Production of poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyvalerate) P(3HB-<Emphasis Type="Italic">co</Emphasis>-3HV) with a broad range of 3HV content at high yields by <Emphasis Type="Italic">Burkholderia sacchari</Emphasis> IPT 189

The biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from sucrose and propionic acid by Burkholderia sacchari IPT 189 was studied using a two-stage bioreactor process. In the first stage, this bacterium was cultivated in a balanced culture medium until sucrose exhaustion. In the second stage, a solution containing sucrose and propionic acid as carbon source was fed to the bioreactor at various sucrose/propionic acid (s/p) ratios at a constant specific flow rate. Copolymers with 3HV content ranging from 40 down to 6.5 (mol%) were obtained with 3HV yield from propionic acid (Y _3HV/prop) increasing from 1.10 to 1.34 g g⁻¹. Copolymer productivity of 1 g l⁻¹ h⁻¹ was obtained with polymer biomass content rising up to 60% by increasing a specific flow rate at a constant s/p ratio. Increasing values of 3HV content were obtained by varying the s/p ratios. A simulation of production costs considering Y _3HV/prop obtained in the present work indicated that a reduction of up to 73% can be reached, approximating US$ 1.00 per kg which is closer to the value to produce P3HB from sucrose (US$ 0.75 per kg).     

Performance,kinetics, and substrate utilization in a continuous yeast fermentation with cell recycle by ultrafiltration membranes

Summary A continuous single stage yeast fermentation with cell recycle by ultrafiltration membranes was operated at various recycle ratios. Cell concentration was increased 10.6 times, and ethanol concentration and fermentor productivity both 5.3 times with 97% recycle as compared to no recycle. Both specific growth rate and specific ethanol productivity followed the exponential ethanol inhibition form (specific productivity was constant up to 37.5 g/l of ethanol before decreasing), similar to that obtained without recycle, but with greater inhibition constants most likely due to toxins retained in the system at hight recycle ratios.By analyzing steady state data, the fractions of substrate used for cell growth, ethanol formation, and what which were wasted were accounted for. Yeast metabolism varied from mostly aerobic at low recycle ratios to mostly anaerobic at high recycle ratios at a constant dissolved oxygen concentration of 0.8 mg/kg. By increasing the cell recycle ratio, wasted substrate was reduced. When applied to ethanol fermentation, the familiar terminology of substrate used for Maintenance must be used with caution: it is not the same as the wasted substrate reported here.A general method for determining the best recycle ratio is presented; a balance among fermentor productivity, specific productivity, and wasted substrate needs to be made in recycle systems to approach an optimal design.Nomenclature B Bleed flow rate, l/h - C _T Concentration of toxins, arbitrary units - D Dilution rate, h^-1 - F Filtrate or permeate flow rate, removed from system, l/h - F _o Total feed flow rate to system, l/h - K _s Monod form constant, g/l - P Product (ethanol) concentration, g/l - P _o Ethanol concentration in feed, g/l - PP} Adjusted product concentration, g/l - PD Fermentor productivity, g/l-h - R Recycle ratio, F/F _o - S Substrate concentration in fermentor, g/l - S _o Substrate concentration in feed, g/l - V Working volume of fermentor, l - V _MB Viability based on methylene blue test - X Cell concentration, g dry cell/l - X _o Cell concentration in feed, g/l - Y _ATP Cellular yield from ATP, g cells/mol ATP - Y _ATPS Yield of ATP from substrate, mole ATP/mole glucose - Y _G True growth yield or maximum yield of cells from substrate, g cell/g glucose - Y _P Maximum theoretical yield of ethanol from glucose, 0.511 g ethanol/g glucose - Y _P/S Experimental yield of product from substrate, g ethanol/g glucose - Y _x/s Experimental yield of cells from substrate, g cell/g glucose - S _NP/X Non-product associated substrate utilization, g glucose/g cell - k ₁, k₂, k₃, k₄ Constants - k ₁ ^APP , k ₂ ^APP Apparent k ₁, k₃ - k ₁ ^TRUE True k ₁ - m Maintenance coefficient, g glucose/g cell-h - m ^* Coefficient of substrate not used for growth nor for ethanol formation, g glucose/g cell-h - Specific growth rate, g cells/g cells-h, reported as h^-1 - _m Maximum specific growth rate, h^-1 - v Specific productivity, g ethanol/g cell-h, reported as h^-1 - v _m Maximum specific productivity, h^-1     

Methane Metabolism of the Obligate Methane-utilizing Bacterium,Methylomonas flagellata,in Methane-limited and Oxygen-limited Chemostat Culture

The effect of a limited supply of methane and oxygen on growth of Methylomonas flagellata was analyzed in chemostat culture. A significant decrease in Y_CH4 was observed under oxygen-limitation. Accumulation of formaldehyde is supposed to cause a decrease of Y_CH4. A low level of formaldehyde oxidizing activity in cells grown under oxygen-limited steady-states suggests that formaldehyde dehydrogenase is repressed under oxygen deficiency. In spite of the suppression of respiration, methane oxidation progressed independently under oxygen-limitation. These facts support the idea that methanol oxidation couples with methane oxidation (a mono-oxygenase system) in this microorganism.     

Energetics of the growth of Methanococcus thermolithotrophicus

Methanococcus thermolithotrophicus was grown in a mineral salts medium at 65° C in a fermenter gassed with H₂ and CO₂, which were the sole carbon and energy sources. Evolution of growth parameters during batch culture experiments showed the existence of an uncoupling phenomenon. The growth was then studied using a continuous technique and steady states for various gas flow rates were obtained. Y _{CH 4}and the maintenance coefficient varied with the gas input. The maximum Y _{CH 4} determined for Methanococcus thermolithotrophicus was 3.33 g·mol^-1 CH₄. An excess of energy and carbon sources induced uncoupling of growth.     

Pyruvate — a novel substrate for growth and methane formation in Methanosarcina barkeri

Methanosarcina barkeri strain Fusaro was found to grow on pyruvate as sole carbon and energy source after an incubation period of 10–12 weeks in the presence of high pyruvate concentrations (100 mM). Growth studies, cell suspension experiments and enzymatic investigations were performed with pyruvate-utilizing M. barkeri. For comparison acetate-adapted cells of M. barkeri were analyzed.

1. Pyruvate-utilizing M. barkeri grew on pyruvate (100 mM) with an initial doubling time of about 25 h (37 °C, pH 6.5) up to cell densities of about 0.8 g cell dry weight/l. The specific growth rate was linearily dependent on the pyruvate concentration up to 100 mM indicating that pyruvate was taken up by passive diffusion. Only CO₂ and CH₄ were detected as fermentation products. As calculated from fermentation balances pyruvate was converted to CH₄ and CO₂ according to following equation: Pyruvate^-+H⁺+0.5 H₂O » 1.25 CH₄+1.75 CO₂. The molar growth yield (Ych ₄) was about 14 g dry weight cells/mol CH₄. In contrast the growth yield (Ych ₄) of M. barkeri during growth on acctate (Acetate^-+H⁺ » CH₄+CO₂) was about 3 g/mol CH₄.
2. Cell suspensions of pyruvate-grown M. barkeri catalyzed the conversion of pyruvate to CH₄, CO₂ and H₂ (5–15 nmol pyruvate consumed/min x mg protein). At low cell concentrations (0.5 mg protein/ml) 1 mol pyruvate was converted to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂. At higher cell concentration less H₂ and CO₂ and more CH₄ were formed due to CH₄ formation from H₂/CO₂. The rate of pyruvate conversion was linearily dependent on the pyruvate concentration up to about 30 mM. Cell suspensions of acetate-grown M. barkeri also catalyzed the conversion of 1 mol pyruvate to 1 mol CH₄, 2 mol CO₂ and 1 mol H₂ at similar rates and with similar affinity for pyruvate as pyruvate-grown cells.
3. Cell extracts of both pyruvate-grown and acetate-grown M. barkeri contained pyruvate: ferredoxin oxidoreductase. The specific activity in pyruvate-grown cells (0.8 U/mg) was 8-fold higher than in acetate-grown cells (0.1 U/mg). Coenzyme F₄₂₀ was excluded as primary electron acceptor of pyruvate oxidoreductase. Cell extracts of pyruvate-grown M. barkeri contained carbon monoxide dehydrogenase activity and hydrogenase activity catalyzing the reduction by carbon monoxide and hydrogen of both methylviologen and ferredoxin (from Clostridium).

This is the first report on growth of a methanogen on pyruvate as sole carbon and energy source, i.e. on a substrate more complex than acetate.     

Quantification of methane oxidation in the rhizosphere of emergent aquatic macrophytes: defining upper limits

Rates of rhizospheric methane oxidation were evaluated by aerobic incubations of subcores collected in flooded anoxic soils populated by emergent macrophytes, by greenhouse whole plant incubations, and by CH₄ stable isotopic analysis. Subcore incubations defined upper limits for rhizospheric methane oxidation on an areal basis which were equal to or greater than emission rates. These rates are considered upper limits because O₂ did not limit CH₄ uptake as is likely to occur in situ. The ratio of maximum potential methane oxidation (MO) to methane emission (ME) ranged from 0.7 to 1.9 in Louisiana rice (Oryza sativa), from 1.0 to 4.0 in a N. Florida Sagittaria lancifolia marsh, and from 5.6 to 51 in Everglades Typha domingensis and Cladium jamaicense areas. Methane oxidation/methane emission ratios determined in whole plant incubations of Sagittaria lancifolia under oxic and anoxic conditions ranged from 0.5 to 1.6. Methane oxidation activity associated with emergent aquatic macrophytes was found primarily in fine root material. A weak correlation was observed between live root biomass and CH₄ uptake in Typha. Rhizomes showed small or zero rates of methane uptake and no uptake was associated with plant stems. Methane stable isotope data from a S. lancifolia marsh were as follows: CH₄ emitted from plants: −61.6 ± 0.3%; CH₄ within stems: −42.0 ± 0.2%; CH₄ within sedimentary bubbles: −51.7 ± 0.3%). The ¹³C enrichment observed relative to emitted CH₄ could be due to preferential mobilization of CH₄ containing the lighter isotope and/or the action of methanotrophic bacteria.     

Kinetic studies of constitutive human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression in continuous culture of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Extracellular human granulocyte-macrophage colony stimulating factor (hGM-CSF) expression was studied under the control of the GAP promoter in recombinant Pichia pastoris in a series of continuous culture runs (dilution rates from 0.025 to 0.2 h⁻¹). The inlet feed concentration was also varied and the steady state biomass concentration increased proportionally demonstrating efficient substrate utilization and constancy of the biomass yield coefficient (Y_x/s) for a given dilution rate. The specific product formation rate (q_P) showed a strong correlation with dilution rates demonstrating growth associated product formation of hGM-CSF. The volumetric product concentration achieved at the highest feed concentration (4×) and a dilution rate of 0.2 h⁻¹ was 82 mg l⁻¹ which was 5-fold higher compared to the continuous culture run with 1× feed concentration at the lowest dilution rate thus translating to a 40 fold increase in the volumetric productivity. The specific product yield (Y_P/X) increased slightly from 2 to 2.5 mg g⁻¹, with increasing dilution rates, while it remained fairly invariant, for all feed concentrations demonstrating negligible product degradation or feed back inhibition. The robust nature of this expression system would make it easily amenable to scale up for industrial production.     

Determinants of spatial variability of methane emissions from wet grasslands on peat soil

Methane (CH₄) emissions from soils, representing the consequence of CH₄ production, CH₄ consumption and CH₄ transport, are poorly characterised and show a large spatial variability. This study aimed to assess the determinants of field-scale spatial variability of CH₄ emissions from wet grasslands on peat soil. Mean CH₄ emission rates of a three-year experiment at 18 plots distributed over three sites in the nature preserve Nieuwkoopse Plassen on peat soil in the Netherlands were related to CH₄ production and CH₄ consumption capacities of soil layers, and to soil and vegetation characteristics. Spatial variability of CH₄ emissions and possible determining factors was high. Annual CH₄ emissions ranged from 3 to 37 g CH₄ m^–2 yr^–1. Coefficients of variation (CV) of CH₄ emissions were on average 37% among sites and 83% within sites. Most important determinants of spatial variability were CH₄ production capacity (average: 211 ng CH₄ g^–1 dry soil h^–1; CV: 131%) and aboveground biomass of sedges (Carex spp.) (average: 0.45 g dm^–2; CV: 127%) (P<0.01). Sedges may affect CH₄ emissions by stimulating CH₄ transport from anaerobic layers to the surface via their vascular system and/or by serving as substrate for methanogens. For extrapolation of CH₄ emissions to larger areas, best results will be obtained by using factors that are easy to determine, like vegetation.     

Diel methane emissions in stands of Spartina alterniflora and Suaeda salsa from a coastal salt marsh

In this study, we aimed to understand the influence of plant type on the monthly variations of diel CH₄ fluxes from Spartina alterniflora and Suaeda salsa of coastal salt marshes at three growth stages (July, August and September). Dissolved CH₄ concentrations in porewater and sediment redox potentials were monitored, as were aboveground plant biomass and stem densities. CH₄ fluxes exhibited clear monthly variations and peaked in September in the S. alterniflora and S. salsa mesocosms. However, no discernible diel variation was observed in the CH₄ flux in the S. salsa mesocosm, probably due to its weak gas transport capacity. By contrast, notable diel variations of CH₄ flux with the peak of 1.42 and 3.67 mg CH₄ m⁻² h⁻¹ at 12:00 and the lowest of 0.75 and 2.11 mg CH₄ m⁻² h⁻¹ at 3:00 or 6:00 were observed in the S. alterniflora mesocosm on 11 August and 11 September, respectively, but not in July mainly due to low plant biomass masking diel variations in the porewater CH₄ concentration. The ratios of the maximum flux to minimum flux over the course of the day in the S. alterniflora mesocosm on 10 July, 11 August and 11 September were 1.28, 1.89 and 1.76, respectively, and corresponding values for porewater CH₄ concentration were 1.31, 1.39 and 1.17, respectively. CH₄ flux significantly correlated with CH₄ concentration in porewater, and both were significantly related to air temperature. These findings indicate that CH₄ production and CH₄ flux at the middle growth stage (August) exhibited greater responses to changes in air temperature, which in turn induced the higher diel variation. The higher diel cycle for CH₄ flux in August than in September was likely due to the higher proportion of CH₄ oxidized during diffusion within the aerenchyma system. Our results suggest that the extent of diel variations in CH₄ flux may have depended on the gas transport capacity of plants, and the highest diel variation occurred at the middle growth stage.     

Pelagic denitrification and methane oxidation in oxygen-depleted waters of the Louisiana shelf

Anthropogenic nutrient inputs fuel eutrophication and hypoxia ([O₂]?<?2 mg L^?1), threatening coastal and near shore environments across the globe. The world’s second largest anthropogenic coastal hypoxic zone occurs annually along the Louisiana (LA) shelf. Springtime loading of dissolved inorganic nitrogen (DIN) from the Mississippi River, combined with summertime stratification and increased water residence time on the shelf, promotes establishment of an extensive hypoxic zone that persists throughout the summer. We investigated the patterns of pelagic denitrification and methane (CH₄) oxidation along the LA shelf. Microbial activity rates were determined along with concentrations of dissolved nutrients and greenhouse gases, nitrous oxide (N₂O) and CH₄, during summer in 2013, 2015, and 2016. We documented denitrification rates up to 1900 nmol N L^?1 d^?1 and CH₄ oxidation rates as high as 192 nmol L^?1 d^?1 in hypoxic waters characterized by high concentrations of N₂O (range: 1 to 102 nM) and CH₄ (range: 3 to 641 nM). Ecosystem scaling estimates suggest that pelagic denitrification could remove between 0.1 and 47% of the DIN input from the Mississippi River, whereas CH₄ oxidation does not function as an effective removal process with CH₄ escaping to the atmosphere. Denitrification and CH₄ oxidizing bacteria within the LA shelf hypoxic zone were largely unable to keep up with the DIN and CH₄ inputs to the water column. Rates were variable and physiochemical dynamics appeared to regulate the microbial removal capacity for both nitrate/nitrite and CH₄ in this ecosystem.

     

Biochemical methane potential (BMP) test for thickened sludge using anaerobic granular sludge at different inoculum/substrate ratios

The biochemical methane potential (BMP) test for thickened sludge was evaluated at three different inoculum/substrate (I:S) ratios. The cumulative methane yield was 51.4 mL CH₄/g VS_added at an I:S ratio of 1:1, 76.3 mL CH₄/g VS_added at an I:S ratio of 1:3, and 21.9 mL CH₄/g VS_added at an I:S ratio of 1:8. The greatest ultimate methane yield and methane production rate constant were achieved at an I:S ratio of 1:3, whereas the least was obtained at an I:S ratio of 1:8. The maximum methane production rate constant was 0.38/day and the minimum methane production rate constant was 0.0016/day. For the case of a lower I:S ratio, the biomass activity may be affected due to the low substrate concentration. On the other hand, for the case of higher I:S ratios, anaerobic digestion of thickened sludge was inhibited by higher concentrations of volatile fatty acids and lower pH.     

Seasonal variation in pathways of CH4 production and in CH4 oxidation in rice fields determined by stable carbon isotopes and specific inhibitors

Flooded rice fields, which are an important source of the atmospheric methane, have become a model system for the study of interactions between various microbial processes. We used a combination of stable carbon isotope measurements and application of specific inhibitors in order to investigate the importance of various methanogenic pathways and of CH₄ oxidation for controlling CH₄ emission. The fraction of CH₄ produced from acetate and H₂/CO₂ was calculated from the isotopic signatures of acetate, carbon dioxide (CO₂) and methane (CH₄) measured in porewater, gas bubbles, in the aerenchyma of the plants and/or in incubation experiments. The calculated ratio between both pathways reflected well the ratio determined by application of methyl fluoride (CH₃F) as specific inhibitor of acetate‐dependent methanogenesis. Only at the end of the season, the theoretical ratio of acetate: H₂ = 2 : 1 was reached, whereas at the beginning H₂/CO₂‐dependent methanogenesis dominated. The isotope discrimination was different between rooted surface soil and unrooted deep soil. Root‐associated CH₄ production was mainly driven by H₂/CO₂. Porewater CH₄ was found to be a poor proxy for produced CH₄. The fraction of CH₄ oxidised was calculated from the isotopic signature of CH₄ produced in vitro compared to CH₄ emitted in situ, corrected for the fractionation during the passage from the aerenchyma to the atmosphere. Isotope mass balances and in situ inhibition experiments with difluoromethane (CH₂F₂) as specific inhibitor of methanotrophic bacteria agreed that CH₄ oxidation was quantitatively important at the beginning of the season, but decreased later. The seasonal pattern was consistent with the change of potential CH₄ oxidation rates measured in vitro. At the end of the season, isotope techniques detected an increase of oxidation activity that was too small to be measured with the flux‐based inhibitor technique. If porewater CH₄ was used as a proxy of produced CH₄, neither magnitude nor seasonal pattern of in situ CH₄ oxidation could be reproduced. An oxidation signal was also found in the isotopic signature of CH₄ from gas bubbles that were released by natural ebullition. In contrast, bubbles stirred up from the bulk soil had preserved the isotopic signature of the originally produced CH₄.     

über den Einfluß von Diäthylstilboestrol und anderer Wirkstoffe auf den Stoffwechsel männlicher Ratten

The presented study aimed at investigating the residual feed intake (RFI) of Sahiwal calves, nutrient utilisation as affected by RFI and its relationship with methane (CH₄) emissions and some blood metabolites. Eighteen male Sahiwal calves (10–18 months of age; mean body weight 133 kg) were fed ad libitum with a total mixed ration. After calculating RFI for individual calves (?0.40 to +0.34 kg DM/d), they were divided into three groups with low, medium and high RFI, respectively. Dry matter intake (DMI) was higher (p < 0.05) in Group High RFI, whereas digestibility of all nutrients except crude protein and ether extract was significantly higher in Group Low RFI. Nitrogen balance was also significantly higher in Group Low RFI (20.2 g/d) than in Group High RFI (17.0 g/d). Average daily gain and feed conversion ratio were similar among the groups. With exception of glucose, concentrations of all measured blood metabolites were higher in Group High RFI (p < 0.05). Compared with Group High RFI, the CH₄ emission of Group Low RFI was significantly lower (on the basis g/d and g/kg DMI by 11% and 19%, respectively). Furthermore, the CH₄ emission [g/d] was significantly correlated with RFI (r = 0.77). Because higher feed efficiency and less CH₄ production were observed in Group Low RFI, it was concluded that RFI can be used as a measure of feed efficiency, which has a potential to select Sahiwal calves for lowered CH₄ emissions.     

The mechanism of sex determination in Rumex acetosella

Summary Cytogenetic studies were made with particular emphasis on the sex-determining mechanism in Rumex acetosella (6 x = 42) and its hybrids (F ₁, F ₂, BC ₁ and BC ₂) with R. hastatulus (synthetic 4 x = 16 = 4 A +4 X = and 4 x = 18 = 4 A + 2 (X Y ₁ Y ₂) = ). Rumex acetosella was almost strictly dioecious with 5050 male and female. Breeding tests revealed that the males were heterogametic. The longest chromosomes (S), usually two, are the sex chromosomes of this hexaploid species. The S chromosomes are homomorphic in both male and female. The sex chromosome: autosome ratios, and the strong epistatic male effect of the S ^M chromosome in the polyploid dioecious species and in the hybrids, are evidence of an X/Y Melandrium type sex-determining mechanism controlled by a single pair of homomorphic sex chromosomes. Thus, the sex chromosome formula of the males was S ^F S ^M and that of females was S ^F S ^F. The present approach is a new method for resolving the sex-determining mechanism in a dioecious species.     

Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal

n‐Butanol was produced continuously in a two‐stage fermentor system with integrated product removal from a co‐feed of n‐butyric acid and glucose. Glucose was always required as a source of ATP and electrons for the conversion of n‐butyrate to n‐butanol and for biomass growth; for the latter it also served as a carbon source. The first stage generated metabolically active planktonic cells of Clostridium saccharoperbutylacetonicum strain N1‐4 that were continuously fed into the second (production) stage; the volumetric ratio of the two fermentors was 1:10. n‐Butanol was removed continuously from the second stage via gas stripping. Implementing a two‐stage process was observed to dramatically dampen metabolic oscillations (i.e., periodical changes of solventogenic activity). Culture degeneration (i.e., an irreversible loss of solventogenic activity) was avoided by periodical heat shocking and re‐inoculating stage 1 and by maintaining the concentration of undissociated n‐butyric acid in stage 2 at 3.4 mM with a pH‐auxostat. The system was successfully operated for 42 days during which 93% of the fed n‐butyrate was converted to n‐butanol at a production rate of 0.39 g/(L × h). The molar yields Y_{n‐butanol/n‐butyrate} and Y_{n‐butanol/glucose} were 2.0, and 0.718, respectively. For the same run, the molar ratio of n‐butyrate to glucose consumed was 0.358. The molar yield of carbon in n‐butanol produced from carbon in n‐butyrate and glucose consumed (Y_{n‐butanol/carbon}) was 0.386. These data illustrate that conversion of n‐butyrate into n‐butanol by solventogenic Clostridium species is feasible and that this can be performed in a continuous system operating for longer than a month. However, our data also demonstrate that a relatively large amount of glucose is required to supply electrons and ATP for this conversion and for cell growth in a continuous culture. Biotechnol. Bioeng. 2012; 109:913–921. © 2011 Wiley Periodicals, Inc.     

Cytogenetic,hybridization and molecular evidence of four cytological forms of Anopheles nigerrimus (Hyrcanus Group) in Thailand and Cambodia

Thirteen isoline colonies of Anopheles nigerrimus were established from individual wild‐caught females collected from cow‐baited traps at locations in Thailand and Cambodia. Three types of X (X₁, X₂, X₃) and 4 types of Y (Y₁, Y₂, Y₃, Y₄) chromosomes were recovered, according to differing amounts of extra heterochromatin. Four karyotypic forms were designed depending upon apparently distinct figures of X and Y chromosomes, i.e., Form A (X₁, X₂, X₃, Y₁), B (X₂, X₃, Y₂), C (X₁, Y₃), and D (X₃, Y₄). Forms C and D were new metaphase karyotypes discovered in this study. Form A appeared to be common in both Thailand and Cambodia. Forms B and D were found to be rather specific to southern and northeastern Thailand, respectively, whereas Form C was confined to Cambodia. Hybridization experiments among the eight isoline colonies, which were representative of four karyotypic forms of An. nigerrimus, demonstrated genetic compatibility in giving viable progenies and synaptic salivary gland polytene chromosomes through F₂‐generations. These results elucidated the conspecific relationship, comprising four cytological forms within this taxon. Supportive evidence was confirmed further by very low intraspecific sequence variations (average genetic distance = 0.002–0.007) of the nucleotide sequences in ribosomal DNA [second internal transcribed spacer (ITS2)] and mitochondrial DNA [cytochrome c oxidase subunit I (COI) and subunit II (COII)].     

Synthesis and Insecticidal Activity of 4-Substituted 1-Indanyl Chrysanthemates

A variety of 4-substituted 1-indanyl chrysanthemates were prepared and their insecticidal activity was tested on American cockroachs. The activity of the chrysanthemates decreased in the following order: CH₂=CHCH₂>CH₃OCH₂?CH₃CH₂?HC≡CCH₂>PhCH₂, which was similar to that of p-substituted benzyl chrysanthemates against houseflies with the exception of the propargyl group. Formulation of the quantative structure-activity relationship by the Hansch’s program indicated that Van der Waals interaction between the chemical substance and the macromolecular in vivo play an important role in the 1-indanyl chrysanthemates.     

Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440

Four automatic substrate feeding strategies were developed and investigated in this study to obtain rapid, repeatable, and reliable high cell densities of Pseudomonas putida KT2440 from glucose. Growth yield data of the key nutrients, Y _X/Glucose, Y _X/NH4, Y _X/PO4, Y _X/Mg, and Y _CO2/Glucose, were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g g⁻¹, respectively. Although standard exponential feeding strategy worked well when the predetermined μ was set at 0.25 h⁻¹, an exponential glucose feeding strategy with online μ _max estimation resulted in a higher average biomass productivity (3.4 vs 2.8 g l⁻¹ h⁻¹). A CO₂ production rate based pulse glucose feeding strategy also resulted in good overall productivity (3.0 g l⁻¹ h⁻¹) and can be used as an alternative to pH-stat or DO-stat feeding. A cumulative CO₂ production based continuous feed with real-time cumulative glucose consumption estimation resulted in much higher biomass productivity (4.3 g l⁻¹ h⁻¹) and appears to be an excellent and reliable approach to fully automating high-cell-density fed-batch cultivation of P. putida.     

Formate and glucose stimulation of methane and hydrogen production in rumen liquor

Membrane-inlet mass spectrometry was used to investigate the effects of increasing the concentration of the rumen metabolites, formate and glucose, upon CH₄ and H₂ production during fermentation by unfractionated rumen liquor. Additions of formate up to 3.6 mM stimulated CH₄ and then excess H₂ production. Each addition caused a large accumulation of H₂ (>40 µM), which returned to in situ concentrations after periods of more than 1 h. Glucose additions up to 2.0 mM gave linear increases in CH₄ and H₂ production. The conversion of substrate carbon into CH₄ was found to decrease from 34% to 9% for formate, as concentrations were increased (1.6–3.6 mM); approximately 13.5% of the glucose carbon was converted to CH₄.