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.     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

Cellulase production by trichoderma harzianum in static and mixed solid-state fermentation reactors under nonaseptic conditions

Cellulase production from lignocellulosic materials was studied in solid-state cultivation by both static and mixed techniques under nonaseptic conditions. The effects of fermentation conditions, such as moisture content, pH, temperature, and aeration, on cellulase production by Trichoderma harzianum using a mixture of wheat straw (80%) and bran (20%) were investigated. With a moisture content of 74% and a pH of 5.8., 18 IU filter paper activity and 198 IU endoglucanase activity/g initial substrate content were obtained in 66 h. The extension from static column cultivation to stirred tank reactor of 65 L capacity gave similar yields of cellulase.     

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.     

High cell density cultivation of recombinant yeasts and bacteria under non-pressurized and pressurized conditions in stirred tank bioreactors

This study demonstrates the applicability of pressurized stirred tank bioreactors for oxygen transfer enhancement in aerobic cultivation processes. The specific power input and the reactor pressure was employed as process variable. As model organism Escherichia coli, Arxula adeninivorans, Saccharomyces cerevisiae and Corynebacterium glutamicum were cultivated to high cell densities. By applying specific power inputs of approx. 48kWm(-3) the oxygen transfer rate of a E. coli culture in the non-pressurized stirred tank bioreactor was lifted up to values of 0.51moll(-1)h(-1). When a reactor pressure up to 10bar was applied, the oxygen transfer rate of a pressurized stirred tank bioreactor was lifted up to values of 0.89moll(-1)h(-1). The non-pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities of more than 40gl(-1) cell dry weight (CDW) of E. coli, whereas the pressurized stirred tank bioreactor was able to support non-oxygen limited growth of cell densities up to 225gl(-1) CDW of A. adeninivorans, 89gl(-1) CDW of S. cerevisiae, 226gl(-1) CDW of C. glutamicum and 110gl(-1) CDW of E. coli. Compared to literature data, some of these cell densities are the highest values ever achieved in high cell density cultivation of microorganisms in stirred tank bioreactors. By comparing the specific power inputs as well as the k(L)a values of both systems, it is demonstrated that only the pressure is a scaleable tool for oxygen transfer enhancement in industrial stirred tank bioreactors. Furthermore, it was shown that increased carbon dioxide partial pressures did not remarkably inhibit the growth of the investigated model organisms.     

Cultivation of transgenicNicotiana tabacum suspension cells in bioreactors for the production of mGM-CSF

TransgenicNicotiana tabacum cells were cultivated for the production of murine granulocyte macrophage-colony stimulating factor (mGM-CSF) in both a stirred, tank biore|actor and an airlift bioreactor with draft tube. Cell growth and mGM-CSF production in the airlift bioreactor were found to be better than those achieved in the stirred tank bioreactor. In the airlift bioreactor. 9.0 g/L of cells and 2.2 ng/mL of mGM-CSF were obtained (11.0 g/L and 2.4 ng/mL, respectively in shake flasks). Although the lag period was prolonged and mGM-CSF production was lowered by 33% in the stirred tank bioreactor as compared to the control culture, the maximum cell density was increased up to 12.0 g/L due to better mixing by agitation at the higher cell density.     

Pilot-plant production of prednisolone using calcium alginate immobilized Arthrobacter simplex

The maximum conversion of hydrocortisone suspensions at initial substrate concentrations greater than 4 g/L by immobilized Arthrobacter simplex in a batch reactor was 80-85%. By feeding hydrocortisone suspensions continuously to either a fed-batch-operated stirred tank reactor or to a continuous-flow airlift loop reactor, at a rate such that the soluble hydrocortisone concentration in the reactor remained ca. 0.05 g/L, 95% conversion of substratewas obtained at final product concentrations exceeding 4 g/L.     

Unstable steady state operations of substrate inhibited cultures by dissolved oxygen control

Microorganism kinetic growth characterized by substrate inhibition was investigated by means of a continuous stirred tank reactor equipped with a feedback controller of the medium feeding flow rate. The aerobic growth of Pseudomonas sp. OX1 with phenol as carbon/energy source was adopted as a case study to test a new control strategy using dissolved oxygen concentration as a state variable. The controller was successful in steadily operating bioconversion under intrinsically unstable conditions. A simple model of the controlled system was proposed to set the feedback controller. The specific growth rate of Pseudomonas sp. OX1 was successfully described by means of the Haldane model. The regression of the experimental data yielded μ(M)=0.26 h(-1), K(Ph)=5×10(-3)g/L and K(I)=0.2g/L. The biomass-to-substrate fractional yield as a function of the specific growth rate did not change moving from substrate-inhibited to substrate-deficient state. The data was modelled according to the Pirt model: m=1.7×10(-2)g/(gh), Y(X/Ph)(Th)=1.3g/g. The specific growth rates calculated for batch and continuous growth were compared.     

Growth and biomass production with enhanced β‐glucan and dietary fibre contents of Ganoderma australe ATHUM 4345 in a batch‐stirred tank bioreactor

In this study we maximized biomass production by the basidiomycete Ganoderma australe ATHUM 4345, a species of pharmaceutical interest as it is a valuable source of nutraceuticals, including dietary fibers and glucans. We used the Biolog FF MicroPlate to screen 95 different carbon sources for growth monitoring. The pattern of substrate catabolism forms a substrate assimilation fingerprint, which is useful in selecting components for media optimization of maximum biomass production. Response surface methodology, based on the central composite design was applied to explore the optimum concentrations of carbon and nitrogen sources of culture medium in shake flask cultures. When the improved culture medium was tested in a 20‐L stirred tank bioreactor, using 13.7 g/L glucose and 30.0 g/L yeast extract, high biomass yields (10.1±0.4 g/L) and productivity of 0.09 g L^?1 h^?1 were obtained. The yield coefficients for total glucan and dietary fibers on biomass formed were 94.82±6 and 341.15±12.3 mg/g mycelium dry weight, respectively.     

Design of an efficient fermentation process for the production of Metarhizium acridum blastospores

Using a sequential approach, we described efficient blastospore production in a stirred tank bioreactor (3?L capacity). We used the response surface methodology to optimise the media ingredients and fermentation parameters to obtain the maximum production of blastospores by a locally collected isolate of Metarhizium acridum (Ascomycota: Hypocreales). The results showed that a liquid culture medium supplemented with monopotassium phosphate (15.17?g/L), corn steep liquor (69.25?g/L), and casamino acids (80.68?g/L) in a stirred tank bioreactor under operating conditions constant at 635?rpm, a temperature of 26°C, and pH 3.3 produced 1.25?×?10⁸?blastospores (bls)/ml, with 93% viability after 120?h of fermentation. This bioreactor yield compares favourably with the yields obtained by shake flask production and confirms the suitability of the media and production parameters for the potential scale-up fermentation production of M. acridum.     

Start‐up of Anaerobic Hydrogen Producing Reactors Seeded with Sewage Sludge

The procedure for starting‐up continuously stirred tank reactors (CSTR) for acclimating anaerobic hydrogen‐producing microorganisms with sewage sludge was investigated. Initially, feeding with glucose and sucrose as well as mixing were carried out in semicontinuous mode; hydraulic retention time (HRT) was in an order of 20, 15, 10, 5, 2.5 and 2 days. When the pH declined to its lowest value (pH 5.18), it was adjusted to 6.7 using sodium hydroxide (1 N). At the same time, the semi‐continuous operation was changed to a continuous one. Finally, the pH was continuously regulated at approximately 6.7. The results indicate that this procedure can be used to cultivate seed sludge for hydrogen production from sewage sludge resulting in a large hydrogen production in less than 60 days. When the substrate was glucose, a hydrogen yield of 1.63 mol H₂/mol glucose and a specific hydrogen production rate of 321 mmol H₂/g VSS day at an HRT of 13.3 h was achieved. When the substrate was sucrose with the same HTR, a hydrogen yield of 4.45 mol H2/mol sucrose and a specific hydrogen production rate of 707 mmol H₂/g VSS day was obtained.     

On oxygen limitation in a whole cell biocatalytic Baeyer-Villiger oxidation process

In this article, a recombinant cyclohexanone monooxygenase (CHMO), overexpressed in Escherichia coli has been used to study the oxidation of bicyclo[3.2.0]hept-2-en-6-one to its two corresponding lactones at very high enantiomeric excess. The reaction is a useful model for the study of biocatalytic oxidations to create optically pure molecules. The major limitations to a highly productive biocatalytic oxidation in this case are oxygen supply, product inhibition, and biocatalyst stability. In this article, we investigate the effects of whole cell biocatalyst concentration on the rate of reaction at a range of scales from shake flasks to 75 L bioreactors. At low cell concentrations (<2 g(dcw)/L) the maximum specific rate (0.65 g/g(dcw).h) is observed. However, at higher cell concentrations (> 2 g(dcw)/L), the reaction becomes oxygen limited and both the specific rate and absolute rate decrease with further increases in cell concentration. The role of oxygen limitation in reducing the rate of reaction with scale was investigated by increasing the maximum oxygen transfer rate in the reactor at a high cell concentration and observing the increase in product formation rate. We propose a qualitative model demonstrating the relationship between oxygen limitation, biocatalyst concentration, and the rate of reaction. This conceptual model will be a useful guide in the industrial scale-up of whole cell mediated Baeyer-Villiger biocatalysis.     

Factors affecting the growth and the oil accumulation of marine microalgae, Tetraselmis suecica

Biomass production and oil productivity in microalgae culture are the most important key factors for algal biodiesel production. However, proper culture condition for the biomass production of microalgae is different from that for the oil production of microalgae. A study on the biomass production of Tetraselmis suecica using various light intensities and nitrate concentrations as growth factors was carried out to evaluate proper culture conditions in 20-L batch culture. The effect of nitrate depletion on the oil accumulation was also evaluated with two-stage culture. It took 5 days to reach the stationary phase for the cultures of T. suecica on the light intensities of 108.9 and 133.1 μmol m(-2 )s(-1) with biomass of 0.89 and 0.88 g dcw L(-1), respectively. Biomass productions of 1.07 and 1.00 g dcw L(-1) were obtained with the nitrate concentrations of 18.6 and 24.7 mg L(-1), respectively. The two-stage culture increased oil contents from 7.6 to 17.3% (w/w) and contents of C(16)-C(18) fatty acids from 540.2 to 720.5 mg g(-1) oil. The predominant fatty acid was palmitic acid (C(16:0)) in nitrate depletion group, however, oleic acid (C(18:1)) was predominated in nitrate added groups. The two-stage culture enhanced overall oil productivity of 18.7 mg g(-1) day(-1) which is higher than that of 12.2 mg g(-1) day(-1) in single-stage culture.     

Scale-up of enzymatic production of lactobionic acid using the rotary jet head system

Enzymatic oxidation of lactose to lactobionic acid (LBA) by a carbohydrate oxidase from Microdochium nivale was studied in a pilot-scale batch reactor of 600 L working volume using a rotary jet head (RJH) for mixing and mass transfer (Nordkvist et al., 2003, Chem Eng Sci 58:3877-3890). Both lactose and whey permeate were used as substrate, air was used as oxygen source, and catalase was added to eliminate the byproduct hydrogen peroxide. More than 98% conversion to LBA was achieved. Neither enzyme deactivation nor enzyme inhibition was observed under the experimental conditions. The dissolved oxygen tension (DOT) was constant throughout the tank for a given set of operating conditions, indicating that liquid mixing was sufficiently good to avoid oxygen gradients in the tank. However, at a given oxygen tension measured in the tank, the specific rate of reaction found in the RJH system was somewhat higher than previously obtained in a 1 L mechanically stirred tank reactor (Nordkvist et al., 2007, in this issue, pp. 694-707). This can be ascribed to a higher pressure in the recirculation loop which is part of the RJH system. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient, k(L)a, were obtained when lactose was used as substrate, especially at low values of the specific power input and the superficial gas velocity. k(L)a was lower for experiments with whey permeate than with lactose due to addition of antifoam. The importance of mass transfer and of the saturation concentration of oxygen on the volumetric rate of reaction was demonstrated by simulations.     

Tracking the acetate threshold using DO-transient control during medium and high cell density cultivation of recombinant Escherichia coli in complex media

DO-transient nutrient controllers use the dissolved oxygen signal to attempt acetate threshold tracking during fed-batch cultivation of recombinant E. coli. Here we apply DO-transient control to the production of Jembrana disease virus protein in complex Super Luria medium and compare performance against a high-limit pH-stat controller. For induction at medium cell density (harvest between 31 and 32.5 g dcw L) a total productivity of 0.27 g L h was achieved as compared to 0.24 g L h with the high-limit pH-stat. For induction at high cell density (harvest at 60 g dcw L), decreased productivity (0.12 g L h) was attributed to the effect of acetate accumulation on recombinant protein formation and a concomitant lowering of the critical growth rate. Our results suggest that complex media provides a difficult environment for the application of acetate threshold tracking DO-transient control because of difficulties in re-oxidizing acetate, and apparent localized production of acetate below the production threshold (as detected by the DO-transient controller as SPOUR(crit)). Configuring the DO-transient controller to avoid aggressive threshold probing is suggested as a means to improve performance and reduce acetate accumulation in complex media.     

Microbial degradation of quinoline: Kinetic studies with Comamonas acidovorans DSM 6426

The microbial degradation of quinoline by Comamonas acidovorans was studied in a laboratory scale stirred tank reactor. In continuous culture experiments using quinoline as a sole source of carbon and nitrogen, it was shown by means of mass balances that quinoline was converted completely to biomass, carbon dioxide, and ammonia. Degradation rates up to 0.7 g/L h were obtained. Measured yield coefficients Y(x/s) for quinoline were about 0.7 g/g, which is in agreement with the theoretical value for complete mineralization. Kinetic constants based on Haldane substrate inhibition were evaluated. The values were mu(max) = 0.48 h(-1), K(i) = 69 mg/L, and K(s) < 1.45 mg/L. (c) 1993 John Wiley & Sons, Inc.     

重组大肠杆菌生物转化甘油生产3-羟基丙酸

目的:以甘油为底物构建高效的3-羟基丙酸生产菌株。方法:以自身携带乙醛脱氢酶的E.coli BL21(DE3)plysS作为宿主,异源表达源自Klebsiella pneumoniae的甘油脱水酶基因dhaB。结果:重组菌E.coli HP获得的甘油脱水酶比活力在1.0mmol/L IPTG的诱导下达到了77.2 U/mg,摇瓶条件下,3-HP的最大产量为5.44 g/L,摩尔转化率为53%,该产量比目前报道的最高水平(4.4 g/L)提高了23.6%。结论:重组菌株E.coli HP实现了甘油向3-羟基丙酸(3-HP)的高效生物转化。     

Optimization of bio-hydrogen production from biodiesel wastes by Klebsiella pneumoniae

Biodiesel wastes containing glycerol were utilized by Klebsiella pneumoniae DSM 2026 to produce hydrogen. The optimization of medium components was performed using both Plackett-Burman and uniform design methods. Using the Plackett-Burman design, glycerol, yeast extract, NH(4)Cl, KCl and CaCl2 were found to be the most important components, which were further investigated by uniform design and second-order polynomial stepwise regression analysis. The optimized medium containing 20.4 g.L(-1) glycerol, 5.7 g.L(-1) KCl, 13.8 g.L(-1) NH(4)Cl, 1.5 g.L(-1) CaCl(2) and 3.0 g.L(-1) yeast extract resulted in 5.0-fold increased level of hydrogen (57.6 mL/50 mL medium) production compared to initial level (11.6 mL/50 mL medium) after 24 h of fermentation The optimization of fermentation condition (pH, temperature and inoculum) was also conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5-L stirred tank bioreactor for batch production, hydrogen yield and production reached 0.53 mol/mol and 117.8 mmol/L, respectively. The maximum hydrogen evolution rate was 17.8 mmol/(L.h). Furthermore, 1,3-propanediol (6.7 g.L(-1)) was also obtained from the liquid medium as a by-product.     

Biotransformation of 4-halophenols to 4-halocatechols using Escherichia coli expressing 4-hydroxyphenylacetate 3-hydroxylase

Escherichia coli cells, expressing 4-hydroxyphenylacetate 3-hydroxylase, fully transformed 4-halogenated phenols to their equivalent catechols as single products in shaken flasks. 4-Fluorophenol was transformed at a rate 1.6, 1.8, and 3.4-fold higher than the biotransformation of 4-chloro-, 4-bromo-, and 4-iodo-phenol, respectively. A scale-up from shaken flask to a 5 L stirred tank bioreactor was undertaken to develop a bioprocess for the production of 4-substituted halocatechols at higher concentrations and scale. In a stirred tank reactor, the optimized conditions for induction of 4-HPA hydroxylase expression were at 37 °C for 3 h. The rate of biotransformation of 4-fluorophenol to 4-fluorocatechol by stirred tank bioreactor grown cells was the same at 1 and 4.8 mM (5.13 μmol/min/g CDW) once the ratio of biocatalyst (E. coli CDW) to substrate concentration (mM) was maintained at 2:1. At 10.8 mM 4-fluorophenol, the rate of 4-fluorocatechol formation decreased by 4.7-fold. However, the complete transformation of 1.3 g of 4-fluorophenol (10.8 mM) to 4-fluorocatechol was achieved within 7 h in a 1 L reaction volume. Similar to 4-fluorophenol, other 4-substituted halophenols were completely transformed to 4-halocatechols at 2 mM within a 1–2 h period. An increase in 4-halophenol concentration to 4.8 mM resulted in a 2.5–20-fold decrease in biotransformation efficiency depending on the substrate tested. Organic solvent extraction of the 4-halocatechol products followed by column chromatography resulted in the production of purified products with a final yield of between 33% and 38%.     

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.