Production of statins by filamentous fungi

Several Monascus and Aspergillus strains were screened for statins production. Lovastatin, monacolin J, pravastatin and mevastatin were produced, with higher yields from the A. terreus strains than from Monascus species. Of all the strains investigated M. paxii AM12M, an isolated spontaneous mutant, yielded 127 mg lovastatin/l and 53 mg pravastatin/l at 21 days, and 18 mg pravastatin/l at 16 days employing a whole soybean flour medium; A. terreus BST yielded 230 mg lovastatin/l and 118 mg pravastatin/l at 14 days employing a defatted soybean flour medium. Statins recovery showed that pravastatin was, in both strains, mostly found in both the mycelium and the culture filtrate, while lovastatin remained closely associated (83%) to the A. terreus mycelium or was mainly released into the culture filtrate (64%) of M. paxii culture.     

Production and purification of statins from Aspergillus terreus strains

Lovastatin, mevastatin, pravastatin and monacolin J were produced using Aspergillus terreus strains. Mevastatin (170 mg/l) was obtained at 14 days from the A1 strain, lovastatin (256 mg/l) at 21 days from the A2 strain and pravastatin (270-300 mg/l) at 14 days from both the A1 and A2 strains grown on defatted soybean flour. Similar yields of monacolin J (5-10 mg/l) were detected for both strains. Fermentation carried out by adding glycerol to A1 7-d old cultures gave 244 mg lovastatin/l at 14 days employing whole soybean flour. A new extraction procedure was applied to an A2 19-d old culture on the mycelium and the culture filtrate separately. Recovery yield showed that 83% lovastatin was associated with the mycelium and 17% was free in the culture filtrate. © Rapid Science Ltd. 1998     

Statistical optimization of lovastatin production by Omphalotus olearius (DC.) singer in submerged fermentation

In this study, culture conditions were optimized to improve lovastatin production by Omphalotus olearius, isolate OBCC 2002, using statistical experimental designs. The Plackett–Burman design was used to select important variables affecting lovastatin production. Accordingly, glucose, peptone, and agitation speed were determined as the variables that have influence on lovastatin production. In a further experiment, these variables were optimized with a Box–Behnken design and applied in a submerged process; this resulted in 12.51 mg/L lovastatin production on a medium containing glucose (10 g/L), peptone (5 g/L), thiamine (1 mg/L), and NaCl (0.4 g/L) under static conditions. This level of lovastatin production is eight times higher than that produced under unoptimized media and growth conditions by Omphalotus olearius. To the best of our knowledge, this is the first attempt to optimize submerged fermentation process for lovastatin production by Omphalotus olearius.     

In vitro Clonal Propagation and Organogenesis in Spilanthes acmella (L) Murray: A Herbal Pesticidal Plant of North-East India

Multiple shoots were regenerated in MS medium using different concentrations of BAP and Kn and different combinations of BAP with IAA, NAA and IBA. Highest multiplication of shoots was obtained with BAP (0.75 mg l^?1) with 28.4 shoots per explant after 60 days of culture. Shoots rooted best on IBA (0.5 mg l^?1), numbering 48.8 per explant. Organogenesis was maximum in callus cultured on MS medium supplemented with BAP (2.0 mg l^?1) and IAA (1.0 mg l^?1).     

In vitro plant regeneration from leaf callus of Grindelia robusta Nutt

Abstract

A regeneration protocol from leaf explants of Grindelia robusta Nutt. was developed. The combination of 0.5 mg l^?1 IBA plus 0.5 mg l^?1 or 1 mg l^?1 BA added to Murashige-Skoog (MS) medium resulted in the best callus induction frequency; the combination of 0.4 or 0.9 mg l^?1 BA plus 1.2 mg l^?1 GA₃ resulted in the best shoot regeneration. Rooting was successful on MS medium supplemented with 0.5 mg l^?1 IBA. Hardening of G. robusta plants was accomplished in 30 days with 85% survival rate.     

Substrate concentration dependence of voltage and power production characteristics in two-chambered mediator-less microbial fuel cells with acetate and peptone substrates

Objectives

Power production characteristics and substrate concentration dependence of voltage have been investigated together with the determination of kinetic constants in two-chambered mediator-less microbial fuel cells (MFC) for acetate and peptone substrates.

Results

At 500 mg DOC l^?1 (dissolved organic carbon), power densities normalized to the anode surface of 112 mW m^?2 with acetate and 114 mW m^?2 with peptone as electron donor were attained by applying cathodes with a Pt catalyst layer. Related anode surface specific substrate removal rate was 44 g DOC m^?2 h^?1 for acetate and 52 g DOC m^?2 h^?1 for peptone. Substrate concentration dependency of the voltage suggests Monod-like kinetics with extremely low, <1 mg DOC l^?1, half saturation constants and with final DOC concentrations of 6–10 mg l^?1.

Conclusions

Acetate and peptone are equivalent substrates for the exoelectrogenic bacteria both from the point of view of biodegradation kinetics and power production characteristics.

     

Efficient organogenesis and plantlet regeneration in the timber species <Emphasis Type="Italic">Cunninghamia lanceolata</Emphasis> (Lamb.) Hook

Two efficient regeneration systems were developed in Cunninghamia lanceolata, the most important conifer for industrial wood production in China. Cotyledons and hypocotyls derived from greenhouse-grown seedlings were used as initial explants in our research. A high frequency (95.1?±?1.84%) of adventitious buds were initiated directly from cotyledons cultured on Douglas-fir cotyledon revised (DCR) medium supplemented with 1 mg l^?1 benzyladenine (BA), 0.1 mg l^?1 α-naphthaleneacetic acid (NAA), and 0.004 mg l^?1 thidiazuron (TDZ) with a maximum mean number of adventitious buds per cotyledon explant of 3.76?±?0.08. In contrast, a high percentage (93.73?±?0.55%) of adventitious buds regenerated via callus produced from hypocotyls cultured on DCR medium supplemented with plant growth regulators with a maximum number of adventitious buds per explant (16.71?±?0.34). Adventitious buds elongated on DCR medium supplemented with 0.2 mg l^?1 BA and 0.02 mg l^?1 NAA. After liquid pretreatment with 50 mg l^?1 indole-3-butyric acid (IBA), over 95% of the shoots successfully rooted on ½ DCR medium supplemented with 0.3 mg l^?1 IBA. The innovated systems reported in this study will be useful tools for future genetic manipulation of C. lanceolata and may be adapted for large-scale propagation in other conifers.     

In Vitro Plant Regeneration from Immature Leaflets Derived Callus of Acacia confusa Merr via Organogenesis

An efficient plant regeneration system was established from immature leaflet-derived callus of Acacia confusa Merr, through organogenesis. Under optimized culture conditions, the high rate of callus induction and proliferation was obtained in 35 days on MMS medium supplemented with 2,4-D (3 mg l^?1) + NAA (0.01 mg l^?1) + Kin (0.05 mg l^?1). The highest percentage of shoot regeneration response (95%) and greatest number of shoots (52.9) were obtained after the 46-day transfer of green nodular calli onto the shoot regeneration medium (WPM) supplemented with the BA 3 mg l^?1 + NAA 0.05 mg l^?1 + Zeatin 0.1 mg l^?1 + AdSO₄ 5 mg l^?1 combination. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoot buds to medium (half-strength MS) containing GA, (1 mg l^?1) and BA (0.05 mg l^?1), within 30 days. The elongated shoots were rooted on half-strength MS medium supplemented with 4 mg l^?1 IBA and 0.05 mg l^?1 Kin in the 42-day culture. Rooted plantlets were hardened and successfully established in soil. The field-established plants were morphologically normal and fertile.     

Isolation and identification of a yeast strain capable of degrading four and five ring aromatic hydrocarbons

A yeast strain AEH was isolated from oil contaminated soil and identified by analysis of 18S and 26S ribosomal DNA sequences asPichia anomala. Strain AEH was capable of degrading naphthalene, phenanthrene and chrysene, singly, and benzo(a)pyrene in combination. The yeast degraded 5.36 mg naphthalene l^?1 within 2 days, and 5.04 mg phenanthrene l^?1 and 1.54 mg chrysene 1^?1 within 10 days. When a mixture of the four polycyclic aromatic hydrocarbons (PAHs) was treated at a concentration between 2.98 mg l^?1 and 6.89 mg l^?1, degradation rates were delayed for naphthalene and phenanthrene (3.79 mg l^?1 and, 4.20 mg l^?1 within 10 days, respectively), but enhanced for chrysene and benzo(a)pyrene (3.37 mg l^?1 and, 1.91 mg l^?1 within 10 days, respectively). In a binary system, all of the other 3 PAHs could be utilized as the carbon source for the cometabolic degradation of benzo(a)pyrene with naphthale ne as the best one.     

In Vitro Propagation and Reintroduction of the Endangered Renanthera imschootiana Rolfe

Renanthera imschootiana Rolfe is an endangered tropical epiphytic orchid that is threatened with extinction due to over-collection and the loss of suitable habitats. In vitro propagation is a useful way to mass produce plants for re-establishment in the wild and for commercial propagation. Seeds collected 150 days after pollination (DAP) were the optimum stage for in vitro culture. Seed germination reached 93.1% on quarter-strength MS (i.e., MS containing a quarter of macro- and micronutrients) medium containing 0.5 mg l⁻¹ α-naphthaleneacetic acid (NAA), 20% coconut water (CW), 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 1.0 g l⁻¹ activated charcoal (AC). Quarter-strength MS medium supplemented with 1.0 mg l⁻¹ BA, 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 10 g l⁻¹ sucrose and 20% CW was suitable for the sub-culture of protocorm-like bodies (PLBs) in which the PLB proliferation ratio was 2.88. Quarter-strength MS medium containing 1.0 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 100 g l⁻¹ banana homogenate (BH), and 1.0 g l⁻¹ AC was suitable for plantlet formation and 95.67% of plantlets developed from PLBs within 60 days of culture. Hyponex N016 medium supplemented with 0.5 mg l⁻¹ NAA, 1.0 g l⁻¹ peptone, 20 g l⁻¹ sucrose, 150 g l⁻¹ BH, and 1.0 g l⁻¹ AC was suitable for the in vitro growth of plantlets about 2-cm in height. Plantlets 3-cm in height or taller were transplanted to Chilean sphagnum moss, and 95% of plantlets survived after 60 days in a greenhouse. Three hundred transplanted of seedlings 360-days old were reintroduced into three natural habitats. Highest percentage survival (79.67%) was observed in Yuanjiang Nature Reserve two years after reintroduction, followed by Huolu Mountain forest park (71.33%). This protocol is an efficient means for the large-scale propagation and in vitro and in vivo germplasm conservation of R. imschootiana.     

An Improved Method for In Vitro Large Scale Propagation of Piper betle L

A protocol for large-scale propagation of Piper betle cvs Desawari and Desi Bangla was developed through axillary shoot proliferation. Due to systemic infection as well as high phenol content the crop was very difficult to establish in aseptic condition. But a mixture of 5 mg l^?1 each of chloramphenicol and oxytetracyclin and 100 mg l^?1 each of citric acid and ascorbic acid used in MS medium for 2 days helped in establishment. After 48 h, the explants were transferred to the antibiotic free medium having PVP and ascorbic acid (100 mg l^?1 each), citric acid (50 mg l^?1), and glutathione (20 mg l^?1). Regular subculturing of the explants into liquid medium, use of antioxidants and incubation of the cultures in the dark for initial 7–10 days played a crucial role for keeping them fresh and green. Maximum numbers of axillary shoots were obtained with 2 mg l^?1 BA and 0.2 mg l^?1 NAA as growth supplements. The plants were rooted in 0.25 mg l^?1 IBA and hardened in the soil. Phenolic compound analysis showed almost the same results in tissue-raised and in vivo grown plants in Desawari.     

Induction and Proliferation of Callus Derived from Fruits of Different Varieties of Capsicum annuum

Callus was initiated on Murashige and Skoog (MS) medium containing different combinations of growth regulators or different concentrations of vitamins from pericarp of six varieties of Capsicum annuum differing in their capsaicin content. Callus derived from pericarp of low capsaicin containing varieties was snowy white, friable and showed excellent growth. Callus initiation was delayed (10-15 days) in Punjab Lal, which had very high fruit capsaicin content (7.0 mg g^?1 DW). It also showed relatively slow proliferation although callus obtained was white and friable. Several different media were tried to improve the initiation and the proliferation of the callus in this variety. Callus growth improved greatly by doubling the concentration of MS salts. Initiation time was reduced to 4-6 days by adding 10 mg l^?1 NAA and 0.5 mg l^?1 Kin in MS medium. Other combinations of growth regulators or increase in concentration of vitamins or activated charcoal (0.1%) resulted in poor callus growth and callus texture. Of all media tried, MS medium containing 2 mg l^?1 2,4 D and 0.5 mg l^?1 Kin was the best for callus initiation and proliferation.     

Rapid In Vitro Propagation of Orchid Zygopetalum intermedium

The influence of plant growth substances, medium and potting mixture on protocorm development, differentiation, growth and establishment of Zygopetalum intermedium was assessed. Embryo from mature green but unripe capsule cultured on half strength Murashige and Skoog medium containing 1.5 g l^?1 AC with 0.25 mg l^?1 PBZ and 0.1 mg l^?1 of NAA swollen in 59.7 days, followed by formation of globular bodies in 64 days and protocorm development in 70.7 days. Nitsch medium in combination with 0.5 mg l^?1 of BAP and 0.25 mg l^?1 of Triacontanol resulted in shoot and root differentiation and maximum plant growth in vitro. Plantlets with 4–5 well-developed leaves with roots pre hardened in medium supplemented with 0.25 mg l^?1 each of PBZ and Triacontanol transferred to community pots filled with potting mixture of coco peat and tree fern (1:1) resulted in 72.3% survival ex vitro.     

Evaluation of waste chicken feathers as peptone source for bacterial growth

Aims: Peptones are one of the most expensive constituents of microbial media. This study was undertaken to prepare the peptone from waste chicken feathers through a new process. Methods and Results: The chemical analysis of chicken feather peptone (CFP) was performed. The ability of CFP to support the growth of the three test bacteria in liquid and agar media was comparable to those of three commercial peptones [tryptone peptone (TP), fish peptone and protease peptone (PP)]. Conclusions: CFP was found to be rich in ash (42·1 g 100 g^?1), protein (55·8 g 100 g^?1) and mineral contents. The maximum biomass yield (3·13 g l^?1) and colony number (83 × 10⁸ CFU ml^?1) for bacterium Bacillus subtilis were attained with CFP. The maximum biomass yields and colony numbers for Lactobacillus delbrueckii ssp. bulgaricus and Escherichia coli were reached in TP medium. Second high biomass yield (2·64 g l^?1) and colony number (75 × 10⁸ CFU ml^?1) for E. coli were achieved using CFP. Third high biomass yield (1·29 g l^?1) and colony number (90 × 10⁷ CFU ml^?1) for Lact. delbrueckii ssp. bulgaricus were obtained in CFP medium. Significance and Impact of the Study: Usability of waste chicken feathers as substrate for bacteria was investigated for the first time in the present study. The peptone may be used in industrial fermentations for production of antibiotics, organic acids, enzymes and biopolymer. It may be also used in clinical microbiology. A new chemical process was developed for peptone preparation. This process may be also employed for peptone preparation from other organic materials, especially fibrose protein‐containing materials.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

In Vitro Multiplication of Paedaria foetida L — A Rare Medicinal Plant

Paedaria foetida L, which has great medicinal value is facing danger of extinction. For its conservation, in vitro multiplication may prove one of the best techniques. Micropropagation of P. foetida has been achieved through the culture of nodal explants. The explants produced shoots on MS medium with 0.8% agar. This medium, however, caused high degree of browning of the explants and death of sprouted shoots. Agar free liquid MS medium with polyvinylpyrrolidone (PVP) proved better. Maximum shoot proliferation, free from callus and vitrification but with poor rooting could be obtained in liquid MS medium with PVP (0.8%), NAA (0.5 mg l^?1) and BA (2.0 mg l^?1). The best rooting occurred on semisolid MS medium containing 0.8% agar and 0.5 mg l^?1 IBA.     

Effects of the principal nutrients on lovastatin production by Monascus pilosus

Lovastatin production is dependent on the substrates provided. We investigated how several carbon and nitrogen sources in the medium affect lovastatin production by Monascus pilosus. M. pilosus required a suitable concentration of organic nitrogen peptone for high lovastatin production. As sole carbon source with peptone, although glucose strongly repressed lovastatin production, maltose was responsible for high production. Interestingly, glycerol combined with maltose enhanced lovastatin production, up to 444 mg/l in the most effective case. Moreover, an isolated mutant, in which glucose repression might be relieved, easily produced the highest level of lovastatin, 725 mg/l on glucose-glycerol-peptone medium. These observations indicate that lovastatin production by M. pilosus is regulated by strict glucose repression and that an appropriate release from this repression by optimizing medium composition and/or by a mutation(s) is required for high lovastatin production.     

Micropropagation of Withania coagulans (Stocks) Dunal: A Critically Endangered Medicinal Herb

An efficient micropropagation protocol has been developed for Withania coagulans, a highly endangered medicinal herb and an important natural source of withanolides. Prolific multiplication of axillary buds occurred from the nodal segments taken from adult plant, and cultured on MS medium enriched with BA (0.5 mg l^?1), Kn (0.5 mg l^?1) and PG (0.5 mg l^?1). Nodal segments and shoot tips of elongated microshoots also behaved the same way in cultures and formed multiple shoots through axillary bud multiplication. Addition of PG (0.5 mg l^?1) in the regeneration medium significantly improved induction and elongation of shoot buds. Elongated shoots were placed on filter paper bridges soaked in MS medium with CC (10 mg l^?1) and PG (0.5 mg l^?1) for the initial 7 days’ pulse treatment and thereafter, they were transferred to rooting medium containing IBA (0.25 mg l^?1) + PAA (0.5 mg l^?1) + CC (2 mg l^?1). This protocol has the capacity of producing 1000 plants from one nodal segment after 4 subcultures of 2 weeks each.     

Is an organic nitrogen source needed for cellulase production by Trichoderma reesei Rut-C30?

The effect of organic and inorganic nitrogen sources on Trichoderma reesei Rut-C30 cellulase production was investigated in submerged cultivations. Stirred tank bioreactors and shake flasks, with and without pH control, respectively, were employed. The experimental design involved the addition of individual organic nitrogen sources (soy peptone, glutamate, glycine and alanine) within a basal medium containing Avicel (i.e. micro crystalline cellulose) and ammonium sulphate. It was found that in the shake flask experiments, the highest cellulase activities (~0.1 ± 0.02 FPU ml^?1) were obtained with media containing soy peptone (3–6 g l^?1) and glutamate (3.6 g l^?1). However, these improvements in the cellulase titers in the presence of the organic nitrogen sources appeared to be related to smaller changes in the pH of the medium. This was confirmed using stirred tank bioreactors with pH control. No significant differences were observed in the highest cellulase titers and the protein pattern (according to the SDS-PAGE) of supernatants from pH controlled stirred tank bioreactor cultivations, when different nitrogen sources were used in the medium. Here the cellulase activities (~1.0 ± 0.2 FPU ml^?1) were also much greater (8–150 times) than in shake flask cultivation. Consequently, the addition of ammonium sulphate as sole nitrogen source to Avicel basal medium is recommended when performing cultivations in stirred tank bioreactors with strict pH controlled conditions.     

Thidiazuron-induced Shoot Multiplication and Plant Regeneration in Bamboo (Dendrocalamus strictus Nees)

Thidiazuron (TDZ) stimulated shoot proliferation from different seedling explants (i.e., shoot, basal node, node and apical segment) of bamboo (Dendrocalamus strictus) when incorporated in half-strength Murashige and Skoog (MS) medium having 2% (w/v) sucrose. All the concentrations of TDZ (0.01 to 1.0 mg l^?1) tried were effective in shoot proliferation. Maximum shoots (14.8 ± 1.0) were obtained from the shoot explants cultured in 0.5 mg l^?1 TDZ supplemented halfstrength MS liquid medium for 21 days and subsequently transferred to the same medium devoid of TDZ. The longer culture period (i.e. 28 and 35 days) in TDZ medium caused reduction in shoot proliferation. The shoots regenerated with lower concentrations of TDZ treatment (i.e. 0.01 to 0.1 mg l^?1) rooted in half-strength MS liquid medium. The shoots formed with 0.5 mg l^?1 TDZ treatment did not root in basal medium and required auxin supplementation in the medium for rooting and about 55% shoots produced roots in 1.0 mg l^?1 IBA supplemented medium. The shoots formed with 1.0 mg l^?1 TDZ did not root even after auxin treatment. The well rooted shoots transplanted to plastic pots filled with sand and garden soil (1:1) mixture showed 98% establishment.