Growth and exopolysaccharide production during free and immobilized cell chemostat culture of Lactobacillus rhamnosus RW-9595M

AIMS: Biomass and exopolysaccharide (EPS) production were studied during chemostat cultures in whey permeate medium with Lactobacillus rhamnosus RW-9595M-free cells and cells immobilized on solid porous supports (ImmobaSil). METHODS AND RESULTS: A continuous culture with free cells was conducted for 9 days at dilution rates (D) between 0.3 and 0.8 h(-1) in yeast extract (YE)/mineral supplemented whey permeate. Maximum EPS production (1808 mg l(-1)) and volumetric productivity (542.6 mg l(-1) h(-1)) were obtained for a low D of 0.3 h(-1). A continuous fermentation in a two-stage bioreactor system, composed of a first stage with immobilized cells and a second stage inoculated with free cells produced in the first reactor, was carried out for 32 days. The influence of YE concentration, temperature and dilution rate, and their interactions on biomass, EPS and lactic acid production was investigated. A statistically significant model was found only for lactic acid production. Marked cell morphological and physiological changes led to the formation of very large cell-containing aggregates and a low mean soluble EPS production (138 mg l(-1)). Aggregate volumetric productivity of the two-stage system varied between 5.7 and 49.5 g l(-1) h(-1) for different fermentation conditions and times. Aggregates contained a very high biomass concentration, estimated at 74% of aggregate dry weight by nitrogen analysis and 4.3 x 10(12) CFU g(-1) by a DNA extraction method and a high nonsoluble polysaccharide content (14.2%). At age 24 days, insoluble EPS concentration and volumetric productivity were 1250 mg l(-1) and 2240 mg l(-1) h(-1) respectively. The physiological changes were shown to be reversible when cells were incubated during three successive batch cultures. CONCLUSIONS: EPS production and volumetric productivity during continuous free-cell chemostat cultures with L. rhamnosus RW-9595M are among the highest values reported for lactobacilli in literature. Immobilization and continuous culture resulted in low soluble EPS production and large morphological and physiological changes of L. rhamnosus RW-9595M, with formation of macroscopical aggregates mainly composed of biomass and nonsoluble EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study on continuous EPS production by immobilized LAB. Immobilization and culture time-induced cell aggregation and could be used to produce new synbiotic products with very high viable cell and EPS concentrations.     

Coupling two sizes of CSTR-type bioreactors for sequential lactic acid and xylitol production from hemicellulosic hydrolysates of vineshoot trimmings

This study develops a system for the efficient valorisation of hemicellulosic hydrolysates of vineshoot trimmings. By connecting two reactors of 2L and 10L, operational conditions were set up for the sequential production of lactic acid and xylitol in continuous fermentation, considering the dependence of the main metabolites and fermentation parameters on the dilution rate. In the first bioreactor, Lactobacillus rhamnosus consumed all the glucose to produce lactic acid at 31.5°C, with 150rpm and 1L of working volume as the optimal conditions. The residual sugars were employed for the xylose to xylitol bioconversion by Debaryomyces hansenii in the second bioreactor at 30°C, 250rpm and an air-flow rate of 2Lmin(-1). Several steady states were reached at flow rates (F) in the range of 0.54-5.33mLmin(-1), leading to dilution rates (D) ranging from 0.032 to 0.320h(-1) in Bioreactor 1 and from 0.006 to 0.064h(-1) in Bioreactor 2. The maximum volumetric lactic acid productivity (Q(P LA)=2.908gL(-1)h(-1)) was achieved under D=0.266h(-1) (F=4.44mLmin(-1)); meanwhile, the maximum production of xylitol (5.1gL(-1)), volumetric xylitol productivity (Q(P xylitol)=0.218gL(-1)h(-1)), volumetric rate of xylose consumption (Q(S xylose)=0.398gL(-1)h(-1)) and product yield (0.55gg(-1)) were achieved at an intermediate dilution rate of 0.043h(-1) (F=3.55mLmin(-1)). Under these conditions, ethanol, which was the main by-product of the fermentation, was produced in higher amounts (1.9gL(-1)). Finally, lactic acid and xylitol were effectively recovered by conventional procedures.     

Effect of operating parameters on specific production rate of a cloned-gene product and performance of recombinant fermentation process

A two-stage continuous system in combination with a temperature-sensitive expression system were used as model systems to maximize the productivity of a cloned gene and minimize the problem associated with the plasmid instability for a high-expression recombinant. In order to optimize the two-stage fermentation process, the effects of such operational variables as temperature and dilution rate on productivity of cloned gene were studied using the model systems and a recombinant, Escherichia coli K12 DeltaH1 Deltatrp/pPLc23trp A1. When the expression of cloned gene is induced by raising the operating temperature above 38 degrees C, a significant decrease in the colony-forming-units (CFU) of the plasmid-harboring cell was observed, and the decrease was related to the product concentration. In order to describe this phenomenon, a new kinetic parameter related to the metabolic stress (metabolic stress factor) was introduced. It is defined as the ratio of the rate of change of pheno-type from colony-forming to non-colony-forming cells to the product accumulation per unit cell mass. At a fixed temperature of 40 degrees C, the varying dilution rate D in the range of 0.35-0.90 h(-1) did not affect the metabolic stress factor significantly. At a fixed dilution rate of D = 0.35 h(-1), this factor remained practically constant up to 41 degrees C but increased rapidly beyond 41 degrees C. The effects of temperature and dilution rate in the second stage on the specific production rate were also studied while maintaining the apparent specific growth rate (mu(2) (app)) of the second stage constant at or near mu(2) (app) = 0.26 h(-1). Under a constant dilution rate, D(2) = 0.35 h(-1), the maximum specific production rate obtained was about q(p, max) = 38 units TrpA/mg cell/h at 41 degrees C. At a constant temperature, T(2) = 40 degrees C, specific production rate increased with decreasing dilution rate with in the dilution rate range of D(2) = 0.35-0.90 h(-1). Based on the results of our study, the optimal operating conditions found were dilution rate D(2) = 0.35 h(-1) and operating temperature T(2) = 41 degrees C at the apparent specific growth rate of 0.26 h(-1). Under the optimal operating conditions, about threefold increase in productivity was achieved compared to the best batch culture result. In addition, the fermentation period could be extended for more than 100 h.     

Operational patterns affecting lactic acid production in ultrafiltration cell recycle bioreactor

Lactic acid production with cell recycling on an ultrafiltration tubular membrane reactor was studied; higher lactic acid concentrations as well as productivities were obtained under long-term fermentations compared with other high cell density systems. Different operational conditions, namely dilution rates and start-up modes, were assessed. Performances were very different at the three different dilution rates tested (D = 0.20 h(-1), D = 0.40 h(-1), or D = 0.58 h(-1)). The different behaviours are discussed and factors responsible for them are presented. The best way to operate for lactic acid production is chosen, the dilution rate of D = 0.40 h(-1) being the one providing the best overall performance. On the other hand, results show that of the two start-up modes tested, continuous start (membrane open) permits higher permeabilities throughout the operational runs than batch start (membrane closed). Operational stability was found to be directly associated with membranes that work at "steady state," the membrane permeability being kept around 15 L/m(2) h. Optimized cell bleed can improve time of operation if such membrane permeability can be maintained for a longer time. A comparison of results with those obtained in other lactic acid production systems is presented; such comparison shows that this tubular ultrafiltration membrane cell recycle reactor presents three important advantages: (1) concomitant lactic acid concentrations and productivities; (2) long periods of operation at reasonable permeabilities; and (3) good mechanical stability permitting the use of steam sterilization. (c) 1995 John Wiley & Sons, Inc.     

Effects of light intensity and dilution rate on the semicontinuous cultivation of Arthrospira (Spirulina) platensis. A kinetic Monod-type approach

Semicontinuous cultures were carried out at different dilution rates (D) and light intensities (I) to determine the maximum productivity of Arthrospira platensis cultivated in helicoidal photobioreactor up to the achievement of pseudo-steady-state conditions. At I=108 μmol photons m(-2) s(-1), the semicontinuous regime ensured the highest values of maximum cell concentration (X(m)=5772±113 mg L(-1)) and productivity (P(XS)=1319±25 mg L(-1) d(-1)) at the lowest (D=0.1 day(-1)) and the highest (D=0.3 day(-1)) dilution rates, respectively. A kinetic model derived from that of Monod was proposed to determine the relationship between the product of light intensity to dilution rate (ID) and the cell productivity, which were shown to exert a combined influence on this parameter. This result put into evidence that pseudo-steady-state conditions could be modified according to circumstances, conveniently varying one or other of the two independent variables.     

Continuous growth kinetics of Candida utilis in pineapple cannery effluent

Candida utilis was grown on a pineapple cannery effluent as the sole carbon and energy source in a chemostat at dilution rates between 0.10 and 0.62 h(-1) to determine the growth kinetics. The principal sugars in the effluent were sucrose, glucose, and fructose. The cell yield coefficient on carbohydrate varied with dilution rate and a maximum value of 0.63 was observed at a dilution rate of 0.33 h(-1). The steady-state concentrations of carbohydrate, reducing sugar, and chemical oxygen demand (COD) appeared to follow Monod saturation kinetics with increasing dilution rate, although none of the measured parameters represented a pure substrate. The maximum specific growth rate and reducing sugar saturation constant were 0.64 h(-1) and 0.060 g/L, respectively. A maximum cell mass productivity of 2.3 g/L h was observed at a dilution rate of 0.51 h(-1). At this dilution rate, only 68% of the COD was removed. A 95% COD removal was attained at a dilution rate of 0.10 h(-1). Optimal yeast productivity and COD reduction occurred at a dilution rate of 0.33 h(-1).     

Production of biomass and beta-D-galactosidase by Candida pseudotropicalis grown in continuous culture on whey

The production of biomass and beta-D-galactosidase by the lactose-utilizing yeast Candida pseudotropicalis NCYC 744 in whey medium was studied. Apparent optimization of growth conditions and medium was done in continuous culture. Optimaql pH and temperature were 2.6 and 36-38 degrees C, respectively, Limitations in Cu, Zn, and possbily Mn were detected in deproteinized whey medium. Additions of tryptophan estimulated growth of the yeast. Under optimal conditions in medium supplemented with excess tryptophan, Cu, Zn, and Mn the maximum values obtained: yeast concentration, 4.6 g/L; yeast productivity, 1.4 g/L h (at D = 0.35 h(-1)); enzyme volumetric productivity, 2100 U/L h (at D = 0.25 h(-1)); maintenance coefficient, 5-10 mg lactose/g cell h; saturation constant (K(s)) for lactose, 4.76mM; maximum specific growth rate, (mu(max)), 0.47 h(-1). No significant increase in specific enzyme activity (U/mg cell) was observed after medium optimiztion evidencing the importance of regulatory controls in enzyme synthesis.     

High-rate continuous production of lactic acid by Lactobacillus rhamnosus in a two-stage membrane cell-recycle bioreactor

It is important to produce L(+)-lactic acid at the lowest cost possible for lactic acid to become a candidate monomer material for promising biodegradable polylactic acid. In an effort to develop a high-rate bioreactor that provides high productivity along with a high concentration of lactic acid, the performance of membrane cell-recycle bioreactor (MCRB) was investigated via experimental studies and simulation optimization. Due to greatly increased cell density, high lactic acid productivity, 21.6 g L(-1) h(-1), was obtained in the reactor. The lactic acid concentration, however, could not be increased higher than 83 g/L. When an additional continuous stirred tank reactor (CSTR) was attached next to the MCRB a higher lactic acid concentration of 87 g/L was produced at significant productivity expense. When the two MCRBs were connected in series, 92 g/L lactic acid could be produced with a productivity of 57 g L(-1) h(-1), the highest productivity among the reports of L(+)-lactic acid that obtained lactic acid concentration higher than 85 g/L using glucose substrate. Additionally, the investigation of lactic acid fermentation kinetics resulted in a successful model that represents the characteristics of lactic acid fermentation by Lactobacillus rhamnosus. The model was found to be applicable to most of the existing data with MCRBs and was in good agreement with Levenspiel's product-inhibition model, and the Luedeking-Piret equation for product-formation kinetics appeared to be effective in representing the fermentation kinetics. There was a distinctive difference in the production potential of cells (cell-density-related parameter in Luedeking-Piret equation) as lactic acid concentration increases over 55 g/L, and this finding led to a more precise estimation of bioreactor performance.     

Air-bubbling, hollow-fiber reactor with cell bleeding and cross-flow filtration

Continuous asymmetric reduction of dyhydrooxoisophorone (DOIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) was achieved by a thermophilic bacterium Bacillus stearothermophilus NK86-0151. Three reactors were used: an air-bubbling hollow-fiber reactor with cell bleeding and cross-flow filtration, an air-lift reactor, and a CSTR with PAA immobilized cells. The maximum cell concentration of 11.1 g dry wt L(-1) was obtained in an air-bubbling hollow-fiber reactor, while in the other reactors the cell densities were between 3.5 and 4.1 g dry wt L(-1) The optimum bleed ratio was 0.1 at the dilution rate 0.3 h(-1) in the hollow-fiber reactor. The highest viable cell concentration was maintained in the dilution range of 0.4-0.7 h(-1) by a combination of proper cell bleeding and cross-flow filtration. The maximum volumetric productivity of 4-HTMCH reached 826 mg L(-1) h(-1) at the dilution rate 0.54 h(-1). This value was 4 and 2 times higher than those in the air-lift reactor and CSTR, respectively. The increasing viable cell concentration increased the volumetric productivity of 4-HTMCH. A cell free product solution was continuously obtained by cross-flow filtration.     

Acetic acid production using a fermentor equipped with a hollow fiber filter module

The continuous production of acetic acid by Acetobacter aceti M23 was carried out using a fermentor equipped with a hollow fiber filter module. The culture continued for 830 h with various dilution rates, which were changed stepwisely from low to high. The final cell concentration was 21.9 g dry cell/L and the maximum productivity of acetic acid was 12.7 g/L.h for the exit acetic acid concentration of about 50 g/L. The productivity was higher than any literature's values surveyed so far. The cell concentration was 62.8 times and the productivity was 4.6 times as high as those of the fermentor without the filter module. The productivity increased with the increase of dilution rate up to 0.3 h(-1). It is interesting to note that the viable cell concentration was kept almost constant about 1.1 x 10(9) cells/ml in spite of the increase of dilution rate. Use of oxygen-rich air was indispensable to establish the high productivity of acetic acid.     

甲醇抑制时重组毕赤酵母发酵的特性研究

本文对毕赤酵母进行了恒化培养研究。以甲醇为唯一碳源时,在稀释率较低时(D<0.048 h^-1),连续培养系统操作很稳定。但在稀释率高时(D>0.048h^-1),连续培养系统的定态点不止一个,实验不能维持,故采用比生长速率恒定的分批流加培养进行研究。结果表明,毕赤酵母的生长符合Andrew普遍化底物抑制模型。综合考虑水蛭素的生成、底物的消耗,在生产中维持甲醇浓度为限制性浓度(0.5 g/L),且维持比生长速率为0.02 h^-1时,水蛭素Hir65的比生成速率达到最大值0.2 mg/(g·h)且甲醇的比消耗速率为0.04 g/(g·h)。     

Bacteriocin production by strain <Emphasis Type="Italic">Lactobacillus delbruecki</Emphasis>i ssp. <Emphasis Type="Italic">bulgaricus</Emphasis> BB18 during continuous prefermentation of yogurt starter culture and subsequent batch coagulation of milk

By screening for bacteriocin-producing lactic acid bacteria of 1,428 strains isolated from authentic Bulgarian dairy products, Lb. bulgaricus BB18 strain obtained from kefir grain was selected. Out of 11 yogurt starters containing Lb. bulgaricus BB18 and S. thermophilus strains resistant to bacteriocin secreted by Lb. bulgaricus BB18 a yogurt culture (S. thermophilus 11A+Lb. bulgaricus BB18) with high growth and bacteriocinogenic activity in milk was selected. Continuous (pH-stat 5.7) prefermentation processes were carried out in milk at 37 degrees C in a 2l MBR bioreactor (MBR AG, Zurich, Switzerland) with an IMCS controller for agitation speed, temperature, dissolved oxygen, CO2 and pH. Prefermented milk with pH 5.7 coagulated in a thermostat at 37 degrees C until pH 4.8-4.9. S. thermophilus 11A and Lb. bulgaricus BB18 grew independently in a continuous mode at similar and sufficiently high-dilution rates (D=1.83 h(-1)-S. thermophilus 11A; D=1.80 h(-1)-Lb. bulgaricus BB18). The yogurt cultures developed in a stream at a high-dilution rate (D=2.03-2.28 h(-1)). The progress of both processes (growth and bacteriocin production) depended on the initial ratio between the two microorganisms. The continuous prefermentation process promoted conditions for efficient fermentation and bacteriocinogenesis of the starter culture during the batch process: strong reduction of the times for bacteriocin production and coagulation of milk (to 4.5-5.0 h); high cell productivity (lactobacilli-4x10(12) CFU ml(-1), streptococci-6x10(12) CFU ml(-1)); high productivity of bacteriocins (4,500 BU ml(-1))-1.7 times higher than the bacteriocinogenic activity of the batch starter culture.     

Continuous cultivation of the diatom Nitzschia laevis for eicosapentaenoic acid production: physiological study and process optimization

The continuous cultures of the diatom Nitzschia laevis were performed at different dilution rates (D) and feed glucose concentrations (S(0)) to investigate cellular physiological responses and its production potential of eicosapentaenoic acid (EPA). Steady-state cell dry weight, residual glucose concentration, cell growth yield, specific glucose consumption rate, and fatty acid profiles were investigated within the range of D from 0.1 to 1.0 day(-1) (S(0) fixed at 20 g/L) and the range of S(0) from 5 to 35 g/L (D fixed at 0.3 day(-1)), respectively. The highest EPA productivity of 73 mg L(-1) day(-1) was obtained at D = 0.5 day(-1) and S(0) = 20 g/L. However, when the continuous culture achieved high productivities of EPA at certain dilution rates and feed glucose concentrations, glucose in the feed could not be consumed completely. Accordingly, the continuous culture was evaluated in terms of both EPA productivity (P) and glucose assimilation efficiency (E). The parameter eta, defined as the product of P and E, was used as an overall performance index. Since eta is a function of the two independent variables D and S(0), we employed a central composite design to optimize D and S(0) for the highest eta value. Based on the experimental results of the design, a second-order polynomial equation was established to represent the relationship between eta and D and S(0). The optimal values of D and S(0) were subsequently determined as 0.481 day(-1) and 15.56 g/L, respectively by the empirical model. The verification experiment confirmed the validity of the model. Under the optimal conditions, eta value reached 46.5 mg L(-1) day(-1), suggesting a considerably high efficiency of the continuous culture of N. laevis in terms of EPA production and glucose utilization.     

Growth and lactic acid production in batch culture of Lactobacillus rhamnosus in a defined medium

To facilitate metabolic analysis, batch fermentations of Lactobacillus rhamnosus were carried out in a new defined medium. Biomass at 10.5 g/l and lactic acid at 67 g/l with a YP/S of 0.84 were achieved. The maximum specific growth rate and the average productivity were 0.49/h and 2.48 g/l.h, respectively. These are comparable to those of this organism and related organisms in complex media. Preliminary amino acid studies were also conducted, highlighting the importance of serine, asparagine, glutamine and cysteine. Kinetic analysis revealed that lactic acid production was predominantly growth-associated with growth associated and non-growth associated lactic acid constants of 0.389 mol/g-cell and 0.0025 mol/g-cell.h, respectively. Finally a kinetic model has been included to describe the fermentation of L. rhamnosus.     

Bifidobacterium longum ATCC 15707 cell production during free- and immobilized-cell cultures in MRS-whey permeate medium

Bifidobacterium longum ATCC 15707 cell production was studied in MRS medium supplemented with whey permeate (MRS-WP) during free-cell batch fermentations and continuous immobilized-cell cultures. Very high populations were measured after 12 h batch cultures in MRS-WP medium controlled at pH 5.5 (1.7+/-0.5x10(10) cfu/ml), approximately 2-fold higher than in non-supplemented MRS. Our study showed that WP is a low-cost source of lactose and other components that can be used to increase bifidobacteria cell production in MRS medium. Continuous fermentation in MRS-WP of B. longum immobilized in gellan gum gel beads produced the highest cell concentrations in the effluent (4.9+/-0.9x10(9) cfu/ml) at a dilution rate (D) of 0.5 h(-1). However, maximal volumetric productivity (6.9+/-0.4x10(9) cfu ml(-1)h(-1)) during continuous cultures was obtained at D =2.0 h(-1), and was approximately 9.5-fold higher than during free-cell batch cultures at an optimal pH of 5.5 (7.2x10(8) cfu ml(-1)h(-1)).     

Growth of Bacteroides fragilis in Continuous Culture and in Batch Cultures at Controlled pH

Bacteroides fragilis NCTC 9343 has been grown in continuous cultures with glucose as growth-limiting factor. At pH 7.0 and at a dilution rate of 0.07 per h, glucose limited growth in concentrations up to 0.6%. Maximal cell yield and productivity were obtained with 0.87% glucose in the inflowing medium. A pH of 7.0 was optimal for growth. With 0.6% glucose in the fresh medium and at pH 7.0, cell yield and productivity were highest at a dilution rate of 0.07 per h and 0.11 per h, respectively. At dilution rates higher than 0.07 per h, glucose was no longer growth limiting, and at dilution rates above 0.11 per h, another compound seemed to have replaced glucose also as energy source. When grown in batch cultures at pH 7.0, the best yields of B. fragilis was achieved with 0.6% glucose in the fresh medium. The highest specific growth rate (mum) determined from viable counts was 0.45, corresponding to a mean generation time of 92 min.     

Continuous cultivation of Lactobacillus brevis NCL912 for production of gamma-aminobutyric acid

A continuous cultivation method for Lactobacillus brevis NCL912 to synthesize gamma-aminobutyric acid was developed in this work. Different dilution rates were evaluated for obtaining steady state in continuous cultivation. The results showed that steady state could be achieved at dilution rates from 0.08 to 0.12 h^?1. The highest gamma-aminobutyric acid productivity (5.11 g L^?1?h^?1) was obtained at dilution rate of 0.09 h^?1. The kinetic models were established for continuous gamma-aminobutyric acid production by using the Monod equation for microbial growth, and the Luedeking–Piret equation for product formation. The microbial growth and product formation can be described by equations $ \mu = {{{0.1234{C_S}}} \left/ {{\left( {0.9338+{C_S}} \right)}} \right.} $ and $ {Q_P}=6.86\,\mathrm{g}\,{{\mathrm{g}}^{-1 }}\mathrm{cell}\,{{\mathrm{h}}^{-1 }} $ , respectively. The production of gamma-aminobutyric acid by L. brevis NCL912 was non-growth-associated.     

A rational approach to improving productivity in recombinant Pichia pastoris fermentation

A Mut(S) Pichia pastoris strain that had been genetically modified to produce and secrete sea raven antifreeze protein was used as a model system to demonstrate the implementation of a rational, model-based approach to improve process productivity. A set of glycerol/methanol mixed-feed continuous stirred-tank reactor (CSTR) experiments was performed at the 5-L scale to characterize the relationship between the specific growth rate and the cell yield on methanol, the specific methanol consumption rate, the specific recombinant protein formation rate, and the productivity based on secreted protein levels. The range of dilution rates studied was 0. 01 to 0.10 h(-1), and the residual methanol concentration was kept constant at approximately 2 g/L (below the inhibitory level). With the assumption that the cell yield on glycerol was constant, the cell yield on methanol increased from approximately 0.5 to 1.5 over the range studied. A maximum specific methanol consumption rate of 20 mg/g. h was achieved at a dilution rate of 0.06 h(-1). The specific product formation rate and the volumetric productivity based on product continued to increase over the range of dilution rates studied, and the maximum values were 0.06 mg/g. h and 1.7 mg/L. h, respectively. Therefore, no evidence of repression by glycerol was observed over this range, and operating at the highest dilution rate studied maximized productivity. Fed-batch mass balance equations, based on Monod-type kinetics and parameters derived from data collected during the CSTR work, were then used to predict cell growth and recombinant protein production and to develop an exponential feeding strategy using two carbon sources. Two exponential fed-batch fermentations were conducted according to the predicted feeding strategy at specific growth rates of 0.03 h(-1) and 0.07 h(-1) to verify the accuracy of the model. Cell growth was accurately predicted in both fed-batch runs; however, the model underestimated recombinant product concentration. The overall volumetric productivity of both runs was approximately 2.2 mg/L. h, representing a tenfold increase in the productivity compared with a heuristic feeding strategy.     

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates)

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.     

Continuous bioconversion of n-octane to octanoic acid by recombinant Escherichia coli (alk(+)) growing in a two-liquid-phase Chemostat

Escherichia coli is able to grow on sugars in the presence of a bulk n-alkane phase. When E. coli is equipped with the alk genes from Pseudomonas oleovorans, the resulting recombinant strain converts n-alkanes into the corresponding alkanoic acids. To study the effects of growth rate and exposure to a bulk apolar phase on the physiology and the productivity of E. coli, we have grown this microorganism in two-liquid-phase continuous cultures containing 5% (v/v) n-octane.In contrast to batch cultures of wild-tape E. coli grown in the presence of n-octane, cells remained viable during the entire continuous culture, which lasted 200 h. Bioconversion of n-octane to n-octanoic acid by a recombinant E. coli (alk(+)) in a two-liquid-phase continuous culture was made possible by optimizing both the recombinant host strain and the conditions of culturing the organism. Continuous production in such two-phase systems has been maintained for the least 125 h without any changes in the product concentration in the fermentation medium. The volumetric productivity was determined as a function of growth rate and showed a maximum at a dilution rate D = 0.32 h(-1), reaching a continuous production rate of 0.5 g octanoate/L . h (4 tons/m(3) . year). (c) 1993 John Wiley & Sons, Inc.