Growth, morphogenesis, and essential oil production in Mentha spicata L. plantlets in vitro

To date, plantlet culture has not been explored as a means to obtain secondary metabolites in vitro. However, plantlets readily produce desirable secondary metabolites, which may not be produced in cell suspension or callus cultures. To optimize plantlet growth in vitro, the influences of various physical environments on the growth (fresh weight), morphogenesis (leaf, root, and shoot number), and volatile carbon metabolites (i.e. monoterpene, (−)-carvone) of Mentha spicata L. (spearmint) plants were studied. The carvone content in different portions of sterile plantlets was analyzed. Carvone was only produced from the foliar regions of cultured plantlets and was absent in the callus and roots. The influence of physical support (e.g., agar, glass gravel, liquid, platform or sponge), frequency of media replacement, and culture vessel capacity on spearmint plantlets growth and carvone production was tested. A comparative study was conducted testing the growth, morphogenesis, and secondary metabolism occurring with three different spearmint cultivars grown in either culture tubes containing 25 ml agar medium or in an automated plant culture system (APCS; a sterile hydroponics system) employing a 1-l medium reservoir. Increasing the number of media immersions (4, 8, 12 or 16 immersions d⁻¹) of plantlets growing in the APCS increased growth and morphogenesis responses. Generally, higher culture growth rates resulted in lower carvone treatment⁻¹ (mg carvone g-FW⁻¹); however, overall total carvone ((mg carvone g-FW⁻¹) × g culture FW) increased because of the production of greater biomass obtained per vessel.     

Residual phosphate concentration under nitrogen-limiting conditions regulates curdlan production in Agrobacterium species

We investigated the influence of inorganic phosphate concentration on the production of curdlan by Agrobacterium species. A two-step culture method was employed where cells were first cultured, followed by curdlan production under nitrogen-limiting conditions. In the curdlan production step, cells did not grow but metabolized sugar into curdlan. Shake-flask experiments showed that the optimal phosphate concentration for curdlan production was in the range of 0.1–0.3 g l⁻¹. As the cell concentration increased from 0.42 to 1.68 g l⁻¹ in shake-flask cultures, curdlan production increased from 0.44 to 2.80 g l⁻¹. However, the optimal phosphate concentration range was not dependent upon cell concentration. The specific production rate was about 70 mg curdlan g-cell⁻¹ h⁻¹ irrespective of cell concentration. When the phosphate concentration was maintained at 0.5 g l⁻¹ under nitrogen-limiting conditions, as high as 65 g l⁻¹ of curdlan was obtained in 120 h. Journal of Industrial Microbiology & Biotechnology (2000) 25, 180–183. Received 25 October 1999/ Accepted in revised form 21 July 2000     

Desulfurization of dibenzothiophene by Bacillus subtilis recombinants carrying dszABC and dszD genes

The desulfurization (dsz) genes from Rhodococcus erythropolis DS-3 were successfully integrated into the chromosomes of Bacillus subtilis ATCC 21332 and UV1 using an integration vector pDGSDN, yielding two recombinant strains, B. subtilis M29 and M28 in which the integrated dsz genes were expressed efficiently under the promoter, Pspac. The dibenzothiophene (DBT) desulfurization efficiency of M29 was 16.2 mg DBT l⁻¹ h⁻¹ at 36 h, significantly higher than that of R. erythropolis DS−3 and B. subtilis M28 and also showed no product inhibition. The interfacial tension of the supernatant fermented by M29 varied from 48 mN m⁻¹ to 4.2 mN m⁻¹, lower than that of the recombinant strain, M28, reveals that the biosurfactant secreted from M29 may have an important function in the DBT desulfurization process.     

Optimizing conditions for poly(β-hydroxybutyrate) production by <Emphasis Type="Italic">Halomonas boliviensis</Emphasis> LC1 in batch culture with sucrose as carbon source

Halomonas boliviensis LC1 is able to accumulate poly(β-hydroxybutyrate) (PHB) under conditions of excess carbon source and depletion of essential nutrients. This study was aimed at an efficient production of PHB by growing H. boliviensis to high cell concentrations in batch cultures. The effect of ammonium, phosphate, and yeast extract concentrations on cell concentration [cell dry weight (CDW)] and PHB content of H. boliviensis cultured in shake flasks was assayed using a factorial design. High concentrations of these nutrients led to increments in cell growth but reduced the PHB content to some extent. Cultivations of H. boliviensis under controlled conditions in a fermentor using 1.5% (w/v) yeast extract as N source, and intermittent addition of sucrose to provide excess C source, resulted in a polymer accumulation of 44 wt.% and 12 g l⁻¹ CDW after 24 h of cultivation. Batch cultures in a fermentor with initial concentrations of 2.5% (w/v) sucrose and 1.5% (w/v) yeast extract, and with induced oxygen limitation, resulted in an optimum PHB accumulation, PHB concentration and CDW of 54 wt.%, 7.7 g l⁻¹ and 14 g l⁻¹, respectively, after 19 h of cultivation. The addition of casaminoacids in the medium increased the CDW to 14.4 g l⁻¹ in 17 h but reduced the PHB content in the cells to 52 wt.%.     

Long-term production of soluble human Fas ligand through immobilization of <Emphasis Type="Italic">Dictyostelium discoideum</Emphasis> in a fibrous bed bioreactor

The production of recombinant glycoproteins in Dictyostelium discoideum by conventional cell culture methods was limited by low cell density as well as low growth rate. In this work, cotton towel with a good adsorption capability for D. discoideum cells was used as the immobilization matrix in an external fibrous bed bioreactor (FBB) system. With batch cultures in the FBB, the concentration of immobilized cells in the cotton fiber carrier increased to 1.37 × 10⁸ cells per milliliter after 110-h cultivation, which was about tenfold higher than the maximal cell density in the conventional free-cell culture. Correspondingly, a high concentration of soluble human Fas ligand (hFasL; 173.7 μg l⁻¹) was achieved with a high productivity (23 μg l⁻¹ h⁻¹). The FBB system also maintained a high density of viable cells for hFasL production during repeated-batch cultures, achieving a productivity of 9∼10 μg l⁻¹ h⁻¹ in all three batches studied during 15 days. The repeated-batch culture using immobilized cells of D. discoideum in the FBB system thus provides a good method for long-term and high-level production of hFasL.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

An improved screening method for microorganisms able to convert crude glycerol to 1,3-propanediol and to tolerate high product concentrations

A new screening method was developed and established to find high-performance bacteria for the conversion of crude glycerol to 1,3-propanediol. Three soil samples from palm oil-rich habitats were investigated using crude glycerol of a German biodiesel plant. Nine promising 1,3-propanediol producers could be found. Because of a special pH buffer system, a fast evaluation on microscale and high 1,3-propanediol concentrations up to 40 g L⁻¹ could be achieved. Three strains demonstrated very high product tolerance and were identified as Clostridium butyricum. Two strains, AKR91b and AKR102a, grew and produced 1,3-propanediol in the presence of 60 g L⁻¹ initial 1,3-propanediol, the strain AKR92a even in the presence of 77 g L⁻¹ 1,3-propanediol. The strains AKR91b and AKR102a tolerated up to 150 g L⁻¹ crude glycerol and produced 80% of the 1,3-propanediol attained from pure glycerol of the same concentration. Further criteria for the choice of a production strain were the pathogenicity (risk class), ability to grow on low-cost media, e.g., with less yeast extract, and robustness, e.g., process stability after several bioconversions. Overall, the strain C. butyricum AKR102a was chosen for further process optimization and scale-up due to its high productivity and high final concentration in a pH-regulated bioreactor.     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Improved biodesulfurization of hydrodesulfurized diesel oil using Rhodococcus erythropolis and Gordonia sp.

Substituted benzothiophenes (BTs) and dibenzothiophenes (DBTs) remain in diesel oil following conventional desulfurization by hydrodesulfurization. A mixture of washed cells (13.6 g dry cell wt l⁻¹) of Rhodococcus erythropolis DS-3 and Gordonia sp. C-6 were employed to desulfurize hydrodesulfurized diesel oil; its sulfur content was reduced from 1.26 g l⁻¹ to 180 mg l⁻¹, approx 86% (w/w) of the total sulfur was removed from diesel oil after three cycles of biodesulfurization. The average desulfurization rate was 0.22 mg sulfur (g dry cell wt)⁻¹ h⁻¹. A bacterial mixture is therefore efficient for the practical biodesulfurization of diesel oil.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Optimization of nutrient components for enhanced phenazine-1-carboxylic acid production by <Emphasis Type="Italic">gacA</Emphasis>-inactivated <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18G using response surface method

The nutritional requirements for phenazine-1-carboxylic acid (PCA) production using Pseudomonas sp. M18G, a gacA chromosomal-inactivated mutant of the strain M18, with a high PCA yield, were optimized statistically in shake flask experiments. Based on a single-factor experiment design, we implemented the two-level Plackett–Burman (PB) design with 11 variables to screen medium components that significantly influence PCA production. Soybean meal, glucose, soy peptone, and ethanol were identified as the most important significant factors (P < 0.05). Response surface methodology based on the Center Composite Design (CCD) was applied to determine these factors’ optimal levels and their mutual interactions between components for PCA production. The predicted results showed that 1.89 g l⁻¹ of PCA production was obtained after a 60-h fermentation period, with optimal concentrations of soybean meal powder (33.4 g l⁻¹), glucose (12.7 g l⁻¹), soy peptone (10.9 g l⁻¹), and ethanol (13.8 ml l⁻¹) in the flask fermentations. The validity of the model developed was verified, and the optimum medium led to a maximum PCA concentration of 2.0 g l⁻¹, a nearly threefold increase compared to that in the basal medium. Furthermore, the experiment was scaled up in the 10 l fermentor and 2 g l⁻¹ PCA productions were achieved in 48 h based on optimization mediums which further verified the practicability of this optimum strategy.     

Towards a high-yield bioconversion of ferulic acid to vanillin

Natural vanillin is of high interest in the flavor market. Microbial routes to vanillin have so far not been economical as the medium concentrations achieved have been well below 1 g l⁻¹. We have now screened microbial isolates from nature and known strains for their ability to convert eugenol or ferulic acid into vanillin. Ferulic acid, in contrast to the rather toxic eugenol, was found to be an excellent precursor for the conversion to vanillin, as doses of several g l⁻¹ could be fed. One of the isolated microbes, later identified as Pseudomonas putida, very efficiently converted ferulic acid to vanillic acid. As vanillin was oxidized faster than ferulic acid, accumulation of vanillin as an intermediate was not observed. A completely different metabolic flux was observed with Streptomyces setonii. During the metabolism of ferulic acid, this strain accumulated vanillic acid only to a level of around 200 mg l⁻¹ and then started to accumulate vanillin as the principal metabolic overflow product. In shake-flask experiments, vanillin concentrations of up to 6.4 g l⁻¹ were achieved with a molar yield of 68%. This high level now forms the basis for an economical microbial production of vanillin that can be used for flavoring purposes. Received: 15 October 1998 / Received revision: 13 January 1999 / Accepted: 18 January 1999     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g␣wet␣weight cells)⁻¹ day⁻¹ was too low to be practical. C.␣famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)⁻¹ day⁻¹]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations. Received: 8 July 1996 / Received revision: 9 September 1996 / Accepted: 18 September 1996     

Production of curdlan using sucrose or sugar cane molasses by two-step fed-batch cultivation of Agrobacterium species

Maltose and sucrose were efficient carbon sources for the production of curdlan by a strain of Agrobacterium sp. A two-step, fed-batch operation was designed in which biomass was first produced, followed by curdlan production which was stimulated by nitrogen limitation. There exists an optimal timing for nitrogen limitation for curdlan production in the two-step, fed-batch operation. Maximum curdlan production (60 g L⁻¹) was obtained from sucrose with a productivity of 0.2 g L⁻¹ h⁻¹ when nitrogen was limited at a cell concentration of 16.0 g L⁻¹. It was also noted that the curdlan yield from sucrose was as high as 0.45 g curdlan g⁻¹ sucrose, and the highest specific production rate was 1.0 g curdlan g⁻¹ cells h⁻¹ right after nitrogen limitation. Of particular importance was the use of molasses as a cheap carbon source to produce curdlan in the two-step, fed-batch cultivation. As high as 42 g L⁻¹ of curdlan with a yield of 0.35 g curdlan g⁻¹ total sugar was obtained after 120 h of fed-batch cultivation. Received 20 August 1996/ Accepted in revised form 26 November 1996     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration

The effect of glucose concentration on erythritol production by Torula sp. was investigated. The maximum volumetric productivity of erythritol was obtained at an initial glucose concentration of 300 g l⁻¹ in batch culture. The volumetric productivity was maximal at a controlled glucose concentration of 225 g l⁻¹, reducing the lag time of the erythritol production. A fed-batch culture was established with an initial glucose concentration of 300 g l⁻¹ and with a controlled glucose concentration of 225 g l⁻¹ in medium containing phytic acid as a phosphate source. In this fed-batch culture, a final erythritol production of 192 g l⁻¹ was obtained from 400 g l⁻¹ glucose in 88 h. This corresponded to a volumetric productivity of 2.26 g l⁻¹ h⁻¹ and a 48% yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 248–252. Received 26 September 2000/ Accepted in revised form 16 January 2001     

Extensive desulfurization of diesel by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The resting cells of a new isolate of Rhodococcus erythropolis FSD-2 were used to desulfurize diesel fuels. About 97% of the total sulfur content in the hydrodesulfurized diesel was removed by the two consecutive biodesulfurization (BDS) processes with the majority (∼94%) being removed in the first treatment, resulting in diesel with a sulfur content of 5.7 μg ml⁻¹.     

Production of lipase by high cell density fed-batch culture of Candida cylindracea

Candida cylindracea NRRL Y-17506 was grown to produce extracellular lipase from oleic acid as a carbon source. Through flask cultures, it was found that the optimum initial oleic acid concentration for cell growth was 20 g l⁻¹. However, high initial concentrations of oleic acid up to 50 g l⁻¹ were not inhibitory. The highest extracellular lipase activity obtained in flask culture was 3.0 U ml⁻¹ after 48 h with 5 g l⁻¹ of initial oleic acid concentration. Fed-batch cultures (intermittent and stepwise feeding) were carried out to improve cell concentration and lipase activity. For the intermittent feeding fed-batch culture, the final cell concentration was 52 g l⁻¹ and the extracellular lipase activity was 6.3 U ml⁻¹ at 138.5 h. Stepwise feeding fed-batch cultures were carried out to simulate an exponential feeding and to investigate the effects of specific growth rate (0.02, 0.04 and 0.08 h⁻¹) on cell growth and lipase production. The highest final cell concentration obtained was 90 g l⁻¹ when the set point of specific growth rate (μ_set) was 0.02 h⁻¹. High specific growth rate (0.04 and 0.08 h⁻¹) decreased extracellular lipase production in the later part of fed-batch cultures due to build-up of the oleic acid oversupplied. The highest extracellular lipase activity was 23.7 U ml⁻¹ when μ_set was 0.02 h⁻¹, while the highest lipase productivity was 0.31 U ml⁻¹ h⁻¹ at μ_set of 0.08 h⁻¹.     

Improvement in lactic acid production from starch using α-amylase-secreting <Emphasis Type="Italic">Lactococcus lactis</Emphasis> cells adapted to maltose or starch

To achieve direct and efficient lactic acid production from starch, a genetically modified Lactococcus lactis IL 1403 secreting α-amylase, which was obtained from Streptococcus bovis 148, was constructed. Using this strain, the fermentation of soluble starch was achieved, although its rate was far from efficient (0.09 g l⁻¹ h⁻¹ lactate). High-performance liquid chromatography revealed that maltose accumulated during fermentation, and this was thought to lead to inefficient fermentation. To accelerate maltose consumption, starch fermentation was examined using L. lactis cells adapted to maltose instead of glucose. This led to a decrease in the amount of maltose accumulation in the culture, and, as a result, a more rapid fermentation was accomplished (1.31 g l⁻¹ h⁻¹ lactate). Maximum volumetric lactate productivity was further increased (1.57 g l⁻¹ h⁻¹ lactate) using cells adapted to starch, and a high yield of lactate (0.89 g of lactate per gram of consumed sugar) of high optical purity (99.2% of l-lactate) was achieved. In this study, we propose a new approach to lactate production by α-amylase-secreting L. lactis that allows efficient fermentation from starch using cells adapted to maltose or starch before fermentation.