50 °C. The most prominent organic products during fermentation of steam-exploded corn stalks were formic (0.86 g/l), acetic (0.59 g/l), propanoic (0.27 g/l), butanoic (0.62 g/l), and lactic acid (0.64 g/l) after 3 days of fermentation; ethanol (0.18 g/l), ethanediol (0.68 g/l), and glycerin (3.06 g/l) were also produced. These compounds would be suitable substrates for conversion to methane by anaerobic digestion.     

Improved (<Emphasis Type="Italic">R</Emphasis>)-phenylacetylcarbinol production with <Emphasis Type="Italic">Candida utilis</Emphasis> pyruvate decarboxylase at decreased organic to aqueous phase volume ratios

The effect of decreasing the organic (octanol) to aqueous phase volume ratio was evaluated in a two-phase enzymatic process for (R)-phenylacetylcarbinol (PAC) production. Decreasing the ratio from 1:1 to 0.43:1 at 4°C increased PAC in the organic phase from 112 g/l to 183 g/l with a 10% improvement in overall productivity. Interestingly, the rate of enzyme (pyruvate decarboxylase) activity loss was unaffected by the reduced phase ratio over the reaction period (48 h). At 20°C and 0.43:1 phase ratio the organic phase PAC concentration increased to 212 g/l and the overall productivity increased by 30% although the PAC yield (based on pyruvate) declined by about 10% due to greater byproduct acetoin formation at the higher temperature. Product recovery in such a system is facilitated both by the higher PAC concentration and the reduced organic phase volume.     

Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

Nutrient removal from slaughterhouse wastewater in an intermittently aerated sequencing batch reactor

The performance of a 10 L sequencing batch reactor (SBR) treating slaughterhouse wastewater was examined at ambient temperature. The influent wastewater comprised 4672+/-952 mg chemical oxygen demand (COD)/L, 356+/-46 mg total nitrogen (TN)/L and 29+/-10 mg total phosphorus (TP)/L. The duration of a complete cycle was 8 h and comprised four phases: fill (7 min), react (393 min), settle (30 min) and draw/idle (50 min). During the react phase, the reactor was intermittently aerated with an air supply of 0.8L/min four times at 50-min intervals, 50 min each time. At an influent organic loading rate of 1.2g COD/(Ld), average effluent concentrations of COD, TN and TP were 150 mg/L, 15 mg/L and 0.8 mg/L, respectively. This represented COD, TN and TP removals of 96%, 96% and 99%, respectively. Phase studies show that biological phosphorus uptake occurred in the first aeration period and nitrogen removal took place in the following reaction time by means of partial nitrification and denitrification. The nitrogen balance analysis indicates that denitrification and biomass synthesis contributed to 66% and 34% of TN removed, respectively.     

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.     

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal. Received: 16 March 1998 / Received revision: 20 May 1998 / Accepted: 21 May 1998     

Treatment of recycled wastewaters from fishmeal factory by an anaerobic filter

Fishmeal industries processes produce effluents with high load organic matter. These effluents, after recycling and physical-chemical pretreatment, have a high organic content (5-6 g COD/l), proteins (3-5 g/l), salinity close to sea water, sodium chloride (30 g/l) and sulphate (1-3.3 g/l). An anaerobic filter was used for the treatment of this wastewater, with marine sediment as anaerobic inoculum. Anaerobic filter removed up to 70% of the influent COD concentrations at organic loading rates (OLR) of 9.5 and 14.3 (g/l d) and sulphate up to 80% at OLR of 7.1 and 14.3 (g/l d) whereas the pH ranged between 7.0 and 7.5. These results show that anaerobic filter systems are applicable to recycled wastewaters from fishmeal.     

Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen

The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H₂ were assessed in serum-bottle microcosms. Experiments were performed over a range of metal concentrations (20–200 mg/1) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum. In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration. However, the extent of inhibition varied for the different biotransformations and for the different metals tested. For example, the concentration of CuCl₂ effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/1, respectively. Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic. The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 gm/1 respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect. The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.