Kinetic studies on ammonia and methane oxidation by Nitrosococcus oceanus

The kinetics of ammonia oxidation and the ability of a marine ammonia-oxidizing bacterium, Nitrosococcus oceanus, to metabolize methane were investigated in semicontinuous batch culture. The effects of inhibitors (acetylene and nitrapyrin) and coreactants were determined in order to elucidate the behavior of the ammonia oxygenase enzyme in N. oceanus. Acetylene and nitrapyrin were potent inhibitors and their effects were not mitigated by increased ammonia concentrations. Oxygen concentration had the effect of a mixed-type inhibitor; reduced oxygen inhibited the rate or ammonia oxidation at high substrate concentration but may enhance the rate at low substrate concentrations. Substrate affinity in terms of NH ₄ ⁺ increased (K _m decreased) with increasing pH. Optimal pH was about 8. Methane inhibited ammonia oxidation; the interaction was not simple competitive inhibition and the presence of multiple active sites on the enzyme was indicated by the behaviour of the inhibited treatments. Half-saturation constants for methane (K _i=6.6 M) and ammonia (K _m=8.1 M) were similar. N. oceanus oxidized methanol and methane linearly over time, with CO₂ and cell material being produced at approximately equal rates.     

Production of Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from Gluconate and Glucose by Recombinant Aeromonas hydrophila and Pseudomonas putida

Aeromonas hydrophila 4AK4 and Pseudomonas putida GPp104 were genetically engineered to synthesize poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) using gluconate and glucose rather than fatty acids. A truncated tesA gene, encoding cytosolic thioesterase I of Escherichia coli which catalyzes the conversion of acyl-ACP into free fatty acids, was introduced into A. hydrophila 4AK4. When grown in gluconate, the recombinant A. hydrophila 4AK4 synthesized 10% (w/w) PHBHHx containing 14% (mol/mol) 3-hydroxyhexanoate. If additional PHBHHx synthesis genes, phaPCJ, were over-expressed with the truncated tesA in A. hydrophila 4AK4, the PHBHHx content increased to 15% (w/w) and contained 19% (mol/mol) 3-hydroxyhexanoate. Recombinant P. putida GPp104 harboring phaC encoding PHBHHx synthase of A. hydrophila, phaB encoding acetoacetyl-CoA reductase of Wautersia eutropha and phaG encoding 3-hydroxyacyl-ACP-CoA transferase of P. putida, synthesized 19% (w/w) PHBHHx containing 5% (mol/mol) 3-hydroxyhexanoate from glucose. The results suggest that the engineered pathways were applicable to synthesize PHBHHx from unrelated carbon sources such as gluconate and glucose.     

Influence of pectin and glucose on growth and polygalacturonase production by Aspergillus niger in solid-state cultivation

The solid-state production of endo- and exo-polygalacturonases (PG) by Aspergillus niger was studied in a media containing wheat bran, salts, and different citric pectin and/or glucose concentrations. Kinetic analysis of the process indicated that the formation of PG and the growth of A. niger are associated processes. By increasing citric pectin from 0 to 16% (w/w), the maximum A. niger concentration (X _m) was raised from 94 to 121 mg/g dry medium suggesting that pectin can be used by A. niger as a growth substrate besides its role as an inducer. With 16% (w/w) pectin, 281 U exo-PG/gdm and 152 U endo-PG/gdm were obtained. Otherwise, pectin concentrations from 20 to 30% (w/w) hindered both production and growth. A. niger concentrations of 108–113 mg/gdm were achieved in runs with glucose from 5 to 12% (w/w), whereas at 16 and 20% (w/w) glucose, lower X _m values (ca. 100 mg/gdm) were measured. The addition of glucose to the wheat bran medium, up to 10% (w/w) led to maximum endo-PG titers slightly lower than those found in the absence of glucose. Nevertheless, exo-PG formation in these media was strongly increased and activities over 370 U/gdm were achieved. The results suggest that in experiments with pectin concentrations until 16% (w/w), exo-PG production was repressed by pectin-degradation products although these same substances had favored biomass growth. When glucose concentrations over 10% (w/w) were added to the media, the maximum activities of both enzymes decreased drastically, suggesting that glucose at high concentrations also exerts a repressive effect on PG production.     

Cytochromes in Thiobacillus tepidarius and the respiratory chain involved in the oxidation of thiosulphate and tetrathionate

Thiobacillus tepidarius was shown to contain cytochrome(s) c with absorption maxima at 421, 522 and 552 nm in room temperature reduced minus oxidized difference spectra, present at 1.1–1.2 nmol per mg dry wt and present in both membrane and soluble fractions of the cell. The membrane-bound cytochrome c (1.75 nmol per mg membrane protein) had a midpoint potential (E_m, pH 7.0) of 337 mV, while the soluble fractions appeared to contain cytochrome(s) c with E_m (pH 7.0) values of about 270 and 360 mV. The organism also contained three distinct membrane-bound b-type cytochromes (totalling 0.33 nmol per mg membrane protein), each with absorption maxima in reduced minus oxidized difference spectra at about 428, 532 and 561 nm. The E_m (pH 7.0) values for the three cytochromes b were 8 mV (47.8% of total), 182 mV (13.7%) and 322 mV (38.5%). No a- or d-type cytochromes were detectable spectrophotometrically in the intact organism or its membrane and soluble fractions. Evidence is presented for both CO-binding and CO-unreactive cytochromes b or o, and CO-binding cytochrome(s) c. From redox effects observed with CO it is proposed that a cytochrome c donates electrons to a cytochrome b, and that a high potential cytochrome b or o may be acting as the terminal oxidase in substrate oxidation. This may be the 445 nm pigment, a photodissociable CO-binding membrane haemoprotein. Substrate oxidation was relatively insensitive to CO-inhibition, but strongly inhibited by cyanide and azide. Thiosulphate oxidation couples directly to cytochrome c reduction, but tetrathionate oxidation is linked (probably via ubiquinone Q-8) to reduction of a cytochrome b of lower potential than the cytochrome c. The nature of possible electron transport pathways in Thiobacillus tepidarius is discussed. One speculative sequence is: c^– b₈ b₁₈₂ c₂₇₀ c₃₃₇ b₃₂₂/c₃₆₀ O₂ Abbreviations E_m midpoint electrode potential - E _inf0 ^sup pH 7, standard electrode potential at pH 7.0 - Q-8 coenzyme Q-8 (ubiquinone-40)     

Biotransformation of biphenyl by the filamentous fungus <Emphasis Type="Italic">Talaromyces helicus</Emphasis>

The filamentous fungusTalaromyces helicus , isolated from oil-contaminated sludge, oxidizes biphenyl via 4-hydroxybiphenyl to the dihydroxylated derivatives 4,4-dihydroxybiphenyl and 3,4-dihydroxybiphenyl, which, to a certain extent, are converted to glycosyl conjugates. The sugar moiety of the conjugate formed from 4,4-dihydroxybiphenyl was identified as glucose. Further metabolites: 2-hydroxybiphenyl, 2,5-dihydroxylated biphenyl, and the ring cleavage product 4-phenyl-2-pyrone-6-carboxylic acid accumulated only in traces. From these results the main pathway for biotransformation of biphenyl in T. helicus could be proposed to be the excretion of dihydroxylated derivatives (>75%) and their glucosyl conjugates (<25%).     

Phase partition of gaseous hexane and surface hydrophobicity of Fusarium solani when grown in liquid and solid media with hexanol and hexane

The filamentous fungus, Fusarium solani, was grown in liquid and solid culture with glucose, glycerol, 1-hexanol and n-hexane. The partition coefficient with gaseous hexane (HPC) in the biomass was lower when grown in liquid medium with 1-hexanol (0.4) than with glycerol (0.8) or glucose (1) The HPC for surface growth were 0.2 for 1-hexanol, 0.5 for glycerol, 0.6 for glucose, and 0.2 for F. solani biomass obtained from a biofilter fed with gaseous n-hexane. These values show a 200-fold increase in n-hexane solubility when compared to water (HPC = 42). Lower HPC values can be partially explained by increased lipid accumulation with 1-hexanol, 10.5% (w/w) than with glycerol (8.5% w/w) or glucose (7.1% w/w). The diameter of the hyphae diminished from 3 μm to 2 μm when F. solani was grown on solid media with gaseous n-hexane thereby doubling the surface area for gaseous substrate exchange. The surface hydrophobicity of the mycelia increased consistently with more hydrophobic substrates and the contact angle of a drop of water on the mycelial mat was 113° when grown on n-hexane as compared to 75° with glucose. The fungus thus adapts to hydrophobic conditions and these changes may explain the higher uptake of gaseous hydrophobic substances by fungi in biofilters.     

Fatty acid composition of lipids from mushrooms belonging to the family Boletaceae

The non-polar lipid content and fatty acid (FA) composition of 11 mushroom species of the family Boletaceae were determined. The non-polar lipid content ranged from 2.0 (Leccinum aurantiacum and Boletus erythropus) to 5.4 % (w/w) d.w. (Suillus grevillei) with an average value of 2.9 %. More than 25 different FAs were found in the mushroom lipids. Unsaturated FAs, mainly linoleic and oleic acids, accounted for about 83 % of the total FAs, while palmitic acid was the main saturated FA. Some FAs are identified for the first time in Boletaceae and in higher Basidiomycetes (cis-11,12-methyleneoctadecanoic acid, 7-cis,10-cis hexadecadienoic) or in fungi (cis-11,12-methyleneoctadecanoic acid). There were significant differences (P < 0.05) in the contents of specific FAs between mushroom species.     

Processing and characteristics of soybean-fortified gari

Two cassava cultivars were fermented for 3 days with the incorporation of soybean residue or full-fat flour at 25% (w/w) to produce gari. Unfortified gari was superior in taste (P<0.05) to the soybean-fortified gari, though consumers still accepted the latter. Fortified samples from two of the three procedures used, in which soybean was added at different stages of the fermentation, were rated similarly (P>0.05). The water activity of soybean-fortified gari was <0.7, the swelling capacity was 2.5 to 3.3, pH was 5.1±0.2 and total cyanide was 0.8 to 1.3 mg/100 g. The crude protein content of soybean-fortified gari was 8 to 12% (w/w), much higher than the 1 to 2% (w/w) of the unfortified gari. Soybean-fortified gari is recommended for use in areas where cassava is consumed and protein intake is inadequate.The authors are with the Biology Department, The Polytechnic, Ibadan, Nigeria.     

Laboratory evaluation of some plant essences to control Varroa destructor (Acari: Varroidae)

This research was conducted to evaluate acaricidal effects of some plant essences on Varroa mites and the possibility of their usage for Varroa control. First, live Varroa mites were obtained from adult honeybees with CO₂ in a newly designed apparatus. Thyme, savory, rosemary, marjoram, dillsun and lavender essences at concentrations of 2 and 1g/100g (w/w), caused a mite mortality rate of more than 97% and 95%, respectively. Also spearmint at 2g/100g was able to kill more than 97% of Varroa mites. When sprayed on worker honeybees infected with mites, thyme, savory, spearmint and dillsun essences at 2g/100g (w/w) caused 43–58% Varroa mortality. Toxicity of thyme, savory and spearmint essences for worker honeybees was not significantly different from that of controls (acetone and water), but dillsun essence caused 12% honeybee mortality. These results showed that essences of thyme, savory and spearmint have acaricidal properties that could be used for controlling Varroa in honeybee colonies. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Solid state fermentation for the production of α-amylase from Penicillium chrysogenum using mixed agricultural by-products as substrate

Production of α-amylase from local isolate, Penicillium chrysogenum, under solid-state fermentation (SSF) was carried out in this study. Different agricultural by-products, such as wheat bran (WB), sunflower oil meal (SOM), and sugar beet oil cake (SBOC), were used as individual substrate for the enzyme production. WB showed the highest enzyme activity (750 U/gds). Combination of WB, SOM, and SBOC (1:3:1 w/w/w) resulted in a higher enzyme yield (845 U/gds) in comparison with the use of the individual substrate. This combination was used as mixed solid substrate for the production of α-amylase from P. chrysogenum by SSF. Fermentation conditions were optimized. Maximum enzyme yield (891 U/gds) was obtained when SSF was carried out using WB + SOM + SBOC (1:3:1 w/w/w), having initial moisture of 75%, inoculum level of 20%, incubation period of 7 days at 30°C. Galactose (1% w/w), urea and peptone (1% w/w), as additives, caused increase in the enzyme activity.     

Sugarcane molasses and yeast powder used in the Fructooligosaccharides production by Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611

Different concentrations of sucrose (3–25% w/v) and peptone (2–5% w/v) were studied in the formulation of media during the cultivation of Aspergillus japonicus-FCL 119T and Aspergillus niger ATCC 20611. Moreover, cane molasses (3.5–17.5% w/v total sugar) and yeast powder (1.5–5% w/v) were used as alternative nutrients for both strains’ cultivation. These media were formulated for analysis of cellular growth, β-Fructosyltransferase and Fructooligosaccharides (FOS) production. Transfructosylating activity (U _t ) and FOS production were analyzed by HPLC. The highest enzyme production by both the strains was 3% (w/v) sucrose and 3% (w/v) peptone, or 3.5% (w/v) total sugars present in cane molasses and 1.5% (w/v) yeast powder. Cane molasses and yeast powder were as good as sucrose and peptone in the enzyme and FOS (around 60% w/w) production by studied strains.     

Nematodes from the Strait of Magellan and the Beagle Channel (Chile): the genera Cervonema and Laimella (Comesomatidae: Nematoda)

Five species of Cervonema and four species of Laimella are described from the Strait of Magellan and the Beagle Channel, Chile, six species of which are new to science. Cervonema chilensisn. sp. and Cervonema hermanin. sp. are separated from other known species of Cervonema by a short cervical region (less than one head diameter from the front end to the anterior border of the amphids). Cervonema chilensisn. sp. is characterised by a tail length of 5 anal diameters with posterior half filiform; Cervonema hermani n. sp. is characterised by a tail length of 6–9 anal diameter and posterior part (75%) cylindrical. Cervonema shiaen. sp. is characterised by the cephalic seta 4 m long, amphids 9–10 m in diameter; spicules 16 m long and 0.8–0.9 abd; tail 4.7–5.4 anal diameter and 50% posterior part filiform; 4–5 minute precloacal supplements. Laimella subterminatan. sp. is characterised by the subterminal position of the buccal cavity which separates it from the other species of the genus. Laimella annaen. sp. is characterised by the head diameter 9–11 m, cephalic setae and external labial setae 9 + 5 m long, respectively, amphids 7 m in diameter; spicules 28–30 m long; tail 14–17 anal diameter and posterior part (75%) filiform; 5 precloacal supplements. Laimella sandraen. sp. is very close to Laimella annaen.sp. in having similar cephalic sensilla, amphids and spicules. Laimella sandraen. sp., however, can be separated from L.annaen. sp. by the shape of head and the structure of sperm cells, the total body length and the cylindrical part of tail. Cervonema papillatum Jensen, 1988, C. tenuicauda (Stekhoven, 1950) and L. longicauda Cobb, 1920 are found in this area as well. The key of all known species of Cervonemaand Laimellais presented.     

Lack of interaction between the milky disease bacterium Paenibacillus popilliae and stilbene-derived optical brighteners in Japanese beetle larvae

Optical brighteners cansynergistically enhance nucleopolyhedrovirusinfectivity to lepidopteran larvae by blockingthe sloughing of infected primary midgut cellsand inhibiting the formation of the peritrophicmembrane in the hosts. Because of similaritiesin the route of infection, we investigatedwhether optical brighteners would also enhanceinfection with the milky disease bacterium,Paenibacillus popilliae, of Japanesebeetle, Popillia japonica, larvae. Thelarvae were kept in soil mixed with P.popilliae spore preparations and the opticalbrighteners Blankophor BBH, P167, or RKH withperennial ryegrass provided as food. Noenhancing effect of any of the opticalbrighteners on P. popilliae infection wasobserved at a concentration of 0.1% (w/w). Rather, when mixed into the soil at 0.02, 0.1,or 0.5% (w/w) BBH reduced P. popilliaeinfection at the highest rate.     

Functional role of granule-associated genes, phaP and phaR, in poly-β-hydroxybutyrate biosynthesis in recombinant E. coli harboring phbCAB operon

The expression characteristic of two granule-associated genes, phaP and phaR, in a recombinant E. coli harboring the phbCAB operon was investigated. Polybetamydroxybutyrate (PHB) accumulation increased up from 16% to 57% (w/w) after transformation of the granule-associated genes due to the stabilization of PHB granules rather than by a direct effect on PHB biosynthetic enzymes. The morphology of PHB granules also varied depending on the transformed phaP and phaR genes.     

Growth,sporulation and toxin production byBacillus thuringiensis subsp.israelensis andB. sphaericus in media based on mustard-seed meal

Bacillus thuringiensis subsp.israelensis andB. sphaericus strains 2362 and 1593 were grown in media based on defatted mustard-seed meal (MSM). The meal contains 40% (w/w) protein, with glutamic acid and arginine as the major amino acids. The toxic potencies of the final bacterial powders towardsCulex pipens quinquefasciatus Say, compared with those of the respective international reference standards, were 46% forB. thuringiensis subsp.israelensis, 62% forB. sphaericus 2362 and 88% forB. sphaericus 1593 when 2% (w/v) MSM was used for growth. With 4% (w/v) MSM,B. thuringiensis subsp.israelensis grew better but had undetectable larvicidal activity, whereas theB. sphaericus strains not only grew better but gave a higher degree of sporulation and toxicity. The potencies ofB. sphaericus in medium with 4% MSM were comparable with those of international reference standards.The authors are with the Department of Life Sciences, University of Bombay, Bombay 400 098, India.     

Somaclonal variation within poplar

One thousand and ninety-two poplars were regenerated in vitro from callus of 13 poplar clones representing the Leuce, Aigeiros and Tacamahaca sections. At lest 44 of the regenerants differed in some way from the original clones. Somaclonal variation occurred more frequently in poplars of the Leuce section (8%) than in those of the Aigeiros or Tacamahaca sections (1%). Variation was noticed in growth habit, leaf shape or indentation but not in the reaction to four Melampsora races. However, after one growing season in the field, a few regenerants from calli of two clones (Ogy and Rap) differed in their susceptibility vis à vis the original clones. Cultivation of callus from Leuce poplars that had survived exposure to increasing concentrations of toxins from Hypoxylon mammatum gave rise to a toxin-tolerant line from which toxin tolerant plants were regenerated. Flow cytometry to measure the DNA content of nuclei showed that regenerants tended to be tetraploid.Abbreviations NAA naphthaleneacetic acid - BAP benzylaminopurine - TDZ thidiazuron - MS Murashige & Skoog medium     

Application of novel starter cultures for sourdough bread production

Sourdough application has been extensively increased in the last years due to the consumers demand for food consumption without the addition of chemical preservatives. Several starter cultures have been applied in sourdough bread making targeting the increase of bread self-life and the improvement of sensorial character. More specific, Lactobacillus acidophilus and Lactobacillus sakei as single and mixed cultures were used for sourdough bread making. Various sourdough breads were produced with the addition of sourdough perviously prepared with 10% w/w L. acidophilus, 10% w/w L. sakei and 5% w/w L. acidophilus and 5% w/w L. sakei at the same time. Various chemical parameters were determined such as lactic acid, total titratable acidity and pH. The results revealed that the produced sourdough bread made with sourdough containing the mixed culture was preserved for more days (12 days) than all the other breads produced in the frame of this study, since it contained lactic acid in higher concentrations. The respective total titratable acidity varied between 10.5 and 11 ml NaOH N/10. The same sourdough bread had a firmer texture, better aroma, flavor and overall quality compared to other sourdough breads examined in this study, as shown by sensory evaluation tests and results obtained through SPME GC–MS analysis, which revealed significant differences among the different bread types.     

甘蓝型油菜BnCYP79B1基因的克隆与表达

硫苷是十字花科植物的一种次生代谢产物,其合成途径受细胞色素P450的CYP79家族蛋白的调控,该实验采用同源克隆技术在甘蓝型油菜中克隆到了CYP79B1基因,命名为BnCYP79B1(GenBank登录号为JX535391.1)。BnCYP79B1基因cDNA全长1 625bp,编码一个含有541个氨基酸、理论等电点为8.88。序列对比结果显示,BnCYP79B1与花椰菜CYP79B1在DNA序列上的相似性为98.83%,推测蛋白氨基酸序列的相似性为99.26%。通过不同时期不同部位BnCYP79B1基因表达量的分析,发现BnCYP79B1基因在高秆高硫苷品系的根中表达量较高,而对矮秆高硫苷品系则是叶中表达量较高。在BnCYP79B1表达总量上,高秆品系较矮秆品系高,高硫苷品系较低硫苷品系高。     

Oxytetracycline production by Streptomyces rimosus in solid-state fermentation of corncob

Corn-cob was used as a substrate in the production of oxytetracycline by Streptomyces rimosus TM-55 in a solidstate fermentation. Oxytetracycline was detected on day 4, and reached its maximum on day 8. Optimal conditions for oxytetracycline production were an initial pH of 5.2 to 6.3, an initial moisture content of 64% to 67%, supplementation with 20% (w/w) rice bran or 1.5% to 2.5% (w/w) (NH₄)₂SO₄ as sole N source, 1.0% (w/w) CaCO₃, 2% (w/w) MgSO₄.7H₂O, and 0.5% (w/w) KH₂PO₄, with incubation for 8 days at 25 to 30°C. Each g substrate produced 7 to 8 mg oxytetracycline.