Optimization of the mated fermentation process for the production of lycopene by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and NRRL 2896 (−)

The mated fermentation process for the production of lycopene by Blakeslea trispora NRRL 2895 (+) and NRRL 2896 (−) was systematically optimized in shake flasks. The ratio of the (+) to (−) strains, the lycopene cyclase inhibitors piperidine and creatinine, the trisporic acid structural analog abscisic acid, the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) precursor leucine, and the mevalonate kinase enhancer penicillin were all identified as key factors affecting lycopene biosynthesis. With an optimal ratio of 5:1 for the (+) to (−) strains and the addition of 6 g/L creatinine on day 3, the highest lycopene production was 98.1 ± 15.5 mg/L. Based on the above result, the addition of 0.1 g/L penicillin on day 4, 150 μmol/L abscisic acid on day 3 or 0.5 g/L leucine on day 4 enhanced lycopene production to 119.7 ± 17.2, 120.6 ± 12.3 and 135.2 ± 7.0 mg/L, respectively. Finally, an integrated strategy by combining the above key factors was developed, and the highest lycopene production of 156.2 ± 15.4 mg/L was obtained, which was enhanced by 134.9% comparing with its production of 66.5 ± 3.6 mg/L before the optimization process of this work. The results obtained in this study may be useful for large-scale industrial lycopene production.     

Production of polygalacturonase byByssochlamys fulva

Summary Byssochlamys fulva was grown in two fermentation media using shake flasks, stirred fermentor and disc fermentor under conditions to give maximum production of pectolytic enzymes. Only polygalacturonase activity was detected in the culture filtrates during all fermentations. In all production conditions studied, no evidence of pectin methylesterase, pectin lyase, cellulase or proteinase activities were found. The maximum polygalacturonase activity (4.5 units/ml) was achieved when the microorganism was grown on medium II in shake flasks at pH 4.0–4.5 and 30°C after 12 days of fermentation.     

Production of nisin with continuous adsorption to Amberlite XAD-4 resin using<Emphasis Type="Italic"> Lactococcus lactis</Emphasis> N8 and<Emphasis Type="Italic"> L. lactis</Emphasis> LAC48

The production of nisin, biomass and lactic acid in pH-controlled and uncontrolled batch fermentation and batch fermentation (pH 5.5) with continuous removal of nisin was examined in the parent strain Lactococcus lactis N8 and LAC48. Strain LAC48 in batch fermentor (pH not controlled) gave a maximum nisin concentration of 2.5×10⁶ IU g dcw^–1. The nisin concentration remained high (2.0×10⁶ IU g dcw^–1) after the logarithmic growth phase (10–22 h), whereas nisin production of strain N8 decreased after the logarithmic growth phase. The maximum nisin production of strain LAC48 was not directly related to the biomass formation and not associated with growth. In order to study end product inhibition in nisin production, a system was built for adsorption of nisin during fermentation. The adsorbent Amberlite XAD-4 was found to have an effective binding capacity for nisin. Cells of LAC48 and N8 compensated for the removal of nisin, indicating that nisin production also occurs in the stationary phase.     

Physostigmine production byStreptomyces griseofuscus NRRL 5324: Process development and scale-up studies

A reliable and scalable fermentation process was developed for production of the acetylcholine esterase inhibitor physostigmine employingStreptomyces griseofuscus NRRL 5324. Initial fermentation in small-scale bioreactors reached physostigmine levels of approximately 60 mg L^–1 after 139 h. Optimization of both process operating parameters and production medium composition rapidly yielded a seven-fold increase in physostigmine titer. The scaled up process routinely produced physostigmine titers of approximately 400 mg L^–1 during a fermentation cycle of 180 h, and supported the rapid production of large amounts of physostigmine. A physostigmine production of 500 mg L^–1 representing an eight-fold improvement over the original performance, was achieved using a glucose/ammonium fed-batch process.     

Lycopene production from synthetic medium by <Emphasis Type="Italic">Blakeslea trispora</Emphasis> NRRL 2895 (+) and 2896 (−) in a stirred-tank fermenter

The dissolved oxygen tension of 20% of air saturation, pH-shift from 4.0 to 5.5 on day 3, and a moderate shear stress (calculated as an impeller tip speed, V_\texttip = 0. 9 2 6- 2. 1 6 1  \textm/\texts V_{\text{tip}} = 0. 9 2 6- 2. 1 6 1 \, {\text{m}}/{\text{s}} ) were identified to be the key factors in scaling-up the mated fermentation of Blakeslea trispora NRRL 2895 (+) and 2896 (−) for lycopene production from a shake flask to a stirred-tank fermenter. The maximal lycopene production of 183.3 mg/L was obtained in 7.5-L stirred-tank fermenter, and then the mated fermentation process was successfully step-wise scaled-up from 7.5- to 200-L stirred-tank fermenter. The comparability of the fermentation process was well controlled and the lycopene production was maintained during the process scale-up. Furthermore, with the integrated addition of 150 μmol/L abscisic acid on day 3, 0.5 g/L leucine and 0.1 g/L penicillin on day 4, the highest lycopene production of 270.3 mg/L was achieved in the mated fermentation of B. trispora in stirred-tank fermenter.     

Batch cultures of recombinant Lactococcus lactis subsp. lactis in a stirred fermentor

Summary The transfer of plasmids was studied in a stirred fermentor in the course of mixed batch cultures combining recombinant strains of Lactococcus lactis subsp. lactis (donor strains) with L. lactis subsp. lactis CNRZ 268M3 (recipient strain). Donor strains contained one or two of the following plasmids (coding for erythromycin or chloramphenicol resistance): pIL205 (self-transmissible), pIL252, pIL253 (non-transmissible but mobilizable by pIL205, respectively small and large copy number) and pE194 (inserted in the chromosome). Only self-transmissible plasmid pIL205 was transferred, with frequencies ranging from 10^–7 to 10^–8 after 12 h of fermentation. These frequencies were 60–400 times lower than in unstirred M17 broth and 100 000 times lower than on agar medium. In the latter case, non-transmissible plasmids pIL252 and pIL253 were mobilized by pIL205 with a frequency of about 10^–5–10^–6. Correspondence to: C.-Y. Boquien     

Liquid-culture production of blastospores of the bioinsecticidal fungus<Emphasis Type="Italic"> Paecilomyces fumosoroseus</Emphasis> using portable fermentation equipment

The production of fungal spores using on-site, non-sterile, portable fermentation equipment is technically constrained. Very little information is available on the production requirements, such as medium concentration, inoculum stabilization, required fermentation times, and maintenance of axenic growth. In this study, we developed a two-part, liquid concentrate of the production medium that remains stable and soluble at room temperature. We also examined inoculum stability and showed that freeze- or air-dried blastospore preparations were stable for 7 days after rehydration when stored at 4 °C. The use of a low-pH (pH 4), relatively rich complex medium provided a growth environment deleterious to bacterial growth yet conducive to rapid sporulation by Paecilomyces fumosoroseus. High concentrations of blastospores (7.9×10⁸/ml) of P. fumosoroseus were produced in a 40-h fermentation with very low levels of bacterial contamination when the fermentor was charged with a blastospore production medium with a starting pH of 4 and inoculated with blastospore concentrations greater than 1×10⁶ spores/ml. These studies demonstrate that the use of disinfected, portable fermentation equipment has potential for on-site production of high concentrations of blastospores of the bioinsecticidal fungus P. fumosoroseus.     

A repeated batch process for cultivation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis>

A repeated batch process was performed to culture Bifidobacterium longum CCRC 14634. An on-line device, oxidation-reduction potential (ORP), was used to monitor cell growth and uptake of nutrients in the culture. The ORP of the culture medium decreased substantially during fermentation until nutrients were depleted. Six cycles of batch fermentation using ORP as a control parameter were successfully carried out. As soon as ORP remained constant or increased, three-quarters of the broth was removed, and the same volume of fresh medium was fed to the fermenter for a new cycle of cultivation. Average cell concentrations of 1.9×10⁹ and 3.4×10⁹ cfu ml^–1 for repeated batch fermentation in MRS (Lactobacilli MRS broth) and WY (containing whey hydrolyzates, yeast extract, l-cysteine) medium, respectively, were achieved. Cell mass productivities for batch, fed-batch and repeated batch fermentation using MRS medium were 0.51, 0.41, and 0.64 g l^–1 h^–1, respectively, and those for batch and repeated batch using WY medium were 0.76, 0.99 g l^–1 h^–1, respectively. The results indicate a possible industrial process to culture Bifidobacteria sp.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Large scale production and semi-purification of kedarcidin in a 1000-L fermentor

Summary Actinomycete strain ATCC 53650 was grown in a 1000-L fermentor containing 680 L of medium and the production of kedarcidin was monitored by HPLC. The titers of kedarcidin in the fermentor cultures were 0.49–0.53 mg ml^–1. A quick and efficient purification method involving the use of anion exchange resin DE23 (batch adsorption-desorption) and an ultrafiltration system yielded high recovery (65% yield) of kedarcidin from the fermentor culture. Over 200 grams of lyophilized kedarcidin of 70% purity was recovered from each of two 1000-L fermentor cultures using this process.     

Control of ethanol production and monitoring of membrane performance by mass-spectrometric gas analysis in the coupled fermentation-pervaporation of whey permeate

A coupled fermentation-pervaporation process was operated continuously with on-line mass spectrometric gas analysis monitoring of product accumulation on both the upstream and the downstream sides of the membrane. Efficient coupling of the fermentation with pervaporation was attained when a steady state of ethanol production and removal was achieved with whey permeate containing high concentrations of lactose (>8%) or by controlled lactose additions that also compensated for loss of liquid due to pervaporation. The combined system consists of a tubular membrane pervaporation module, directly connected to a stirred fermentor to form one circulation loop, kept at 38°C, with both units operating under computer control. Mass spectrometric gas analysis of the CO₂ gas evolved in the fermentor and the ethanol and water in the pervaporate on the downstream side of the membrane enabled us to follow the production of ethanol and its simultaneous removal. Membrane selectivity was calculated on-line and served to monitor the functioning of the membrane. Batch-wise-operated fermentation-pervaporation with Candida pseudotropicalis IP-513 yielded over 120 gl^–1 of concentrated ethanol solution using supplemented whey permeate containing 16% lactose. A steady state lasting for about 20 h was achieved with ethanol productivity of 20 g h^–1 (approx. 4 g l^–1 h^–1). Membrane selectivity was over 8. Controlled feeding of concentrated lactose suspension in the whey permeate (350 g l^–1) resulted in the continuous collection of 120–140 g l^–1 of ethanol pervaporate for 5 days, by which time salt accumulation hampered the fermentation. Medium refreshment restored the fermentative activity of the yeast cells and further extended the coupled process to over 9 days (200 h), when reversible membrane fouling occurred. The membrane module was exchanged and the combined process restarted. Correspondence to: Y. Shabtai     

Development of a fermentation method using immobilized cells under unsterile conditions. 2. Ethanol andl-lactic acid production without heat and filter sterilization

A process that was developed for protection of immobilized cells against inhibitory substances in the fermentation medium was applied for ethanol and lactic acid production with neither sterilization of the media, fermentor and other apparatus nor filtration of the aeration gas. The process involves co-immobilization of the fermentation micro-organism with castor oil and suppression of contaminant growth by addition of an anti-microbial substance to the fermentation medium. When 0.1%n-butyl,p-hydroxybenzoate (POBB) was added to the medium, ethanol and lactic acid productions were stable for 60 h and 70 h, respectively, while growth of the contaminants was completely suppressed. Longer process stability was achieved when POBB was replaced with Preventol GD, which has higher partition coefficient between castor oil and water. In this case, both glucose consumption and ethanol production were stable for 140 h. The possibilities of increasing the process stabilities were discussed.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

Unsterile and continuous production of polyhydroxybutyrate by Halomonas TD01

An unsterile and continuous fermentation process was developed based on a halophilic bacterium termed Halomonas TD01 isolated from a salt lake in Xinjiang, China. The strain reached 80 g/L cell dry weight containing 80% poly(3-hydroxybutyrate) (PHB) on glucose salt medium during a 56 h fed-batch process. In a 14-day open unsterile and continuous process, the cells grew to an average of 40 g/L cell dry weight containing 60% PHB in the first fermentor with glucose salt medium. Continuous pumping of cultures from the first fermentor to the second fermentor containing the nitrogen-deficient glucose salt medium diluted the cells but allowed them to maintain a PHB level of between 65% and 70% of cell dry weight. Glucose to PHB conversions were between 20% and 30% in the first fermentor and above 50% in the second one. This unsterile and continuous fermentation process opens a new area for reducing the cost in polyhydroxyalkanoates production.     

Two-stage continuous fermentation of Clostridium acetobutylicum: effects of pH and dilution rate

Summary The kinetics of a two-stage continuous fermentation of Clostridium acetobutylicum have been studied. The pH and the dilution rate have been shown to be two essential factors for process optimization. An increase in pH or dilution rate in the first stage decreased solvent production in the second fermentor. To achieve optimal solvent production, the pH had to be maintained at 4.5 in the first stage and between 4.5 and 5.0 in the second stage. Dilution rates of 0.08 h^–1 and 0.04 h^–1,respectively, in the first and second fermentors allowed a high solvent concentration. When the pH was maintained at 4.5 in each stage and when the dilution rates were 0.08 h^–1 and 0.04 h^–1 in the first and second fermentors respectively, 21 g/l solvent concentration was achieved. A conversion yield of 0.36 g solvents/g glucose consumed was obtained with total consumption of glucose. Biomass was only produced in the first stage together with 40% of the solvents, indicating that solvent production had to be induced in the first fermentor. Offprint requests to: J. M. Engasser     

Determination of lycopene, α-carotene and β-carotene in serum by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry with selected-ion monitoring

A selected-ion monitoring (SIM) determination of serum lycopene, α-carotene and β-carotene by an atmospheric pressure chemical ionization mass spectrometry (APCI–MS) was developed. A large amount of serum cholesterols disturbed the SIM determination of carotenoids by contaminating the segment of interface with the LC–MS. Therefore, separation of carotenoids from the cholesterols was performed using a mixed solution of methanol and acetonitrile (70:30) as the mobile phase on a C₁₈ column of mightsil ODS-5 (75 mm×4.6 mm I.D.). The SIM determination was carried out by introducing only the peak portions of carotenoids and I.S. (squalene) by means of an auto switching valve. In the positive mode of APCI–MS, lycopene, α-carotene and β-carotene were monitored at m/z 537 and I.S. was monitored at m/z 411. This method was linear for all analytes in the range of 15–150 ng for lycopene, 7–70 ng for α-carotene and 25–50 ng for β-carotene. The detection limit of LC–APCI–MS-SIM for carotenoids was about 3 ng per 1 ml of serum (S/N=3). The repeatabilities, expressed as C.V.s, were 10%, 8.4% and 5.3% for lycopene, α-carotene and β-carotene, respectively. The intermediate precisions, expressed as C.V.s, were 11. 2%, 8.8% and 6.5% for lycopene, α-carotene and β-carotene, respectively.     

Effect of redox potential on stationary-phase xylitol fermentations using<Emphasis Type="Italic"> Candida tropicalis</Emphasis>

Redox potential was used to develop a stationary-phase fermentation of Candida tropicalis that resulted in non-growth conditions with a limited decline in cell viability, a xylitol yield of 0.87 g g^–1 (95% of the theoretical value), and a high maximum specific production rate (0.67 g g^–1 h^–1). A redox potential of 100 mV was found to be optimum for xylitol production over the range 0–150 mV. A shift from ethanol to xylitol production occurred when the redox potential was reduced from 50 mV to 100 mV as cumulative ethanol (Y_ethanol) decreased from 0.34 g g^–1 to 0.025 g g^–1 and Y_xylitol increased from 0.15 g g^–1 to 0.87 g g^–1 (=0.05). Reducing the redox potential to 150 mV did not improve the fermentation. Instead, the xylitol yield and productivity decreased to 0.63 g g^–1 and 0.58 g g^–1 h^–1 respectively and cell viability declined. The viable, stationary-phase fermentation could be used to develop a continuous fermentation process, significantly increasing volumetric productivity and reducing downstream separation costs, potentially by the use of a membrane cell-recycle reactor.Electronic supplementary material is available if you access this article at . On that page (frame on the left side), a link takes you directly to the electronic supplementary materialAn erratum to this article can be found at     

Metabolic engineering of Pichia pastoris X-33 for lycopene production

As an alternative carotenoid producer, non-carotenogenic Pichia pastoris was chosen for a reddish carotenoid lycopene production because it can grow to high cell density without accumulation of ethanol and utilize various classes of organic materials such as methanol as carbon sources. Two synthetic lycopene-pathway plasmids, pGAPZB-EBI* and pGAPZB-EpBpI*p, were designed and constructed. The pGAPZB-EpBpI*p plasmid encoded three carotenogenic enzymes that were engineered to be targeted into peroxisomes of P. pastoris whereas the pGAPZB-EBI* plasmid encoded non-targeted enzymes. After both plasmids were transformed into P. pastoris, the lycopene-producing clone containing the pGAPZB-EpBpI*p plasmid, referred to as Ω, was selected and used for further optimization study. Of the carbon sources tested, glucose resulted in the highest level of lycopene production in complex and minimal media. Batch fermentation of the Ω clone resulted in the production of 4.6 mg-lycopene/g-DCW, with a concentration of 73.9 mg/l of lycopene in minimal medium. For the first time non-carotenogenic yeast P. pastoris was metabolically engineered by heterologously expressing lycopene-pathway enzymes and the lycopene concentration of 73.9 mg/l was obtained. This serves as a basis for the development of biological process for carotenoids using P. pastoris at a commercial production level.     

Spaceflight‐induced enhancement of 2‐keto‐L‐gulonic acid production by a mixed culture of Ketogulonigenium vulgare and Bacillus thuringiensis

Two bacterial strains used for industrial production of 2‐keto‐L‐gulonic acid (2‐KLG), Ketogulonigenium vulgare 2 and Bacillus thuringiensis 1514, were loaded onto the spacecraft Shenzhou VII and exposed to space conditions for 68 h in an attempt to increase their fermentation productivities of 2‐KLG. An optimal combination of mutants B. thuringiensis 320 and K. vulgare 2194 (KB2194‐320) was identified by systematically screening the pH and 2‐KLG production of 16 000 colonies. Compared with the coculture of parent strains, the conversion rate of L‐sorbose to 2‐KLG by KB2194‐320 in shake flask fermentation was increased significantly from 82·7% to 95·0%. Furthermore, a conversion rate of 94·5% and 2‐KLG productivity of 1·88 g l^?1 h^?1 were achieved with KB2194‐320 in industrial‐scale fermentation (260 m³ fermentor). An observed increase in cell number of K2194 (increased by 47·8%) during the exponential phase and decrease in 2‐KLG reductase activity (decreased by 46·0%) were assumed to explain the enhanced 2‐KLG production. The results suggested that the mutants KB2194‐320 could be ideal substitutes for the currently employed strains in the 2‐KLG fermentation process and demonstrated the feasibility of using spaceflight to breed high‐yielding 2‐KLG‐producing strains for vitamin C production.

Significance and Impact of the Study

KB2194‐320, a combination of two bacterial strains bred by spaceflight mutation, exhibited significantly improved 2‐KLG productivity and hence could potentially increase the efficiency and reduce the cost of vitamin C production by the two‐step fermentation process. In addition, a new pH indicator method was applied for rational screening of K2, which dramatically improved the efficiency of screening.     

An enzymatic route to produce pyruvate from lactate

A bacterial strain of Acinetobacter sp., which was capable of enzymatic production of pyruvate from lactate, was cultured in a 5-l reactor with a basal salt medium. After 14 h of fed-batch fermentation, 9.56 g l^–1 cell concentration in the broth was obtained with 20 g l^–1 (178 mM) sodium lactate and 4 g l^–1 NH₄Cl in the medium; and the biotransformation ability was 2.51 units ml^–1. The cells were harvested from one reactor and then used for pyruvate production from lactate in the same reactor. l-lactate at a concentration about 527 mM was almost stoichiometrically converted to pyruvate in 28 h. After a total 42 h of cell culture and biotransformation, the transformative yield was about 0.72 g g^–1 pyruvate from lactate and the rate of pyruvate production was calculated as 1.33 g l^–1 h^–1 during the process. The results suggested this simple enzymatic production of pyruvate from lactate should be a promising process and may bring a yield higher than that by microbial fermentation. By this process, the recovery of pyruvate from such a simple reaction liquid is relatively easy and inexpensive to perform.