Net nitrogen mineralization in soils under six indigenous tree species,an abandoned pasture and a secondary forest in the atlantic lowlands of costa rica

Nitrogen mineralization, nitrification potentials, pH, total N, C, extractable P and cations were measured in soils under 4-year-old, mono-specific stands of six fast-growing, native tree species, an abandoned pasture, and a 20-year-old secondary forest, as part of a study on the use of indigenous tree species for rehabilitation of soil fertility on degraded pastures at the La Selva Biological Station in the Atlantic humid lowlands of Costa Rica. Soil net nitrification potential rates were higher under two N-fixing, leguminous species,Stryphnodendron microstachyum Poepp. et Endl. (1.1–1.9 mg kg^–1 day^–1) andDalbergia tucurensis Donn. Smith (0.7–1.5 mg kg^–1 day^–1), than under the non-N-fixing trees in the plantation,Vochysia guatemalesis Don. Sm.,Vochysia ferruginea Mart,Dipteryx panamensis (Pittier) Record and Mell andHyeronima alchorneoides Fr. Allemao (0.2–0.8 mg kg^–1 day^–1). Values under the N-fixing trees were comparable to those found in secondary forest. There were no statistically significant differences in soil total N or in other nurtients between the species. Results of pH measurements done before and after incubation did not show any clear evidence of a pH drop attributable to nitrification.     

Phylogenetic analysis of the <Emphasis Type="Italic">lux</Emphasis> operon distinguishes two evolutionarily distinct clades of <Emphasis Type="Italic">Photobacterium leiognathi</Emphasis>

The luminous marine bacterium Photobacterium mandapamensis was synonymized several years ago with Photobacterium leiognathi based on a high degree of phenotypic and genetic similarity. To test the possibility that P. leiognathi as now formulated, however, actually contains two distinct bacterial groups reflecting the earlier identification of P. mandapamensis and P. leiognathi as separate species, we compared P. leiognathi strains isolated from light-organ symbiosis with leiognathid fishes (i.e., ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1) with strains from seawater originally described as P. mandapamensis and later synonymized as P. leiognathi (i.e., ATCC 27561^T and ATCC 33981) and certain strains initially identified as P. leiognathi (i.e., PL-721, PL-741, 554). Analysis of the 16S rRNA and gyrB genes did not resolve distinct clades, affirming a close relationship among these strains. However, strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 were found to bear a luxF gene in the lux operon (luxABFE), whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 lack this gene (luxABE). Phylogenetic analysis of the luxAB(F)E region confirmed this distinction. Furthermore, ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554 all produced a higher level of luminescence on high-salt medium, as previously described for PL-721, whereas ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1 all produced a higher level of luminescence on low-salt medium, a characteristic of P. leiognathi from leiognathid fish light organs. These results demonstrate that P. leiognathi contains two evolutionarily and phenotypically distinct clades, P. leiognathi subsp. leiognathi (strains ATCC 25521^T, ATCC 25587, lequu.1.1 and lleuc.1.1), and P. leiognathi subsp. mandapamensis (strains ATCC 27561^T, ATCC 33981, PL-721, PL-741 and 554).Electronic Supplementary Material Supplementary material is available for this article at .     

Physiological characterization of starch-utilizing <Emphasis Type="Italic">Saccharomyces</Emphasis> sp. SK0704 isolated from kimchi

Saccharomyces sp. SK0704 (further defined as SK0704) isolated from long-term-ripening kimchi was identified by a biochemical method with an API kit; its physiology was found to be very similar to that of S. cerevisiae ATCC 26603 (further defined as ATCC 26603), except in terms of starch utilization. SK0704 did not excrete extracellular glucoamylase, but utilized starch as a sole carbon source under only aerobic conditions. Crude enzyme excreted from SK0704 catalyzed the saccharification of starch to glucose, but ATCC 26603 did not. The PCR product obtained using the chromosomal DNA of SK0704 and the primers designed on the basis of the extracellular glucoamylase-coding gene of S. diastaticus was homologous with the intracellular sporulation-specific glucoamylase of S. cerevisiae. SDS-PAGE pattern of soluble protein extracted from yeast cells grown on glucose was greatly different from that on starch. From these results, we proposed that the SK0704 may have a specific physiological function for starch catabolism such as membrane transport system and intracellular sac-charification of starch.     

Bioenergetic examination of the heterotrophic nitrifier-denitrifier Thiosphaera pantotropha

The heterotrophic nitrifying-denitrifying bacterium Thiosphaera pantotropha is remarkable as it nitrifies and denitrifies simultaneously. With respect to nitrogenous compounds, whether nitrification or denitrification results in energy conservation is of interest. Proton translocation studies were performed to determine if energy was conserved by the bacterium during heterotrophic nitrification and denitrification. Hydrazine (N₂H _inf5 ^sup+ ) was employed as the heterotrophic nitrification substrate while nitrate, nitrite and nitrous oxide were used as denitrification substrates. Analysis of the data indicate that the bacterium does not conserve energy when hydrazine was the substrate. Conversely, energy was conserved when either nitrate, nitrite or nitrous oxide functioned as the oxidants during denitrification-dependent proton translocation experiments. Thiosphaera pantotropha thus is similar to other heterotrophic nitrifiers-denitrifiers in that it conserves energy while denitrifying but has not been observed to do so when heterotrophically nitrifying.     

Characterization of the trehalosyl dextrin-forming enzyme from the thermophilic archaeon <Emphasis Type="Italic">Sulfolobus solfataricus</Emphasis> ATCC 35092

The trehalosyl dextrin-forming enzyme (TDFE) mainly catalyzes an intramolecular transglycosyl reaction to form trehalosyl dextrins from dextrins by converting the -1,4-glucosidic linkage at the reducing end to an -1,1-glucosidic linkage. In this study, the treY gene encoding TDFE was PCR cloned from the genomic DNA of Sulfolobus solfataricus ATCC 35092 to an expression vector with a T7 lac promoter and then expressed in Escherichia coli. The recombinant TDFE was purified sequentially by using heat treatment, ultrafiltration, and gel filtration. The obtained recombinant TDFE showed an apparent optimal pH of 5 and an optimal temperature of 75°C. The enzyme was stable in a pH range of 4.5–11, and the activity remained unchanged after a 2-h incubation at 80°C. The transglycosylation activity of TDFE was higher when using maltoheptaose as substrate than maltooligosaccharides with a low degree of polymerization (DP). However, the hydrolysis activity of TDFE became stronger when low DP maltooligosaccharides, such as maltotriose, were used as substrate. The ratios of hydrolysis activity to transglycosylation activity were in the range of 0.2–14% and increased when the DP of substrate decreased. The recombinant TDFE was found to exhibit different substrate specificity, such as its preferred substrates for the transglycosylation reaction and the ratio of hydrolysis to transglycosylation of the enzyme reacting with maltotriose, when compared with other natural or recombinant TDFEs from Sulfolobus.     

Nitrification in soils of secondary vegetational successions from Eucalyptus forest and grassland to cool temperate rainforest in Tasmania

Rates of nitrification in well drained granitic soils from forest stands and grassland of differing successional status and from beneath isolated individuals of several tree species were compared in a series of laboratory experiments. Fresh samples were perfused with distilled water or nutrient solution for 10 to 14 weeks at 20°C. The following treatments were applied to the soils singly and in combination: 200 and 400 g N g^–1 as (NH₄)₂SO₄; 100 g P g^–1 as KH₂PO₄; 4000 g CaCO₃ g^–1; inoculation of non-nitrifying soil with nitrifying soil; perfusion of nitrifying soil with leachate from non-nitrifying soil.Nitrification was absent or occurred at only a low rate in many soils; it generally increased as succession proceeded from nature grassland or eucalypt forest towards climax temperate rainforest, but decreased in mature climax forests. However, the influence of individual tree species was often paramount. Nitrification was stimulated by disturbance of a stand by disease. A possible inhibitor of nitrification in a rainforest soil could not be removed by leaching with water, nor transferred via the leachate to a nitrifying soil. Addition of P was without effect on either total amount of nitrate produced or on net mineralisation of soil N, but sometimes increased the rate of nitrification of added ammonium. Non-nitrifying rainforest soil of pH 4.3 was induced to nitrify only after addition of (NH₄)₂SO₄, inoculation with a nitrifying soil, and addition of CaCO₃ to raise pH by 3 units. However, once nitrification had commenced it could continue with little change in rate while pH decreased to a value of 3.4.It was concluded that rate of nitrification is dependent upon the presence of particular tree species in a stand, upon its history of disturbance, and hence in part upon the stand's successional status. It is not limited by pHper se within the range found in these soils, although an increase in pH may be necessary to initiate nitrification. In some soils the rate of nitrification may be limited by the level of ammonium substrate, and nitrifiers are virtually absent from others. Overall microbial activity is limited by lack of utilisable carbon substrate.     

Production and characterization of polygalacturonase fromGeotrichum candidum

An extracellular polygalacturonase (EC 3.2.1.15) fromGeotrichum candidum ATCC 34614 grown onsauerkraut brine was produced and characterized. Polygalacturonic acid markedly increased the enzyme yield in the brine. The fungus produced the highest activity (290 U/l) in brine with 0.3% (w/v) polygalacturonic acid. The pH and temperature optima of the enzymes were 4.5 to 5.0 and 30°C, respectively. It was stable from pH 4.0 to 5.8 and at 30°C but lost its activity at higher temperatures. The K_m and V_max values for polygalacturonic acid were 4.2 mg/ml and 0.19mm galacturonic acid/min, respectively. The enzyme was not substrate inhibited.     

Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an α-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, α-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and α-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.     

Litter decomposition and nitrogen mineralization of soils in subtropical plantation forests of southern China,with special attention to comparisons between legumes and non-legumes

Litter decomposition and nitrogen mineralization were investigated in subtropical plantation forests in southern China. The CO₂ –C release from incubated litter and the forest floor of Acacia mangium, Acacia auriculaeformis, Eucalyptus citriodora, Pinus elliotii and Schima superba stands were used to estimate relative rates of litter decomposition. Decomposition was not positively correlated with litter nitrogen. E. citridora litter decomposed most rapidly and A. mangium litter most slowly, both with and without the addition of exotic nitrogen. Aerobic incubation and intact soil core incubation at 30 °C over a period of 30 days were used to assess nitrogen mineralization of six forest soils. Although there were differences in results obtained using the two methods, patterns between legume and non-legume species were the same regardless of method. All soils had pH values below 4.5, but this did not prevent nitrification. The dominant form of mineral nitrogen was nitrate for legume species and ammonium for non-legume species. The nitrogen mineralization potential was highest for soils in which legumes were growing.     

Nitrification in Dutch heathland soils

A survey was conducted over a range of 17 Dutch heathland locations, subdivided into 41 sites dominated by either dwarf-shrubs (Calluna vulgaris or Erica tetralix) or grass species (Deschampsia flexuosa or Molinia caerulea). Among the habitats of the dominant plant species relatively little differences in general soil properties were observed. The P status of Deschampsia sites was relatively high as well as the NO₃ ^–-N concentrations in the 0–10 cm layer (FH included) at the grass-dominated sites. At sites with a dead or degenerating dwarf-shrub vegetation, NH₄ ⁺-N concentrations reached very high levels.Net production of nitrate was observed during incubation of intact 0–10 cm soil cores (FH-layer included) in the laboratory for all sites, even though in some instances, particularly at Calluna and Erica sites, no nitrate was initially measured. Generally, a higher nitrification rate was found for the grass-dominated sites, and for Deschampsia in particular. The net production of nitrate was highly significantly correlated with net N mineralization, being a reasonable predictor of nitrification in a simple regression model (R²=0.47; P<0.001). Net nitrification was also significantly correlated with the NO₃ ^–-N initially present at the start of the growing season (R=0.65; P<0.001) and with the labile organic P content of the soil (R=0.65; P<0.001). By including initial NO₃ ^–-N and labile organic P, together with net N mineralization and pH, in a multiple regression model, net nitrate production could be predicted with a much higher precision (R²=0.75; P<0.001). Although apparent nitrification was not significantly correlated with pH, the latter contributed significantly to the multiple regression equation for the prediction of the former.The influence of the labile organic P pool may act via its positive correlation with microbial biomass, thus more or less reflecting the potential mineralization/nitrifying capacity of a particular site.     

Screening of chitin deacetylase from Mucoralean strains (Zygomycetes) and its relationship to cell growth rate

Chitin deacetylase (CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-d-glucosamine in chitin, converting it to chitosan in fungal cell walls. In the present study, the activity in batch culture of CDA from six Mucoralean strains, two of them wild type, isolated from dung of herbivores of Northeast Brazil, was screened. Among the strains tested, Cunninghamella bertholletiae IFM 46114 showed a high intracellular enzyme activity of 0.075 U/mg protein after 5 days of culture, and a wild-type strain of Mucor circinelloides showed a high intracellular enzyme activity of 0.060 U/mg protein, with only 2 days of culture, using N-acetylchitopentaose as substrate. This enzyme showed optimal activity at pH 4.5 in 25 mM glutamate-sodium buffer at 50°C, and was stable over 1 h preincubation at the same temperature. The kinetic parameters of CDA did not follow Michaelis-Menten kinetics, but rather Hill affinity distribution, showing probable allosteric behavior. The apparent K_HILL and V_max of CDA were 288±34 nmol/l and 0.08±0.01 U mg protein^–1 min^–1, respectively, using N-acetylchitopentaose as substrate at pH 4.5 at 50°C.     

Cloning of the gene <Emphasis Type="Italic">Lecanicillium psalliotae</Emphasis> chitinase <Emphasis Type="Italic">Lpchi1</Emphasis> and identification of its potential role in the biocontrol of root-knot nematode <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>

The complete genome sequence of Bacillus subtilis reveals that sequences encoding several hemicellulases are co-localised with a gene (xynD) encoding a putative family 43 glycoside hydrolase that has not yet been characterised. In this work, xynD has been isolated from genomic DNA of B. subtilis subsp. subtilis ATCC 6051 and cloned for cytoplasmatic expression in Escherichia coli. Recombinant XynD (rXynD) was purified using ion-exchange chromatography and gel permeation chromatography. The enzyme had a molecular mass of approximately 52 kDa, a pI above 9.0 and releases α-l-arabinose from arabinoxylo-oligosaccharides as well as arabinoxylan polymers with varying degree of substitution. Using para-nitrophenyl-α-l-arabinofuranoside as substrate, maximum activity was observed at pH 5.6 and 45°C. The enzyme retained its activity over a large pH range, while activity was lost after pre-incubation above 50°C. Gas–liquid chromatography and proton nuclear magnetic resonance spectrometry analysis indicated that rXynD specifically releases arabinofuranosyl groups from mono-substituted C-(O)-2 and C-(O)-3 xylopyranosyl residues on the xylan backbone. As rXynD did not display endoxylanase, xylosidase or arabinanase activity and was inactive on arabinan, we conclude that this enzyme is best described as an arabinoxylan arabinofuranohydrolase.     

Xylitol production by Candida species grown on a grass hydrolysate

Xylitol was produced by selected species of the yeast Candida after growth on a medium containing a hydrolysate of the North American perennial prairie grass big bluestem. The grass was hydrolysed by a combination of dilute acid and enzymatic treatments. After growth on the medium for 120 h at 30 °C, Candida tropicalis ATCC 750 produced a 1.4-fold higher level of xylitol than did C. tropicalis ATCC 20215 while biomass production by C. tropicalis ATCC 750 was 1.7-fold higher than Candida guilliermondii ATCC 20216. The xylitol yields observed for C. tropicalis ATCC 750, Candida mogii ATCC 18364 and C. guilliermondii ATCC 20216 were at least 1.4-fold higher than the yield observed for C. tropicalis ATCC 20215 after growth for 120 h at 30 °C.     

Fertilization influences nitrogen dynamics in soils under Dalbergia sissoo

The influence of added ammonium, phosphorus, potassium, and gypsum on net nitrogen mineralization was studied in soil beneath a six-year-old plantation of the N₂-fixing tree Dalbergia sissoo in Pakistan. Soil with and without amendments was placed in polyethylene bags and incubated, buried in the soil, for 30 days. After that time the soil was analyzed and net ammonium and nitrate production and net nitrogen mineralization were calculated. The addition of ammonium stimulated nitrification indicating that the process was substrate limited. The inhibition of nitrification by Nitrapyrin showed that the process is autotrophic in these soils. Gypsum addition lowered soil pH from 8.0 to 7.2 and significantly stimulated ammonification, nitrification and net nitrogen mineralization. The addition of potassium more than tripled the soil K:Na ratio. Net ammonium and nitrate production and net nitrogen mineralization all increased in this treatment. The addition of phosphorus had no significant effect on soil nitrogen dynamics.     

Purification and some properties of a starch debranching enzyme ofHendersonula toruloidea

An extracellular, debranching isoamylase fromHendersonula toruloidea ATCC 64930, grown on starch, was purified 12-fold to an electrophoretically homogeneous state. The purified enzyme (estimated mol wt 83000) was optimally active at pH 6.0 and 50°C and remained active when held at 70°C (30 min) and at pH 6 to 8 for 24 h. Na⁺, Fe²⁺ and Ba²⁺ (at 5mm) enhanced enzyme activity while Hg²⁺, Zn²⁺ and Cu²⁺ (at 5mm) were inhibitory. The enzyme hydrolysed amylopectin (Km, 0.25 mg/ml), forming maltose, maltotriose and maltotetraose and hydrolyzed glycogen (Km, 0.29 mg/ml) and soluble starch (Km, 0.42 mg/ml) forming maltotriose and maltotetraose. Pullulan was not hydrolyzed.     

Characterization of the Actinomyces naeslundii ureolysis and its role in bacterial aciduricity and capacity to modulate pH homeostasis

Ammonia production from urea by ureolytic oral bacteria is believed to have a significant impact on oral health and ecological balance of oral microbial populations. Actinomyces naeslundii is an important ureolytic organism in the oral cavity. In this study, we aimed to investigate the substrate affinity and pH optimum for ureolysis of A. naeslundii (ATCC12104), and expression of urease under different environmental factors. In addition, in vitro acid killing and pH drop experiments were used to detect the role of ureolysis in bacterial aciduricity and capacity to modulate pH homeostasis. We observed the K_s value of the ureolytic activity was 7.5 mM and a pH optimum near 6.5. Urease expression by A. naeslundii (ATCC12104) was affected by multiple factors, including environmental pH, glucose and nitrogen availability. The cells could be protected against acid killing through hydrolysis of physiologically relevant concentrations of urea. A. naeslundii (ATCC12104) demonstrated a significant capacity to temper glycolytic acidification in vitro at urea concentrations normally found in the oral cavity.     

Antibacterial activity associated with Lactobacillus gasseri ATCC 9857from the human female genitourinary tract

The 10-fold concentrated spent MRS culture cell-free supernatant concentrate [(cCFS)] of the human female genitourinary tract isolate Lactobacillus gasseri ATCC 9857 was shown to exhibit antibacterial activity towards gram-positive sporogenous and asporogenous fermentative eubacteria in liquid and on solid media under conditions that eliminated the activity of lactic acid (-glycerophosphate) and hydrogen peroxide (catalase). The antibacterial activity of the cCFS was characterized by automated turbidometry (Bioscreen) and non-linear regression analysis (Gompertz model) using MRS broth cultures of the indicator strain L. acidophilus ATCC 11975. It exhibited a bactericidal mode of action, sensitivity to trypsin and proteinase K, partial sensitivity to pepsin and pronase E, partial heat stability at 121 °C for 15 min, and retained significantly more activity following exposure to pH 3.0 and 5.0 compared with pH 7.2 and 9.0. The inhibitory spectrum included a wide range of Lactobacillus species, Bifidobacterium bifidum, B. infantis and B. catenulatum, Lactococcus cremoris, Leuconostoc cremoris, Pediococcus pentosaceus, Bacillus cereus, Clostridium tyrobutyricum, C. pasteurianum, C. sporogenes, Staphylococcus carnosus, and Enterococcus faecalis. Although partial inhibition of Escherichia coli ATCC 25922 by cCFS was observed in liquid medium, inhibition of freshly isolated human uropathogenic E. coli strains could not be demonstrated on TSB agar plates by agar well diffusion. Following partial resolution by gel permeation FPLC on Superose-12, the fractionated cCFS was shown to comprise at least two inhibitory peptides (3.05 and 5.27 kDa) as well as aggregated inhibitory peptide material (21.65, 41.50, 81.20, and 120.90 kDa). The 3.05 kDa peptide, designated Gassericin D, inhibited L. acidophilus strains ATCC 11975 and ACA-DC 241. The 5.27 kDa peptide, designated Gassericin C, inhibited L. gasseri strain UCSC LF221Snb and En. faecalis DPC 3319. The aggregated 21.65 kDa peptide material strongly inhibited L. acidophilus ATCC 11975 and weakly inhibited Listeria inocua DPC 3306. The aggregated 41.50 kDa peptide material strongly inhibited Ba. cereus DPC 3316 and weakly inhibited L. acidophilus ACA-DC 241. The ability of L. gasseri ATCC 9857 to produce bacteriocin-like activity may be of importance in the biopreservation of nutraceuticals and in the management of female genitourinary and gastrointestinal tract infections involving En. faecalis.     

An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

Bicarbonate is a stimulus in the inter-species induced sporulation of strict anaerobic Syntrophomonas erecta subsp. sporosyntropha

Previously Syntrophomonas species had been described as the bacteria those did not form spores, however, in our previous studies, a newly isolated S. erecta subsp. sporosyntropha JCM13344^T was found to form spores in the co-culture with methanogens, while not in mono-culture or in co-culture with sulfate reducer. In this study, we examined the sporulation stimulus conferred by methanogens in the co-culture. By reducing bicarbonate in mono-culture and sulfate-reducing co-culture, we could induce S. erecta subsp. sporosyntropha JCM13344^T to form spores, so that bicarbonate at lower concentration was determined as another stimulus for sporulation. Based on the substrate degradation by strain JCM13344^T in different concentration of bicarbonate vs at different pHs, it was suggested that bicarbonate could stimulate the sporulation by mediating a nutrient deprivation but not pH drop. To further confirm the sporulation potential of this group of bacteria, spo0A fragments were amplified from strain JCM13344^T as well as all the recognized Syntrophomonas species, confirming that they were members of the spore-forming group. Since sporulation is a kind of response of spore-forming bacteria to environmental stresses, the observation in this work implies that stresses can be created even between the mutual beneficial partners, in this case, inducing sporulation.     

Fungal hydroxylation of (−)-santonin and its analogues

Santonin (1) was incubated with separate growing cultures of Aspergillus niger ATCC 9142, Mucor plumbeus ATCC 4740, Whetzelinia sclerotiorum ATCC 18687, Cunninghamella echinulata var. elegans ATCC 8688a and Phanerochaete chrysosporium ATCC 24725. Three novel metabolites were isolated: 11β,13-dihydroxysantonin (3), 6,7-dehydosantonin (5) and 3,6-dihydroxy-9-keto-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (7). 11β-Hydroxysantonin (2), 14-hydroxysantonin (4) and 3,6,9-trihydroxy-9,10-seco-selina-1,3,5(10)-trien-12-oic acid-12,6-lactone (6) were also isolated. Hydroxylation at C-9 followed by a retro-aldol reaction was postulated to have produced 6 and 7. Through the synthesis and fermentation of the santonin analogues: tetrahydrosantonin (8) and α-desmotroposantonin (12), several new compounds were obtained; the most significant being 9-keto-desmotroposantonin (14), which was indicative of C-9 monohydroxylation.