Production of acetone,butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping

We examined the effect of gas-stripping on the in situ removal of acetone, butanol, and ethanol (ABE) from batch reactor fermentation broth. The mutant strain (Clostridium beijerinckii BA101) was not affected adversely by gas stripping. The presence of cells in the fermentation broth affected the selectivities of ABE. A considerable improvement in the productivity and yield was recorded in this work in comparison with the non-integrated process. In an integrated process of ABE fermentation-recovery using C. beijerinckii BA101, ABE productivities and yield were improved up to 200 and 118%, respectively, as compared to control batch fermentation data. In a batch reactor C. beijerinckii BA101 utilized 45.4 g glucose l^–1 and produced 17.7 g total ABE l^–1, while in the integrated process it utilized 161.7 g glucose l^–1 and produced total ABE of 75.9 g l^–1. In the integrated process, acids were completely converted to solvents when compared to the non-integrated process (batch fermentation) which contained residual acids at the end of fermentation. In situ removal of ABE by gas stripping has been reported to be one of the most important techniques of solvent removal. During these studies we were able to maintain the ABE concentration in the fermentation broth below toxic levels.     

Locus Mapping,Molecular Cloning,and Expression Analysis of <Emphasis Type="Italic">rps6kb2</Emphasis>, a Novel Metamorphosis-Related Gene in Chinese Tongue Sole (<Emphasis Type="Italic">Cynoglossus semilaevis</Emphasis>)

Flatfish metamorphosis denotes the extraordinary transformation of a symmetric pelagic larva into an asymmetric benthic juvenile. This unique process involves eye migration, a 90° rotation in posture, and asymmetrical pigmentation for adaptation to a benthic lifestyle. In the present study, we used genetics to map a metamorphosis-related locus (q-10M) in the male linkage group (LG10M), a small interval of 0.9 cM corresponding to a 1.8 M-bp physical area in chromosome 9 in the Chinese tongue sole (Cynoglossus semilaevis). Combined with single-marker analysis, ribosomal protein S6 kinase 2 (rps6kb2) a member of the family of AGC kinases was identified as a novel metamorphosis-related candidate gene. Its expression pattern during metamorphosis was determined by quantitative RT-PCR and whole-mount in situ hybridization analysis. rps6kb2 gene was significantly expressed in metamorphic climax stage larvae and distributed in all the tissues transforming during metamorphosis, including tail, jaw, eye and skin of larvae. The results suggest that rps6kb2 has a general role in tissue transformations during flatfish metamorphosis including tail changes, skull remodeling, eye migration, and asymmetrical pigmentation.     

Density,distribution and population structure of <Emphasis Type="Italic">Opusia indica</Emphasis> (Ocypodoidae: Camptandriidae) in a coastal mangrove creek in Pakistan

The density, distribution and population structure of Opusia indica were studied through transects method. Two transects were delimited in a mangrove area of Korangi creek (24°79′ N/67°20′ E). On each transect, three 0.25 m² quadrats were sampled at three tidal levels on a monthly basis during low tide. A total of 1919 crabs were obtained, of which 775 were males, 945 were non-ovigerous females and 199 were ovigerous females. Density of crabs varied between 198 m⁻² to 798 m⁻². The density and size distribution showed significant difference from low tide level to high tide level and were positively correlated with the percent moisture, percent organic matter and sediment structure. Based on carapace width (CW) males were significantly larger than the females indicating sexual dimorphism. The monthly size frequency distribution of crabs showed recruitment of juvenile crabs (< than 4 mm) nearly throughout the year except in few months. The monthly sex ratio deviated from 1:1 throughout the year, with female bias (χ ² = 31.633, P = 0.001 and df =11). Breeding was seasonal with peaks in SW monsoon. Size at sexual maturity based on smallest ovigerous female was CW = 4.0 mm. The average number of eggs per female were 2066 ± 479 (n = 25). Positive linear relationship was observed between the size of the female crabs and the egg numbers (r ² = 0.554).     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants with enhanced tolerance to oxidative stress

We have previously reported a method for isolation of mutants with enhanced tolerance to the fungal AAL toxin and given a detailed characterization of atr1 (AAL toxin resistant, Gechev et al. in Biochem Biophys Res Commun 375:639–644, 2008). Herewith, we report eight more mutants with enhanced tolerance to the AAL toxin. Phenotypic analysis showed that six of the mutants were reduced in size compared with their original background loh2. Furthermore, atr2 showed delayed flowering and senescence. The mutants were also evaluated for oxidative stress tolerance by growing them on ROS-inducing media supplemented with either aminotriazole or paraquat, generating, respectively, H₂O₂ or superoxide radicals. Oxidative stress, confirmed by induction of the marker genes, HIGH AFFINITY NITRATE TRANSPORTER At1G08090 and HEAT SHOCK PROTEIN 17 At3G46230, inhibited growth of all lines. However, while the original background loh2 developed necrotic lesions and died rapidly on ROS-inducing plant growth media, atr1, atr2, atr7 and atr9 remained green and viable. The tolerance against oxidative stress-induced cell death was confirmed by fresh weight and chlorophyll measurements. Real-time PCR analysis revealed that the expression of the EXTENSIN gene At5G46890, previously shown to be downregulated by aminotriazole in atr1, was repressed in all lines, consistent with the growth inhibition induced by oxidative stress. Taken together, the data indicate a complex link between growth, development and oxidative stress tolerance and indicates that growth inhibition can be uncoupled from oxidative stress-induced cell death.     

Three homopteran pests of citrus as prey for the convergent lady beetle: suitability and preference

The convergent lady beetle, Hippodamia convergens Guérin-Méneville (Coleoptera: Coccinellidae), is an important predator of soft-bodied insect pests in many regions of the United States, but generally uncommon in Florida citrus. Certain citrus producers in Florida recently initiated releases of commercially available H. convergens from California against the Asian citrus psyllid Diaphorina citri Kuwayama, vector of Huanglongbing or citrus greening disease. However, there is little information on potential efficacy of this predator against the psyllid or other pests of citrus. Preference, development, and reproduction by H. convergens was evaluated on freshly collected nymphs of D. citri, brown citrus aphid Toxoptera citricida Kirkaldy, green citrus aphid Aphis spiraecola Patch, and frozen eggs of the flour moth Ephestia kuehniella Zeller. Larvae preferred D. citri over T. citricida in two-way choice tests and consumed more D. citri or A. spiraecola than T. citricida in no-choice tests. Adults consumed equal numbers of all three species in both tests. Development times of larvae at 25.5±0.05°C on A. spiraecola were longer than on the other three diets. Larval survival and pupation times did not differ among diets. Females lived longer than males irrespective of diet, and longevity of both genders was greatly increased on E. kuehniella compared with D. citri and A. spiraecola. Life table analysis indicated that H. convergens should increase on all three species, with a greater potential on psyllids than aphids. Further studies are warranted to assess establishment and persistence of this potential biological control agent in the Florida citrus environment.     

Oxidative stress elevated DNA damage and homocysteine level in normal pregnant women in a segment of Pakistani population

Maternal oxidative stress during pregnancy may impair fetal growth and help in the development of diseases in adulthood. The aim of current study was to assess total oxidation status (TOS), related parameters and their relationship to DNA damage (%) and homocysteine level in normal pregnant women in low-income participants. In a cross-sectional study healthy women were grouped as normal, while age matched nulliparous and singleton pregnancies were included for first, second and third trimester groups. TOS (P < 0.01), melanodialdehyde (MDA) (P < 0.001), aspartate aminotransferase (AST) (P < 0.01), triiodothyronine (T3) (P < 0.01), thyroxine (T4) (P < 0.01), and homocysteine (P < 0.001), in pregnant women were significantly higher as compared to normal healthy women. While serum total proteins (P < 0.01), albumin (P < 0.01) and total antioxidant status (TAS) (P < 0.001) decreased significantly as compared to normal healthy women. Women in third trimester showed a significantly high level of body temperature (P < 0.01), triglyceride (P < 0.01), LDL-cholesterol (P < 0.05), AST (P < 0.01), T3 (P < 0.01), homocysteine (P < 0.001), TOS (P < 0.01) and MDA (P < 0.001) but a lower concentration of serum proteins, albumin and TAS at the end of the pregnancy. Pearson correlation indicated a positive relationship of homocysteine with triglycerides (P < 0.027), TOS (P < 0.01), MDA (P < 0.035) and had a negative relationship with total protein (P < 0.026). DNA damage was strongly related with T3 (P < 0.008), TOS (P < 0.02), MDA (P < 0.037) and MBI (P < 0.048) profiles of pregnant women. These changes were considered normal for pregnant women having optimum blood pressure and normal child birth. Hormonal influences and hemodilution may contribute towards the observed changes in this study.     

Calculation of chromophore excited state energy shifts in response to molecular dynamics of pigment-protein complexes

The absorption and energy transfer properties of photosynthetic pigments are strongly influenced by their local environment or “site.” Local electrostatic fields vary in time with protein and chromophore molecular movement and thus transiently influence the excited state transition properties of individual chromophores. Site-specific information is experimentally inaccessible in many light-harvesting pigment–proteins due to multiple chromophores with overlapping spectra. Full quantum mechanical calculations of each chromophores excited state properties are too computationally demanding to efficiently calculate the changing excitation energies along a molecular dynamics trajectory in a pigment–protein complex. A simplified calculation of electrostatic interactions with each chromophores ground to excited state transition, the so-called charge density coupling (CDC) for site energy, CDC, has previously been developed to address this problem. We compared CDC to more rigorous quantum chemical calculations to determine its accuracy in computing excited state energy shifts and their fluctuations within a molecular dynamics simulation of the bacteriochlorophyll containing light-harvesting Fenna–Mathews–Olson (FMO) protein. In most cases CDC calculations differed from quantum mechanical (QM) calculations in predicting both excited state energy and its fluctuations. The discrepancies arose from the inability of CDC to account for the differing effects of charge on ground and excited state electron orbitals. Results of our study show that QM calculations are indispensible for site energy computations and the quantification of contributions from different parts of the system to the overall site energy shift. We suggest an extension of QM/MM methodology of site energy shift calculations capable of accounting for long-range electrostatic potential contributions from the whole system, including solvent and ions.     

Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> strain FBR5

Ethanol production by recombinant Escherichia coli strain FBR5 from dilute acid pretreated wheat straw (WS) by separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) was studied. The yield of total sugars from dilute acid (0.5% H₂SO₄) pretreated (160 °C, 10 min) and enzymatically saccharified (pH 5.0, 45 °C, 72 h) WS (86 g/l) was 50.0 ± 1.4 g/l. The hydrolyzate contained 1,184 ± 19 mg furfural and 161 ± 1 mg hydroxymethyl furfural per liter. The recombinant E. coli FBR5 could not grow at all at pH controlled at 4.5 to 6.5 in the non-abated wheat straw hydrolyzate (WSH) at 35 °C. However, it produced 21.9 ± 0.3 g ethanol from non-abated WSH (total sugars, 44.1 ± 0.4 g/l) in 90 h including the lag time of 24 h at controlled pH 7.0 and 35 °C. The bioabatement of WS was performed by growing Coniochaeta ligniaria NRRL 30616 in the liquid portion of the pretreated WS aerobically at pH 6.5 and 30 °C for 15 h. The bacterium produced 21.6 ± 0.5 g ethanol per liter in 40 h from the bioabated enzymatically saccharified WSH (total sugars, 44.1 ± 0.4 g) at pH 6.0. It produced 24.9 ± 0.3 g ethanol in 96 h and 26.7 ± 0.0 g ethanol in 72 h per liter from bioabated WSH by batch SSF and fed-batch SSF, respectively. SSF offered a distinct advantage over SHF with respect to reducing total time required to produce ethanol from the bioabated WS. Also, fed-batch SSF performed better than the batch SSF with respect to shortening the time requirement and increase in ethanol yield.     

Tetra primer ARMS-PCR relates folate/homocysteine pathway genes and ACE gene polymorphism with coronary artery disease

Cardiovascular disorders and coronary artery disease (CAD) are significant contributors to morbidity and mortality in heart patients. As genes of the folate/homocysteine pathway have been linked with the vascular disease, we investigated association of these gene polymorphisms with CAD/myocardial infarction (MI) using the novel approach of tetraprimer ARMS-PCR. A total of 230 participants (129 MI cases, 101 normal subjects) were recruited. We genotyped rs1801133 and rs1801131 SNPs in 5'10' methylenetetrahydrofolate reductase (MTHFR), rs1805087 SNP in 5' methyltetrahydrofolate homocysteine methyltransferase (MTR), rs662 SNP in paroxanse1 (PON1), and rs5742905 polymorphism in cystathionine beta synthase (CBS). Angiotensin converting enzyme (ACE) insertion/deletion polymorphism was detected through conventional PCR. Covariates included blood pressure, fasting blood sugar, serum cholesterol, and creatinine concentrations. Our results showed allele frequencies at rs1801133, rs1801131, rs1805087 and the ACE insertion/deletion (I/D) polymorphism varied between cases and controls. Logistic regression, after adjusting for covariates, demonstrated significant associations of rs1801133 and rs1805087 with CAD in the additive, dominant, and genotype model. In contrast, ACE I/D polymorphism was significantly related with CAD where recessive model was applied. Gene-gene interaction against the disease status revealed two polymorphism groups: rs1801133, rs662, and rs1805087; and rs1801131, rs662, and ACE I/D. Only the latter interaction maintained significance after adjusted for covariates. Our study concludes that folate pathway variants exert contributory influence on susceptibility to CAD. We further suggest that tetraprimer ARMS-PCR successfully resolves the genotypes in selected samples and might prove to be a superior technique compared to the conventional approach.     

The Cry Toxin Operon of Clostridium bifermentans subsp. malaysia Is Highly Toxic to Aedes Larval Mosquitoes

The management and control of mosquito vectors of human disease currently rely primarily on chemical insecticides. However, larvicidal treatments can be effective, and if based on biological insecticides, they can also ameliorate the risk posed to human health by chemical insecticides. The aerobic bacteria Bacillus thuringiensis and Lysinibacillus sphaericus have been used for vector control for a number of decades. But a more cost-effective use would be an anaerobic bacterium because of the ease with which these can be cultured. More recently, the anaerobic bacterium Clostridium bifermentans subsp. malaysia has been reported to have high mosquitocidal activity, and a number of proteins were identified as potentially mosquitocidal. However, the cloned proteins showed no mosquitocidal activity. We show here that four toxins encoded by the Cry operon, Cry16A, Cry17A, Cbm17.1, and Cbm17.2, are all required for toxicity, and these toxins collectively show remarkable selectivity for Aedes rather than Anopheles mosquitoes, even though C. bifermentans subsp. malaysia is more toxic to Anopheles. Hence, toxins that target Anopheles are different from those expressed by the Cry operon.