Utilization of cellulosic waste from tequila bagasse and production of polyhydroxyalkanoate (PHA) bioplastics by Saccharophagus degradans

Utilization of wastes from agriculture is becoming increasingly important due to concerns of environmental impact. The goals of this work were to evaluate the ability of an unusual organism, Saccharophagus degradans (ATCC 43961), to degrade the major components of plant cell walls and to evaluate the ability of S. degradans to produce polyhydroxyalkanoates (PHAs, also known as bioplastics). S. degradans can readily attach to cellulosic fibers, degrade the cellulose, and utilize this as the primary carbon source. The growth of S. degradans was assessed in minimal media (MM) containing glucose, cellobiose, avicel, and bagasse with all able to support growth. Cells were able to attach to avicel and bagasse fibers; however, growth on these insoluble fibers was much slower and led to a lower maximal biomass production than observed with simple sugars. Lignin in MM alone did not support growth, but did support growth upon addition of glucose, although with an increased adaptation phase. When culture conditions were switched to a nitrogen depleted status, PHA production commences and extends for at least 48 h. At early stationary phase, stained inclusion bodies were visible and two chronologically increasing infrared light absorbance peaks at 1,725 and 1,741 cm(-1) confirmed the presence of PHAs. This work demonstrates for what we believe to be the first time, that a single organism can degrade insoluble cellulose and under similar conditions can produce and accumulate PHA. Additional work is necessary to more fully characterize these capabilities and to optimize the PHA production and purification.     

Genomic and proteomic analyses of the agarolytic system expressed by Saccharophagus degradans 2-40

Saccharophagus degradans 2-40 (formerly Microbulbifer degradans 2-40) is a marine gamma-subgroup proteobacterium capable of degrading many complex polysaccharides, such as agar. While several agarolytic systems have been characterized biochemically, the genetics of agarolytic systems have been only partially determined. By use of genomic, proteomic, and genetic approaches, the components of the S. degradans 2-40 agarolytic system were identified. Five agarases were identified in the S. degradans 2-40 genome. Aga50A and Aga50D include GH50 domains. Aga86C and Aga86E contain GH86 domains, whereas Aga16B carries a GH16 domain. Novel family 6 carbohydrate binding modules (CBM6) were identified in Aga16B and Aga86E. Aga86C has an amino-terminal acylation site, suggesting that it is surface associated. Aga16B, Aga86C, and Aga86E were detected by mass spectrometry in agarolytic fractions obtained from culture filtrates of agar-grown cells. Deletion analysis revealed that aga50A and aga86E were essential for the metabolism of agarose. Aga16B was shown to endolytically degrade agarose to release neoagarotetraose, similarly to a beta-agarase I, whereas Aga86E was demonstrated to exolytically degrade agarose to form neoagarobiose. The agarolytic system of S. degradans 2-40 is thus predicted to be composed of a secreted endo-acting GH16-dependent depolymerase, a surface-associated GH50-dependent depolymerase, an exo-acting GH86-dependent agarase, and an alpha-neoagarobiose hydrolase to release galactose from agarose.     

BioPEGylation of polyhydroxyalkanoates: influence on properties and satellite-stem cell cycle

The addition of poly(ethylene glycol), PEG, to bioprocessing systems producing polyhydroxyalkanoates (PHAs), has been reported as a means of their molecular weight control and can also support bioPEGylation, resulting in hybrids with amphiphillic properties. However, the study of such natural-synthetic hybrids of PHA-b-PEG is still in its infancy. In this study, we report the influence of bioPEGylation of polyhydroxyoctanoate (PHO) on its physiochemical, material, and biological properties. Consistent with previous studies, bioPEGylation with diethylene glycol (DEG) showed a significant reduction in PHA molecular weight (57%). In comparison to solvent cast films of PHO, PHO-b-DEG films possessed a noticeable X-ray diffraction peak at 9.82 degrees and increased Young's modulus of 11 Gpa (83%). Potential biocompatibility was investigated by measuring the early phase of apoptosis in myoblastic satellite-stem cells (C2C12). Comparative analysis of cell proliferation and progression in the presence of the mcl-PHA and its hybrid showed that the latter induced significant cell cycle progression: the first time a biomaterial has been shown to do so. Microtopographies of the film surfaces demonstrated that these differences were not due to changes in surface morphology; both polymers possessed average surface rugosities of 1.4 +/- 0.2 microm. However, a slight decrease in surface hydrophobicity (3.5 +/- 0.9 degrees) due to the hydrophilic DEG may have exerted an influence. The results support the further study of bioPEGylated PHAs as potential biomaterials in the field of tissue engineering.     

Effect of one-step sucrose dilution of glycerol on in-vitro development of frozen-thawed mouse embyros

Several glycerol (GLY) dilution treatments were compared using frozen-thawed early blastocysts from Swiss Webster mice. These treatments consisted of 0.00 (0.00S n = 68), 0.25 (0.25S n = 67), 0.50 (0.50S n = 76), 0.75 (0.75S n = 66), 1.00 (1.00S n = 59), and 1.25 (1.25S n = 60) M of sucrose to remove GLY from embryos in one step. Then the one step procedure was compared with a three-step GLY dilution treatment (C n = 66). Embryos were exposed to 1.5 M of GLY in three-steps, frozen according to a standard freezing technique and stored at -196 degrees C. Embryos were thawed in a 37 degrees C water bath, pooled, and those graded good or excellent were randomly assigned to the experimental groups. The blastocysts were cultured in Whitten's medium microdrops under paraffin oil in a water saturated 5% CO(2) air atmosphere at 37 degrees C. The proportion (%) of embryos developing to expanded blastocysts was lowest (P < 0.05) in treatment 0.00S (63.1 +/- 4.0). The 0.25S (72.0 +/- 4.3) and 0.50S (75.0 +/- 3.1) 0.75S (79.0 +/- 4.4) treatments yielded a similar percentage of expanded blastocysts. The 1.00S treatment (87.0 +/- 4.0) was similar to 0.75S and 1.25S (98.3 +/- 4.0) treatments. The C treatment was superior (P < 0.05) to dilutions done with < 0.75 M sucrose, similar to 0.75S and 1.00S, and inferior (P < 0.05) to 1.25S. This later treatment yielded the highest percentage of expanded blastocysts. The percentage of embryos that hatched in treatments 0.00S, 0.25S, 0.50S, 0.75S and C was lower (P < 0.05) compared to 1.00S and 1.25S. The percentage of embryos and hatched blastocysts increased linearly (P < 0.01) from 0.0 to 1.25 M sucrose. Dilution of GLY with 1 or 1.25 M sucrose yielded better results compared with lower sucrose concentrations or the three-step GLY removal procedure.     

Biodegradation of cis-dichloroethene as the sole carbon source by a beta-proteobacterium

An aerobic bacterium capable of growth on cis-dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata, indicating that the isolate was a beta-proteobacterium. At 20 degrees C, strain JS666 grew on cDCE with a minimum doubling time of 73 +/- 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 +/- 0.2 microM, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride, trans-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.     

Characterization of new isolated <Emphasis Type="Italic">Ralstonia</Emphasis><Emphasis Type="Italic">eutropha</Emphasis> strain A-04 and kinetic study of biodegradable copolyester poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-4-hydroxybutyrate) production

A new isolated bacterial strain A-04 capable of producing high content of polyhydroxyalkanoates (PHAs) was morphologically and taxonomically identified based on biochemical tests and 16S rRNA gene analysis. The isolate is a member of the genus Ralstonia and close to Ralstonia eutropha. Hence, this study has led to the finding of a new and unexplored R. eutropha strain A-04 capable of producing PHAs with reasonable yield. The kinetic study of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] production by the R. eutropha strain A-04 was examined using butyric acid and γ–hydroxybutyric acid as carbon sources. Effects of substrate ratio and mole ratio of carbon to nitrogen (C/N) on kinetic parameters were investigated in shake flask fed-batch cultivation. When C/N was 200, that is, nitrogen deficient condition, the specific production rate of 3-hydroxybutyrate (3HB) showed the highest value, whereas when C/N was in the range between 4 and 20, the maximum specific production rate of 4-hydroxybutyrate (4HB) was obtained. Thus, the synthesis of 3HB was growth-limited production under nitrogen-deficient condition, whereas the synthesis of 4HB was growth-associated production under nitrogen-sufficient condition. The mole fraction of 4HB units increased proportionally as the ratio of γ–hydroxybutyric acid in the feed medium increased at any value of C/N ratio. Based on these kinetic studies, a simple strategy to improve P(3HB-co-4HB) production in shake flask fed-batch cultivation was investigated using C/N and substrate feeding ratio as manipulating variable, and was successfully proved by the experiments. The nucleotide sequence 1,378 bp reported in this study will appear in the GenBank nucleotide sequence database under accession number EF988626.     

<Emphasis Type="Italic">Acinetobacter</Emphasis> sp. strain Ths,a novel psychrotolerant and alkalitolerant bacterium that utilizes hydrocarbon

A novel psychrotolerant, alkalitolerant bacterium, strain Ths, was isolated from a soil sample immersed in hot spring water containing hydrocarbons and grown on a chemically defined medium containing n-tetradecane as the sole carbon source. The isolate grew at 0 degrees C but not at temperatures higher than 45 degrees C; its optimum growth temperature was 27 degrees C. It grew in the pH range of 7-9. The strain utilized C(13)-C(30) n-alkane and fluorene at pH 9 and 4 degrees C. To our knowledge, this is the first report on the bacterium that utilizes a wide range of hydrocarbons at a high pH and a low temperature. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain Ths is closely related to genomic species 6 ATCC 17979 (99.1% similarity), genomic species BJ13/TU14 ATCC 17905 (97.8% similarity), genomic species 9 ATCC 9957 (97.6% similarity) belonging to the genus Acinetobacter and to Acinetobacter calcoaceticus JCM 6842(T) (97.5% similarity). DNA-DNA hybridization revealed that the isolate has 62, 25, 18 and 19% relatedness, respectively, to genomic species 6 ATCC 17979, genomic species BJ13/TU14 ATCC 17905, genomic species 9 ATCC 9957 and A. calcoaceticus, respectively.     

Cloning of agarase gene from non-marine agarolytic bacterium Cellvibrio sp

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.     

Synthesis of PHB by recombinant E. coli harboring an approximately 5 kb genomic DNA fragment from Streptomyces aureofaciens NRRL 2209

An approximately 5.0 kb Sau3A I genomic DNA fragment from Streptomyces aureofaciens NRRL 2209 was cloned in a plasmid vector and introduced into Escherichia coli. The recombinant E. coli accumulated polyhydroxyalkanoates (PHAs) as cytoplasmic inclusions. The accumulated PHA was identified as the isotactic homopolymer of PHB with a molecular weight of 2.85x10(5). Purified PHB granules were spherical with an average size of 1.1 microm and of stable configuration. DSC thermogram suggested high crystalline nature of the polymer. Maximum thermal degradation of the biopolymer occurred between 250 and 340 degrees C. Recombinant E. coli cells preferentially utilized glycerol as the carbon source and accumulated 25-28 times more PHB than the native S. aureofaciens.     

Simultaneous production of polyhydroxyalkanoates and rhamnolipids by Pseudomonas aeruginosa

The feasibility of the simultaneous production of polyhydroxyalkanoates (PHAs) and rhamnolipids, as a novel approach to reduce their production costs, was demonstrated by the cultivation of Pseudomonas aeruginosa IFO3924. Fairly large amounts of PHAs and rhamnolipids were obtained from the bacterial cells and the culture supernatant, respectively. Decanoate was a more suitable carbon source than ethanol and glucose for the simultaneous production, although glucose was suitable for cell growth without an induction period under pH control. The kind of carbon source affected PHA monomer composition markedly and PHA molecular weight slightly. Monorhamnolipids and dirhamnolipids were included in the rhamnolipids extracted from the culture supernatant using decanoate, glucose, or ethanol as the carbon source. Both PHAs and rhamnolipids were synthesized after the growth phase. PHA content in the cell reached a maximum when the carbon source was exhausted. After exhaustion of the carbon source, PHA content decreased rapidly, but rhamnolipid synthesis, which followed PHA synthesis, continued. This resulted in a time lag for the attainment of maximum levels of PHAs and rhamnolipids. The reusability of the cells used in rhamnolipid production was evaluated in the repeated batch culture of P. aeruginosa IFO3924 for the simultaneous production of PHAs and rhamnolipids. High concentrations of rhamnolipids in the culture supernatant were attained at the end of both the first and second batch cultures. High PHA content was achieved in the resting cells that were finally harvested after the second batch. Simultaneous production of PHAs and rhamnolipids will enhance the availability of valuable biocatalysts of bacterial cells, and dispel the common belief that the production cost of PHAs accumulated intracellularly is almost impossible to become lower than that of cells themselves.     

Effect of temperature on the development and viability of Gryon gallardoi (Brethes) (Hymenoptera: Scelionidae) parasitizing Spartocera dentiventris (Berg) (Hemiptera: Coreidae) eggs.

The development and viability of Gryon gallardoi (Brethes) (Hym.: Scelionidae) in Spartocera dentiventris (Berg) (Hem.: Coreidae) eggs were studied under four temperatures: 15, 20, 25, and 30 +/- 1 degree C, with a 12-h photophase. No parasitoid developed at 15 degrees C. Otherwise, viability reached 98.8% without varying significantly over the temperature range tested. The duration of development for males and females was inversely proportional to the temperature increase, varying respectively from 46.2 +/- 0.13 and 47.1 +/- 0.11 days (20 degrees C) to 13.3 +/- 0.07 and 13.4 +/- 0.06 days (30 degrees C). Males developed faster than females. The values estimated for the lowest thermic thresholds of development and the thermic constants were 15.5 degrees C and 185.19 DD for males and 15.6 degrees C and 192.31 DD for females, respectively. Given the average weather conditions in Porto Alegre, RS (30 degrees 01' S and 51 degrees 13' W), Brazil, G. gallardoi could annually produce 8.54 and 8.07 generations of males and females, respectively. The low rates of parasitism observed in the field during the first generation of its host may be due to the small number of G. gallardoi generations in this period.     

An estimate of the nearest neighbor base-pair content of 5S RNA using CD and absorption spectroscopy.

We analyzed the CD and uv absorption spectra of 5S RNA from Escherichia coli using the method developed in the preceding paper. The analysis of spectra of 5S RNA at 20 degrees C in 0.1M NaClO4, 2.5 mM Na+ (phosphate), pH 7.0, and 0.5 mM MgSO4 gave 7 +/- 3.6 A.U base pairs, 25 +/- 3.6 G.C base pairs, and 7.5 +/- 3.6 G.U base pairs. Estimates of nearest neighbor base pairs were more consistent with the Pieler-Erdmann and the Gewirth-Moore secondary structure models than with the Fox-Woese or the Burns-Luoma-Marshall models. We also examined the structure of 5S RNA as a function of temperature. The melting profile exhibited two transitions--one at about 35 degrees C and one above 50 degrees C. Our spectral data showed that helices I and II were stable during the first transition, and agreed with other data that helix III was the most likely helix to have melted. The results from this in-depth study of 5S RNA indicate that our method of analysis should be useful for studying the secondary structures of other small, unmodified RNAs.     

Characterization of polyhydroxyalkanoates (PHAs) biosynthesis by isolated Novosphingobium sp. THA_AIK7 using crude glycerol

Biodiesel-contaminated wastewater was used to screen for PHAs-producing bacteria by using crude glycerol as the sole carbon source. A gram-negative THA_AIK7 isolate was chosen as a potential PHAs producer. The 16S rRNA phylogeny indicated that THA_AIK7 isolate is a member of Novosphingobium genus which is supported by a bootstrap percentage of 100% with Novosphingobium capsulatum. The 1,487 bp of 16S rRNA gene sequence of THA_AIK7 isolate has been deposited in the GenBank database under the accession number HM031593. Polymer content of 45% cell dry weight was achieved in 72 h with maximum product yield coefficient of 0.29 g PHAs g?1 glycerol. Transmission electron micrograph results exhibited the PHAs granules accumulated inside the bacterial cell. PHAs polymer production in mineral salt media supplemented with 2% (w/v) of crude glycerol at initial pH 7 was extracted by the sodium hypochlorite method. Polymer film spectrographs from Nuclear magnetic resonance displayed a pattern of signal virtually identical to spectra of commercial PHB. Thermal analysis by Differential scanning calorimeter showed a melting temperature at 179°C. Molecular weight analysis by Gel permeation chromatography showed two main peaks of 133,000 and 700 g mol?1 with weight-average molecular weight value of 23,800 and number-average molecular weight value of 755. Endotoxinfree of PHAs polymer was preliminarily assessed by a negative result of the gel-clot formation, Pyrotell? Single test vial, at sensitivity of 0.25 EU ml?1. To our knowledge, this is the first reported test of endotoxin-free PHAs naturally produced from gram-negative bacteria which could be used for biomedical application.     

Polyhydroxyalkanoates in Gram-positive bacteria: insights from the genera <Emphasis Type="Italic">Bacillus</Emphasis> and <Emphasis Type="Italic">Streptomyces</Emphasis>

Gram-positive bacteria, notably Bacillus and Streptomyces, have been used extensively in industry. However, these microorganisms have not yet been exploited for the production of the biodegradable polymers, polyhydroxyalkanoates (PHAs). Although PHAs have many potential applications, the cost of production means that medical applications are currently the main area of use. Gram-negative bacteria, currently the only commercial source of PHAs, have lipopolysaccharides (LPS) which co-purify with the PHAs and cause immunogenic reactions. On the other hand, Gram- positive bacteria lack LPS, a positive feature which justifies intensive investigation into their production of PHAs. This review summarizes currently available knowledge on PHA production by Gram- positive bacteria especially Bacillus and Streptomyces. We hope that this will form the basis of further research into developing either or both as a source of PHAs for medical applications.     

Production of schizophyllan using Schizophyllum commune NRCM

In the present work, four strains were screened for schizophyllan production, of which Schizophyllum commune NRCM was selected for further work. The fermentation was carried out for 168 h at 28+/-2 degrees C on an orbital shaker at 180 rpm. In the first step, one factor-at-a-time method was used to investigate the effect of media constituents such as carbon and nitrogen sources on schizophyllan production. Subsequently in the second step, concentration of the medium components was optimized using Response Surface Method (RSM). The yield increased from 3.25+/-0.72 g/l in the unoptimized media to 8.03+/-1.12 g/l in the medium optimized by RSM.     

Effect of trypsin inhibitor from Crotalaria pallida seeds on Callosobruchus maculatus (cowpea weevil) and Ceratitis capitata (fruit fly)

A proteinaceous trypsin inhibitor was purified from Crotalaria pallida seeds by ammonium sulfate precipitation, affinity chromatography on immobilized trypsin-Sepharose and TCA precipitation. The trypsin inhibitor, named CpaTI, had M(r) of 32.5 kDa as determined by SDS-PAGE and was composed of two subunits with 27.7 and 5.6 kDa linked by disulfide bridges. CpaTI was stable at 50 degrees C and lost 40% of activity at 100 degrees C. CpaTI was also stable from pH 2 to 12 at 37 degrees C. CpaTI weakly inhibited chymotrypsin and elastase and its inhibition of papain, a cysteine proteinase, were indicative of its bi-functionality. CpaTI inhibited, in different degrees, digestive enzymes from Spodoptera frugiperda, Alabama argillacea, Plodiainterpunctella, Anthonomus grandis and Zabrotes subfasciatus guts. In vitro and in vivo susceptibility of Callosobruchus maculatus and Ceratitis capitata to CpaTI was evaluated. C. maculatus and C. capitata enzymes were strongly susceptible, 74.4+/-15.8% and 100.0+/-7.3%, respectively, to CpaTI. When CpaTI was added to artificial diets and offered to both insect larvae, the results showed that C. maculatus was more susceptible to CpaTI with an LD(50) of 3.0 and ED(50) of 2.17%. C. capitata larvae were more resistant to CpaTI, in disagreement with the in vitro effects. The larvae were more affected at lower concentrations, causing 27% mortality and 44.4% mass decrease. The action was constant at 2-4% (w/w) with 15% mortality and 38% mass decrease.     

Metabolic effects of static magnetic fields on Streptococcus pyogenes

This study aimed to develop a simple experimental system utilising bacterial cells to investigate the dose responses resulting from exposures to static magnetic flux densities ranging from 0.05 to 0.5 T on viability, bacterial metabolism and levels of DNA damage in Streptococcus pyogenes. Exposure of S. pyogenes to a field of 0.3 T at 24 degrees C under anaerobic conditions resulted in a significant (P < 0.05) decrease in growth rate, with an increased mean generation time of 199 +/- 6 min compared to the control cells at 165 +/- 6 min (P < 0.05). Conversely, exposure to magnetic fields of 0.5 T significantly accelerated the growth rate at 24 degrees C compared to control cells, with a decreased mean generation time of 147 +/- 4 min (P < 0.05). The patterns of metabolite release from cells incubated in phosphate buffered saline (PBS) at 24 degrees C and exposed to different magnetic flux densities (0.05-0.5 T) were significantly (P < 0.05) altered, compared to non-exposed controls. Concentrations of metabolites, with the exception of aspartic acid (r = 0.44), were not linearly correlated with magnetic flux density, with all other r < 0.20. Instead, "window" effects were observed, with 0.25-0.3 T eliciting the maximal release of the majority of metabolites, suggesting that magnetic fields of these strengths had significant impacts on metabolic homeostasis in S. pyogenes. The exposure of cells to 0.3 T was also found to significantly reduce the yield of 8-hydroxyguanine in extracted DNA compared to controls, suggesting some possible anti-oxidant protection to S. pyogenes at this field strength.     

High cell density cultivation of Pseudomonas oleovorans: growth and production of poly (3-hydroxyalkanoates) in two-liquid phase batch and fed-batch systems

Pseudomonas oleovorans is able to accumulate poly(3-hydroxyalkanoates) (PHAs) under conditions of excess n-alkanes, which serve as sole energy and carbon source, and limitation of an essential nutrient such as ammonium. In this study we aimed at an efficient production of these PHAs by growing P. oleovorans to high cell densities in fed-batch cultures.To examine the efficiency of our reactor system, P. oleovorans was first grown in batch cultures using n-octane as growth substrate and ammonia water for pH regulation to prevent ammonium limiting conditions. When cell growth ceased due to oxygen limiting conditions, a maximum cell density of 27 g .L(-1) dry weight was obtained. When the growth temperature was decreased from the optimal temperature of 30 degrees -18 degrees C, cell growth continued to a final cell density of 35 g . L(-1) due to a lower oxygen demand of the cells at this lower incubation temperature.To quantify mass transfer rates in our reactor system, the volumetric oxygen transfer coefficient (k(L)a) was determined during growth of P. oleovorans on n-octane. Since the stirrer speed and airflow were increased during growth of the organism, the k(L)a also increased, reaching a constant value of 0.49 s(-1) at maximum airflow and stirrer speed of 2 L . min(-1) and 2500 rpm, respectively. This k(L)a value suggests that oxygen transfer is very efficient in our stirred tank reactor.Using these conditions of high oxygen transfer rates, PHA production by P. oleovorans in fed-batch cultures was studied. The cells were first grown batchwise to a density of 6 g . L(-1), after which a nutrient feed, consisting of (NH(4))(2)SO(4) and MgSO(4), was started. The limiting nutrient ammonium was added at a constant rate of 0.23 g NH(4) (+) per hour, and when after 38 h the feed was stopped, a biomass concentration of 37.1 g . L(-1) was obtained. The Cellular PHA content was 33% (w/w), which is equal to a final PHA yield of 12.1 g . L(-1) and an overall PHA productivity of 0.25 g PHA produced per liter medium per hour. (c) 1993 John Wiley & Sons, Inc.     

Consumption rate of phytonematodes by <Emphasis Type="Italic">Pergalumna</Emphasis> sp. (Acari: Oribatida: Galumnidae) under laboratory conditions determined by a new method

Although several generalist species of Pergalumna are known to be nematode predators, the potential of oribatid mites as natural enemies of phytonematodes has been underestimated. The objective of this work was to estimate the consumption rate of a Pergalumna sp. when feeding on two major pest nematodes, Meloidogyne javanica and Pratylenchus coffeae, under laboratory conditions. A new method was used, in which live nematodes are offered to mites and subsequently consumption is quantified based on the sclerotized, well preserved structures in the mite's fecal pellets. The assay was evaluated during 5 days, at 25 degrees C and 96% relative humidity, with three replicates for each nematode species. Every replicate consisted of a group of four mites isolated in an arena, to which 400 nematodes were transferred daily. The daily produced fecal pellets were mounted in Hoyer's medium for examination under a microscope. The nematode buccal stylets and cephalic frameworks were counted to estimate the number of nematodes consumed. It was estimated that a single mite daily ingested 18.3 +/- 0.8 (mean +/- SE) M. javanica (J(2) juveniles) or 41.6 +/- 7.2 P. coffeae (juveniles + adults), the maximal daily consumption being 34 M. javanica and 73 P. coffeae. The method showed to be practical, precise and suitable for laboratory studies in which nematophagous mites classified as engulfers are included.     

Role of membrane potential on artificial transformation of E. coli with plasmid DNA

The standard method of transformation of Escherichea coli with plasmid DNA involves two important steps: cells are first suspended in 100mM CaCl(2) at 0 degrees C (in which DNA is added), followed by the administration of a heat-pulse from 0 to 42 degrees C for 90s [Cohen, S., Chang, A., Hsu, L., 1972. Nonchromosomal antibiotic resistance in bacteria. Proc. Natl. Acad. Sci. U.S.A., 69, 2110-2114]. The first step makes the cells competent for uptake of DNA and the second step is believed to facilitate the DNA entry into the cells by an unknown mechanism. In this study, the measure of membrane potential of the intact competent cells, at different steps of transformation process, either by the method of spectrofluorimetry or that of flow cytometry, indicates that the heat-pulse step (0-->42 degrees C) heavily decreases the membrane potential. A subsequent cold shock (42-->0 degrees C) raises the potential further to its original value. Moreover, the efficiency of transformation of E. coli XL1 Blue cells with plasmid pUC19 DNA remains unaltered when the heat-pulse step is replaced by the incubation of the DNA-adsorbed competent cells with 10 microM carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 90s at 0 degrees C. Since the CCCP, a well-known protonophore, reduces membrane potential by dissipating the proton-motive-force (PMF) across E. coli plasma membrane, our experimental results suggest that the heat-pulse step of the standard transformation procedure facilitates DNA entry into the cells by lowering the membrane potential.