Improved productivity of <Emphasis Type="Italic">β</Emphasis>-fructofuranosidase by a derepressed mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> from conventional and non-conventional substrates

Summary Wild-type cultures of Aspergillus niger produced a basal level of β-fructofuranosidase on glucose of 1 IU l⁻¹ h⁻¹. In contrast, a catabolite-derepressed mutant strain of the same organism produced a markedly higher level (25 IU l⁻¹ h⁻¹) of this enzyme when grown on the same carbon source. Wheat bran induced both the wild type (252 IU l⁻¹ h⁻¹) and the mutant strain (516 IU l⁻¹ h⁻¹) to produce 252- to 516-fold higher levels of this enzyme than was observed with the wild-type grown on glucose and was the best carbon source. When corn steep liquor served as a nitrogen source, the wild-type organism showed a higher activity of enzyme on monosaccharides and disaccharides comparable to that produced by corncobs in the basal medium and that mutant was a potentially improved (> 2-fold) organism for the production of β-fructofuranosidase on all carbon sources. Enhanced substrate consumption and product formation kinetic parameters suggest that the mutant organism may be exploited for bulk production of this useful enzyme.     

Production of phenylacetylcarbinol in various yeast species

Summary Production of phenylacetylcarbinol (PAC) was measured in various yeast species. The yeast strains tested were cultivated under submerged conditions in a medium containing corn steep and sucrose as the main components; sucrose, acetaldehyde and benzaldehyde were added to the grown cultures. In a first series of experiments the initial rate of PAC production, i.e. the PAC production determined 30 min after the addition of benzaldehyde was determined in 38 yeast strains, mostly of the generaSaccharomyces andCandida. The amount of PAC produced varied from zero (12 strains) to 1.24 mg ml^–1. In a second series of experiments, 15 strains, which in the first series had shown a higher PAC production, were further tested. Sucrose, acetaldehyde and benzaldehyde were added to the cultures until the PAC production ceased. The highest PAC production (6.3 mg ml^–1) was reached in the strainSaccharomyces carlsbergensis Budvar; the production was slightly lower in 4 strains of the generaSaccharomyces, Candida andHansenula.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Production of mannitol by a novel strain of Candida magnoliae

A novel microorganism was isolated which is able to produce mannitol when grown in the presence of fructose and glucose as carbon sources. In flask culture in a medium containing 150 g fructose l^–1, it yielded 67 g mannitol l^–1 after 168 h. In fed-batch culture with 3–12% (w/v) fructose, production reached a maximum of 209 g mannitol l^–1 after 200 h, corresponding to an 83% yield and a 1.03 g l^–1 h^–1 productivity. The isolated strain was identified as Candida magnoliae based on identical sequences in the D1/D2 domain of its 26S rDNA and a similar carbon source utilization pattern with C. magnoliae reference strains.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Attempts to produce solasodine in callus and suspension cultures of Solanum mauritianum Scop.

Callus cultures of Solanum mauritianum Scop. were initiated from green berry explants on a hormone-free Murashige and Skoog (1962) medium excluding glycine, and containing 0.1 g L^–1 myo-inositol and 3% sucrose. Such cultures contained 10.08±0.59 g g^–1 DW of solasodine, which is equivalent to that in the leaves of mature S. mauritianum plants, but far less than that extracted from the green berries (185 g g^–1 DW). In vitro solasodine productivity could be increased by reducing the strength of the medium by half, substituting 3% glucose for 3% sucrose as carbon source, or by the addition of certain combinations of BA and NAA. Phosphate limitation and alterations in the carbon: nitrogen ratio were not able to increase solasodine productivity. Suspension cultures of S. mauritianum were initiated and maintained in a Murashige and Skoog (1962) medium with the RT vitamins of Khanna and Staba (1968), 0.1 g L^–1 myo-inositol, 3% sucrose and 1 mg L^–1 2,4-D. No solasodine was detectable in these cultures, or slight modifications thereof.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog's (1962) medium     

In vitro multiplication and microrhizome induction in Curcuma aromatica Salisb.

Shoot multiplication and plant regeneration was achieved from freshly sprouted shoots of Curcuma aromatica on Murashige and Skoog's medium supplemented with BA alone (1–7 mgL^–1) or a combination of BA(1–5 mgL^–1) and Kn (0.5–1 mgL^–1). A concentration of 5 mgL^–1 BA was optimum for shoot multiplication and rooting of shoots. The regenerated plants grew profusely on transfer to liquid medium.In vitro raised plants were successfully established in the field. Microrhizomes were induced at the base of the in vitro derived shoots upon transfer to medium containingvarious combinations and concentrations of sucrose and BA and grown under varying photoperiods. MS basal medium with 5 mgL^–1 BA, 60 gL^–1 sucrose and an8 h photoperiod was optimum for induction ofmicrorhizomes within 30 days of culture. Harvestedmicrorhizomes stored in moist sand in poly-bagssprouted after 2 months of storage at roomtemperature. For in vitro storage, microrhizomeswere grown in medium containing 0.1 mgL^–1 BA.Microrhizome formation was found to be controlled bythe concentrations of BA and sucrose as well asphotoperiod during culture.     

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 g L^–1 day^–1 after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 g L^–1 day^–1, a productivity of 5 g L^–1 day^–1 was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.     

Development of a fermentation process for production of an alginate G-lyase from Klebsiella pneumoniae

A high-density-cell fermentation process for production of an exracellular alginat lyase from Klebseilla pneumoniae on a defined medium has been developed. The process employs a strategy using two carbon sources. One low-molecular-mass, low-viscosity carbon source (sucrose) with high water solubililty is used as the main carbons source for growth, while the high-molecular-mass and viscoous alginate in low concentration is used as an inducer for enzyme synthesis. The repression of algiante lyase production by sucrose and the growth inhibition that we observed at increased levels of ammonia were circumvented by a computer-assisted fed-batch addition of the carbon sources (succrose and alginate) and by supplying nitrogen source as ammonia in the pH control. No enzyme production was observed when dissolved oxygen limited growth at an oxygen uptake rate of 40%–50% of the maximum uptake rate. An optimal composition of the feeding solution (12.5 g alginate and 587.5 g sucrose 1^–1) was found both for the maximum final concentration of enzyme (1330 U 1^–1) and for the maximum volumetric rate of enzyme production (67 U 1^–1 h^–1). The enzyme production dependes of the growth rate in the linear growth phase, giving a maximum enzyme concentration at the highest growth rate tested. The final enzyme concentration shows a fiveflod increase compare with previously reproted daata where alginate was used as a carbon source. In addition, the ratio of alginate lyase by a factor of apporximately 15. A doubling in extracellular specific activity of the enzyme was observed, a property of significant interest, especially for purification of the enzyme. On the othr hand, the final dry cell weight concentration of the bacteria also increased by a factor of 15–20 thus giving a relatively lower specific productivity of 0.4 U (g cell dry weight)^–1 h^–1.     

Influence of dilution rate on the morphology and technological properties ofLactobacillus delbrueckii subsp.bulgaricus

Lactobacillus delbrueckii subsp.bulgaricus ATCC 11842 was grown in a chemostat at 45°C and pH 5.5 using glucose as the carbon source, with the aim of optimizing biomass production. Cells were grown in a complex medium under nitrogen. At dilution rates lower than 0.18h^–1, it was difficult to keep steady-state conditions and pleomorphic forms were observed. The addition of 30mM Ca²⁺ and Mn²⁺ reverted the cells to normal shape: 30mM Mg²⁺ had no effect. Increasing the dilution rate resulted in normal morphology without the addition of any cations. Under these conditions, a maximum productivity of 1.24g dry biomass 1^–1 h^–1 was obtained. The maximum growth yield, corrected for maintenance, was 30g biomass mol^–1 glucose and the maintenance energy was 0.26g glucose g^–1 biomass h^–1. Lactate was the main fermentation product at all glucose concentrations used in the fed medium. Cells grown at high dilution rates had normal technological properties (acid production and proteolysis) when tested in milk.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Enhanced production of pigment-free pullulan by a morphogenetically arrested Aureobasidium pullulans (ATCC 42023) in a two-stage fermentation with shift from soy bean oil to sucrose

A two-stage fermentation process was established for the production of pigment-free pullulan by the yeast-like fungus Aureobasidium pullulans (ATCC 42023). In the first stage, starting at pH 4.5 with soy bean oil as the carbon source and glutamate as the nitrogen source, a cell mass of about 15 g l^–1 dry cell weight was obtained, the population being restricted mainly to the yeast form of the microorganism (yeast form more than 90% of total cells) and the formation of pigment in the culture being prevented. Small amounts of pullulan (less than 2 g l^–1) are produced at this phase, and the viscosity remained low throughout the entire growth stage. When the oil and glutamate source were nearly exhausted (below 5% of initial amounts), the cells were shifted to a production stage with sucrose as the carbon source with continued nitrogen depletion. Production of pullulan started immediately with no lag period. During 50 h of the production phase more than 35 g l^–1 of pullulan was produced (productivity approx. 0.7 g l^–1), resulting in a large increase in the viscosity of the broth. The production yield of pollulan on the sugar was about 0.6 g g^–1. Morphogenesis from the yeast form of the microorganism to chlamydospores was still restrained and no pigment was formed in the culture during the production stage. A pigment-free polysaccharide, with a molecular mass in the range of 600–750 kDa, was recovered from the supernatant of the broth after solvent precipitation.     

Photoautotrophic micropropagation of Russet Burbank Potato

The photoautotrophic micropropagation of potato cv. Russet Burbank was investigated. Single node microcuttings were grown for four weeks on Murashige and Skoog (MS) medium with or without sucrose (30 g l^–1) in the growth room at 21/19 °C day/night temperature, with 16-h photoperiod at 150 mol m^–2 s^–1, with or without supplemental CO₂ at 1500 l l^–1. A 20% increase in the number of nodes per stem (from 7.5 to 9.4) and a 50% increase in stem dry weight were observed in cultures grown on media with sucrose and in CO₂ enriched atmosphere comparing to the conventionally micropropagated cultures or the cultures grown photoautotrophically on media without sucrose but in air supplemented with 1500 l l^–1CO₂. Stems of these cultures (from media with sucrose in CO₂ enriched air) almost doubled in length the stems of cultures from the other two treatments. No significant differences were observed between Control (MS medium supplemented with sucrose, 30 g l^–1) and photoautotrophic cultures coming from MS medium with no sucrose grown under 1500 l l^–1 of CO₂. Photoautotrophic cultures produced stems averaging 43.3 mm, with 7 nodes and weighing 9.2 mg (dry weight), similar to conventionally grown in vitro cultures (47.9 mm with 7.5 nodes, 9.7 mg dry weight). Growers may consider photoautotrophic culturing of potato in areas where the high sterility levels are difficult to maintain. Supplementing air in the growth room with 1500 l l^–1 of CO₂ could be beneficial for potato plantlet production even on media containing sucrose since it significantly improved quality, size and biomass of produced plantlets, speeding up the multiplication.     

Different nitrogen sources and growth responses of Spirulina platensis in microenvironments

Spirulina platensis was cultivated, in comparative studies, using several sources of nitrogen. The standard source used (sodium nitrate) was the same as that used in the synthetic medium Zarrouk, whereas the alternative nitrogen sources consisted of ammonium nitrate, urea, ammonium chloride, ammonium sulphate or acid ammonium phosphate. The initial nitrogen concentrations tested were 0.01, 0.03 and 0.05 M in an aerated photobioreactor at 30 °C, with an illuminance of 1900 lux, and 12 h-light/12 h-dark photoperiod over a period of 672 h. Maximum biomass was produced in medium containing sodium nitrate (0.01–0.03–0.05 M), followed by ammonium nitrate (0.01 M) and urea (0.01 M). The final biomass concentrations were 1.992 g l^–1 (0.03 M sodium nitrate), 1.628 g l^–1 (0.05 M sodium nitrate), 1.559 g l^–1 (0.01 M sodium nitrate), 0.993 g l^–1 (0.01 M ammonium nitrate) and 0.910 g l^–1 (0.01 M urea). This suggested that it is possible to utilize nitrogen sources other than sodium nitrate for growing S. platensis, in order to decrease the production costs of scaled up projects.     

Production of eleutherosides in in vitro regenerated embryos and plantlets of <Emphasis Type="BoldItalic">Eleutherococcus chiisanensis</Emphasis>

High frequency somatic embryogenesis of Eleutheorcoccus chiisanensis was achieved through suspension culture of embryogenic cells in hormone-free Murashige and Skoog liquid medium supplemented with 30 g sucrose l⁻¹. Cotyledonary somatic embryos were germinated and converted into plantlets using 20 μM gibberellic acid which were then grown in a 10 l airlift bioreactor. HPLC analysis revealed the accumulation of eleutheroside B, E and E1 in the embryos and plantlets. Thus mass production of embryos and plantlets of E. chiisanensis can be achieved in liquid cultures and the biomass produced may become an alternative source of eleutherosides.     

Overproduction of pyoverdins by Fur mutants of Pseudomonas aeruginosa strains PAO1 and Fe10 in stirred bioreactors

Fur mutants FPA12 and FF13 of strains Pseudomonas aeruginosa PAO1 and Fe10, respectively, were prepared and their production of pyoverdin evaluated. The strains were cultivated in stirred bioreactor in iron-deficient and iron-supplemented medium containing Casamino acids (CA) or succinate as a source of carbon and energy. When the pyoverdin production rate reached its maximum, the demand of iron-depleted cultures for O₂ was decreased. Mutant FF13 overproduced pyoverdin in both iron-depleted (862 mg l^–1) and iron-supplemented (428 mg l^–1) CA medium and could also be used to produce pyoverdin when grown in a conventional stirred tank fermenter.     

Production of cyclohexanone monooxygenase from Acinetobacter calcoaceticus for large scale Baeyer–Villiger monooxygenase reactions

Cyclohexanone monooxygenase was produced from Acinetobacter calcoaceticus grown on a medium containing both glutamate (30 g l^–1) and cyclohexanol (1 g l^–1). Productivity was increased to 650 U l^–1, an order of magnitude greater than previous production methods, thereby enhancing the potential commercial utility of this enzyme.     

Preparation and testing of Sardinella protein hydrolysates as nitrogen source for extracellular lipase production by Rhizopus oryzae

Various fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were used as nitrogen sources for the production of extracellular lipase by the filamentous fungus Rhizopus oryzae. The best results were obtained with defatted meat–fish protein hydrolysates (DMFPH), indicating the presence in the lipid fraction of some constituents which may repress lipase synthesis. Furthermore, it was found that the extensive hydrolysis of fish proteins resulted in a higher lipase production. The use of 40 g DMFPH l^–1 for the growth of Rhizopus oryzae in medium R1 resulted in a lipase production of 394 U ml^–1, higher than the yield obtained with standard soy peptone as nitrogen source (373 U ml^–1). The most appropriate medium for the growth and the production of lipase is composed only of 24 g DMFPH l^–1 and 10 g glucose l^–1, indicating that the strain can obtain its nitrogen and salts requirements directly from fish substrate.     

Regulation of biosynthesis of bacilysin byBacillus subtilis

Summary Production of the dipeptide antibiotic bacilysin byBacillus subtilis 168 was growth associated and showed no evidence of repression by glucose or sucrose. Carbohydrates other than glucose and sucrose yielded lower specific titers of bacilysin. Bacilysin production in three such carbon sources (maltose, xylose, ribose) was delayed until growth slowed down. Ammonium salts were poor for bacilysin production when used as the sole nitrogen source. When added to the standard medium containing glutamate, they suppressed antibiotic production. Aspartate was slightly better than glutamate for antibiotic production as sole nitrogen source. No other nitrogen source tested, including inorganic, organic or complex, approached the activity of glutamate or aspartate. When added to glutamate, casamino acids, phenylalanine and alanine (a substrate of bacilysin synthetase) suppressed bacilysin production while stimulating growth. Phosphate provided for optimum growth and production at 7.5 mM and both processes were inhibited at higher concentrations. Ferric citrate stimulated growth and inhibited bacilysin production, the effects being due to both the iron and the citrate components. Elimination of ferric citrate stimulated production as did increasing the concentration of Mn to its optimum concentration of 6.6×10^–4M.