Benzene toxicity and yield improvement in a cis-benzeneglycol production processe

We carried out experiments designed to increase the production of cis-benzeneglycol from benzene by a mutant strain of Pseudomonas. Factors affecting the production were the temperature, the nitrogen source and it's concentration, the concentration of iron ions, and the benzene concentration. At a benzene concentration of 33% of saturation vapor pressure, the cis-benzeneglycol production was not stable, but this benzene concentration was not toxic to the cells. In a culture under 10% of saturation vapor pressure the cells kept producing cis-benzeneglycol for over 100 h.The decrease in cis-benzeneglycol production observed production at high benzen partial pressure (33%) could not be accounted for by the combined effects of an inhibition of enzyme production and a rapid turn-over of the enzyme, but by a specific inactivation of the enzyme. Possible mechanisms of inactivation were discussed which could explain the specific inactivation of the benzene dioxygenase: generation of polyphenols or generation of oxygen radicals.     

Ethanol fermentation by an efficient strain,NRRL B-4286, of Zymomonas mobilis

We studied the effect of the initial substrate concentration over the range of 100–250 g·l⁻¹ on the fermentation kinetics in batch cultures of Zymomonas mobilis NRRL B-4286 on glucose, fructose, and sucrose, using an adapted initial inoculum. With increasing concentrations of substrate, parameters related to growth were more rapidly and strongly affected than those related to ethanol production. This strain produced 94.0 g·l⁻¹, 76.9 g·l⁻¹, and 66.5 g·l⁻¹ of ethanol at glucose, fructose, and sucrose concentrations of 200 g·l⁻¹, respectively, more than the amount produced by the efficient strain ZM4 (NRRL B-14023).     

CO2 flux in alpine wetland ecosystem on the Qinghai-Tibetan Plateau, China

Using the CO₂ flux data measured by the eddy covariance method in the northeast of Qinghai-Tibetan Plateau in 2005, we analyzed the carbon flux dynamics in relation to meteorological and biotic factors. The results showed that the alpine wetland ecosystem was the carbon source, and it emitted 316.02 gCO₂ · m⁻² to atmosphere in 2005 with 230.16 gCO₂ · m⁻² absorbed in the growing season from May to September and 546.18 gCO₂ · m⁻² released in the non-growing season from January to April and from October to December. The maximum of the averaged daily CO₂ uptake rates and release rates was (0.45 ± 0.0012) mgCO₂ · m⁻² · s⁻¹ (Mean ± SE) in July and (0.22 ± 0.0090) mgCO₂ · m⁻² · s⁻¹ in August, respectively. The averaged diurnal variation showed a single-peaked pattern in the growing season, but exhibited very small fluctuation in the non-growing season. Net ecosystem exchange (NEE) and gross primary production (GPP) were all correlated with some meteorological factors, and they showed a negatively linear correlation with aboveground biomass, while a positive correlation existed between the ecosystem respiration (R_es) and those factors.     

Prodigiosin formation by Serratia marcescens in a chemostat

In a study of the control of metabolite formation, prodigiosin production by Serratia marcescens was used as a model. Specific production rates of prodigiosin formation were determined using batch culture technique. Sucrose as carbon source and NH₄NO₃ as nitrogen source resulted in a specific production rate of 0.476 mg prodigiosin (g cell dry weight)⁻¹ h⁻¹. Prodigiosin formation and productivity was inversely correlated to growth rate when the bacterium was grown under carbon limitation on a defined medium in a chemostat culture. The maximum specific growth rate (μ_max) was 0.54 h⁻¹ and prodigiosin was formed in amounts over 1 mg l⁻¹ up to a growth rate (μ) of 0.3 h⁻¹ at steady state conditions. At a dilution rate of 0.1 h⁻¹ growth at steady state with carbon and phosphate limitation supported prodigiosin formation giving a similar specific yield [1.17 mg prodigiosin (g cell dry weight)⁻¹ and 0.94 mg g⁻¹, respectively], however, cells grown with nitrogen limitation [(NH₄)₂SO₄] did not form prodigiosin. Productivity in batch culture was 1.33 mg l⁻¹ h⁻¹ as compared to 0.57 mg l⁻¹ h⁻¹ in the chemostat.     

Growth of Pseudomonas chlororaphis on apolyester–polyurethane and the purification andcharacterization of a polyurethanase–esterase enzyme

A Pseudomonas chlororaphis was found to degrade and utilize apolyester polyurethane as a sole carbon and energy source. Polyurethane utilization by P.chlororaphis followed simple Michaelis–Menten kinetics. The K_s and μ_max values were 0.802 mg·ml⁻¹ and 1.316 doublings·h⁻¹, respectively. The enzymes from P. chlororaphis responsible for polyurethanedegradation were found to be extracellular. Analysis of the polyurethane degrading proteins, usingnon-denaturing polyacrylamide gel electrophoresis, revealed three active protein bands with R_f values of 0.25, 0.417 and 0.917. A polyurethane degrading enzyme was purifiedand displayed esterase activity. This enzyme was inhibited by phenylmethylsulfonyl fluoride andhad a molecular weight of 27,000 daltons.     

Lipid and biomass production by the halotolerant microalga Nannochloropsis salina

The effect of environmental factors on cell-lipid content, on the growth rate and on the overall productivity of Nannochloropsis salina was tested in the laboratory and in outdoor cultures. Under optimum conditions in the laboratory, the maximum growth rate (μ_max) was 0·030 h⁻¹, which corresponds to a doubling time of 23 h. Cellular lipid content was affected by the phase of growth and the temperature, but not by nitrogen starvation, pH or the source of sea water. The most important factor affecting the output rate of biomass was the cell concentration. The maximum biomass productivity obtained in outdoor ponds was 24·5 g·m⁻²·day⁻¹, and the lipid production rate was 4·0 g m⁻²·day⁻¹.     

Isolation and characterization of butachlor‐catabolizing bacterial strain Stenotrophomonas acidaminiphila JS‐1 from soil and assessment of its biodegradation potential

Aims: Isolation, characterization and assessment of butachlor‐degrading potential of bacterial strain JS‐1 in soil. Methods and Results: Butachlor‐degrading bacteria were isolated using enrichment culture technique. The morphological, biochemical and genetic characteristics based on 16S rDNA sequence homology and phylogenetic analysis confirmed the isolate as Stenotrophomonas acidaminiphila strain JS‐1. The strain JS‐1 exhibited substantial growth in M9 mineral salt medium supplemented with 3·2 mmol l^?1 butachlor, as a sole source of carbon and energy. The HPLC analysis revealed almost complete disappearance of butachlor within 20 days in soil at a rate constant of 0·17 day^?1 and half‐life (t_½) of 4·0 days, following the first‐order rate kinetics. The strain JS‐1 in stationary phase of culture also produced 21·0 μg ml^?1 of growth hormone indole acetic acid (IAA) in the presence of 500 μg ml^?1 of tryptophan. The IAA production was stimulated at lower concentrations of butachlor, whereas higher concentrations above 0·8 mmol l^?1 were found inhibitory. Conclusions: The isolate JS‐1 characterized as Stenotrophomonas acidaminiphila was capable of utilizing butachlor as sole source of carbon and energy. Besides being an efficient butachlor degrader, it substantially produces IAA. Significance and Impact of the Study: The bacterial strain JS‐1 has a potential for butachlor remediation with a distinctive auxiliary attribute of plant growth stimulation.     

Bacterium Hafnia alvei secretes l-methioninase enzyme: Optimization of the enzyme secretion conditions

I isolated bacteria from blue cheese in order to find bacterial strains secreting l-methioninase enzyme, and optimized the conditions for the most efficient enzyme secretion. The efficient isolate, identified according to the 16S rRNA gene sequence analysis, was Hafnia alvei belonging to Enterobacteriaceae. I confirmed that the H. alvei strain harbored the methionase gene, mdeA (1194 bp). The environmental (pH, temperature) and nutritional (carbon and nitrogen sources and Mg concentration) factors influencing the l-methioninase production of H. alvei were optimized. The highest yield of l-methioninase enzyme was reached after 48 h of incubation when the acidity of the growing medium was adjusted to pH 7.5 and the temperature was 35 °C. The following concentrations of the supplements increased the l-methioninase yield in the medium: galactose (2.0 g L⁻¹), MgSO₄ (0.25 g L⁻¹), l-methionine as an inducer (2.0 g L⁻¹), and l-asparagine as an additional N source (1.5 g L⁻¹). I introduce a bacterial strain of H. alvei that is previously unreported to secrete l-methioninase enzyme and show that a carbon source is a mandatory supplement whereas l-methionine is not a mandatory supplement for l-methioninase enzyme production of H. alvei.     

A laboratory energy budget for the ormer Haliotis tuberculata L.

Each component of the energy budget (ingestion, egestion,somatic growth, reproductive investment, respiration, excretion, and mucus production) was measured for Haliotis tuberculata L. held at 15°C in a 12-h light: 12-h dark regime. Ulva lactuca L. was used as the food source throughout, and the budget was assessed over the whole size range of the animal. Ingestion rates ranged from 1.94 to 997.2 cal·animal⁻¹·day⁻¹ in 0.01 to 50 g dry wt (3.71 × 10⁻³ to 17.3 g dry flesh wt) animals, respectively. The major component of the energy budget was somatic growth (37.5% of I) in a 0.01-g dry wt animal while it was respiration (31.1% of I) in a 50-g dry wt animal. Mucus production formed a large part of the budget (from 23.3% of I in a 0.01-g dry wt animal to 29.l% of I in a 50-g dry wt animal). Scope for growth, I − (E + R + U + M), was calculated as ranging from 24.5% of ingestion in a 50-g dry wt animal to 36.8% in a 0.01-g dry wt animal.Each component was measured independently and allometric relationships with animal dry weight calculated. Exponents ranged from 0.60 (somatic growth) to 1.06 (reproductive investment).The calorific value of food was 3419 cal·g dry wt⁻¹ and for faeces was 2817 cal·g dry wt⁻¹. Absorption as a percentage of ingestion in terms of dry weight ranged from 78% for 95-mm length animals (50 g dry wt) to 81% in 6-mm animals (0.01-g dry wt⁻¹). Gross and net growth efficiencies (K₁ and K₂) were calculated on an energy basis and both were logarithmically related to animal dry weight.     

Factors affecting l-arginine production in the continuous culture of an l-arginine producer of Corynebacterium acetoacidophilum

The effects of culture conditions on l-arginine production by continuous culture were studied using a stable l-arginine hyperproducing strain of Corynebacterium aceto-acidophilum, SC-190. Strain SC-190 demonstrated a volumetric productivity of 35 g l⁻¹·h⁻¹ at a dilution rate of 0.083h⁻¹ and feeding sugar concentration of 8%, and a product yield of 29.2% at a dilution rate 0.021h⁻¹ and feeding sugar concentration of 15%. The corresponding values for fed-batch culture are 0.85 g·l⁻¹·h⁻¹ and 26%. However, the product yield decreased with an increase in the volumetric productivity. To achieve stable l-arginine production, aeration and agitation conditions sufficient to maintain an optimal level of redox potential (>−100 mV) were necessary. The addition of phosphate to the feeding medium led to a decrease in l-arginine production. It was confirmed in the steady state that growth and l-arginine formation were inhibited by a high concentration of l-arginine.     

Protein hydrolysate from organic fraction of municipal solid waste compost as nitrogen source to produce lactic acid by Lactobacillus fermentum ATCC 9338 and Lactobacillus plantarum NCIMB 8826

In this work a strategy for obtaining free amino-acids concentrate from an organic fraction of municipal solid waste compost and its use as a nitrogen source for lactic acid production, a compound widely used in different industries, using L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 strains is described. Enzymatic digestion is based on the combined action of endoprotease Alcalase 1.5 MG and exoprotease Flavourzyme 500 MG. The highest degree of hydrolysis obtained under the optimal conditions was 41%. The use of glucanase Viscozyme L prior to protein hydrolysis helped to reduce the viscosity of the solution and promote the action of proteases, increasing its hydrolysis degree by 76%. The hydrolysate contained all 21 amino-acids, making it ideal for lactic acid bacteria growth. During shake flask cultivations the culture media was complemented with glucose as carbon source. Finally, with the hydrolysate, a maximum lactic acid concentration of 9.0 ± 0.2 g·L⁻¹ and 11.1 ± 0.1 g·L⁻¹ for L. fermentum ATCC 9338 and L. plantarum NCIMB 8826 respectively was obtained after 27 h. The innovation of the approach lies in exploiting the overproduction of compost for the production of lactic acid.     

Production and purification of 2-phenylethanol by Saccharomyces cerevisiae using tobacco waste extract as a substrate

2-phenylethanol (2-PE), which is extracted naturally from plant or biotechnology processing, is widely used in the food and cosmetics industries. Due to the high cost of 2-PE production, the valorization of waste carbon to produce 2-PE has gained increasing attention. Here, 2-PE was produced by Saccharomyces cerevisiae using tobacco waste extract (TWE) as the substrate. Considering the toxicity of nicotine and its inhibition of 2-PE, the tolerance of S. cerevisiae was first evaluated. The results suggested that the production of 2-PE by S. cerevisiae in TWEs could be carried out at 2·0 mg ml⁻¹ nicotine concentrations and may be inhibited by 1·0 mg ml⁻¹ 2-PE. Thus, the compounds in the TWEs prepared at different temperatures were detected, and the results revealed that the TWEs prepared at 140°C contained 2·18 mg ml⁻¹ of nicotine, had total sugar concentrations of 26·8 mg ml⁻¹ and were suitable for 2-PE production. Due to feedback regulation, the 2-PE production was only 1·11 mg ml⁻¹, and the remaining glucose concentration remained at 13·78 mg ml⁻¹, which indicated insufficient glucose utilization. Then, in situ product recovery was further implemented to remove this inhibition; the glucose utilization (the remaining concentration decreased to 3·64 mg ml⁻¹) increased, and the 2-PE production increased to 1·65 mg ml⁻¹. The 2-PE produced in the fermentation broth was first isolated by elution from the resin with 75% ethanol and then by removing the impurities with 2·5% activated charcoal, and pure 2-PE was identified by gas chromatography mass spectrometry. The results of this study suggest that TWE could be an alternative carbon source for 2-PE production. This could provide an outlet tobacco waste as well as reducing the price of natural 2-PE, although more strategies need to be explored to improve the production yield of 2-PE by using TWE.     

Primary production of the kelp Lessonia corrugata varies with season and water motion: Implications for coastal carbon cycling

Kelp forests provide vital ecosystem services such as carbon storage and cycling, and understanding primary production dynamics regarding seasonal and spatial variations is essential. We conducted surveys at three sites in southeast Tasmania, Australia, that had different levels of water motion, across four seasons to determine seasonal primary production and carbon storage as living biomass for kelp beds of Lessonia corrugata (Order Laminariales). We quantified blade growth, erosion rates, and the variation in population density and estimated both the net biomass accumulation (NBA) per square meter and the carbon standing stock. We observed a significant difference in blade growth and erosion rates between seasons and sites. Spring had the highest growth rate (0.02 g C · blade⁻¹ · d⁻¹) and NBA (1.62 g C · m⁻² · d⁻¹), while summer had the highest blade erosion (0.01 g C · blade⁻¹ · d⁻¹), with a negative NBA (−1.18 g C · m⁻² · d⁻¹). Sites exhibiting lower blade erosion rates demonstrated notably greater NBA than sites with elevated erosion rates. The sites with the highest water motion had the slowest erosion rates. Moreover, the most wave-exposed site had the densest populations, resulting in the highest NBA and a greater standing stock. Our results reveal a strong seasonal and water motion influence on carbon dynamics in L. corrugata populations. This knowledge is important for understanding the dynamics of the carbon cycle in coastal regions.     

Yarrowia lipolytica chassis strains engineered to produce aromatic amino acids via the shikimate pathway

Yarrowia lipolytica is widely used as a microbial producer of lipids and lipid derivatives. Here, we exploited this yeast’s potential to generate aromatic amino acids by developing chassis strains optimized for the production of phenylalanine, tyrosine and tryptophan. We engineered the shikimate pathway to overexpress a combination of Y. lipolytica and heterologous feedback-insensitive enzyme variants. Our best chassis strain displayed high levels of de novo Ehrlich metabolite production (up to 0.14 g l⁻¹ in minimal growth medium), which represented a 93-fold increase compared to the wild-type strain (0.0015 g l⁻¹). Production was further boosted to 0.48 g l⁻¹ when glycerol, a low-cost carbon source, was used, concomitantly to high secretion of phenylalanine precursor (1 g l⁻¹). Among these metabolites, 2-phenylethanol is of particular interest due to its rose-like flavour. We also established a production pathway for generating protodeoxyviolaceinic acid, a dye derived from tryptophan, in a chassis strain optimized for chorismate, the precursor of tryptophan. We have thus demonstrated that Y. lipolytica can serve as a platform for the sustainable de novo bio-production of high-value aromatic compounds, and we have greatly improved our understanding of the potential feedback-based regulation of the shikimate pathway in this yeast.     

Effect and control of glucose feeding on bacitracin production by fed-batch culture of Bacillus licheniformis

The effect of glucose feeding on bacitracin production was investigated by fed-batch culture of Bacillus licheniformis. In batch culture, bacitracin secretion was induced after the glucose initially contained in the medium was completely consumed. The concentration of bacitracin, however, increased to no more than 340 units·ml⁻¹ in the batch cultivations. Therefore, additional glucose was supplied after exhaustion of the initial glucose. The effect of glucose feeding on bacitracin biosynthesss was investigated in two ways, the pH-stat modal feeding method and the CO₂-dependent feeding method. A kinetic study of bacitracin production found that some glucose was necessary, even during the bacitracin production phase. Excessive feeding of glucose, however, caused a reduction in bacitracin biosynthetic activity. When 50 g·l⁻¹ of defatted soy bean meal (SBM) was used, the bacitracin concentration reached 670 units·ml⁻¹ with the pH-stat modal feeding method and 610 units·ml⁻¹ with the CO₂-dependent feeding method, respectively. The yield of bacitracin from consumed glucose was better for the pH-stat method. Using this control strategy, the highest concentration of bacitracin (940 units·ml⁻¹) was obtained with 150 g·l⁻¹ of SBM.     

A phenanthrene-degrading strain Sphingomonas sp. GY2B isolated from contaminated soils

This study systematically characterized an aerobic bacterial strain Sphingomonas sp. GY2B for biotransformation of phenanthrene. The strain was isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs) and was shown to efficiently use phenanthrene as the sole carbon and energy source. The antibiotics discs susceptibility test revealed that the bacterium was susceptible to some commonly used antibiotics, such as cefuroxime, chloramphenicol, erythromycin and tetracycline. It showed better growth at pH 7.4 and 30 °C and in a mineral salts medium (MSM) with phenanthrene at 100 mg L⁻¹ as the substrate. The results indicated that 99.8% of the substrate had been degraded and that salicylate route was likely the metabolic pathway. When added as the second organic chemical, glucose could enhance the bacterial growth at low concentration (10–200 mg L⁻¹), but could inhibit cell growth at high concentration (>500 mg L⁻¹). Further study showed that strain GY2B could also use naphthalene, phenol, 1-hydroxy-2-naphthoic acid, 2-naphthol, salicylic acid and catechol as the sole carbon and energy source, but did not grow on 1-naphthol which could be co-metabolized in the present of phenanthrene or 1-hydroxy-2-naphthoic acid.     

Simultaneous organic carbon removal-nitrification by an activated sludge process with cross-flow filtration

Simultaneous organic carbon removal-nitrification was carried out by an activated sludge process with cross-flow filtration while retaining a higher concentration of activated sludge. Operating the unit at ca. 0.10 gBOD·gVSS⁻¹·d⁻¹ made the sludge retention time very long and simultaneous carbon removal-nitrification was achieved quite well. Compared with conventional activated sludge processes, the present process made possible nearly twice the organic volumetric loading by increasing the sludge concentration five-fold. A nitrification rate of ca. 0.30 gTKN·l⁻¹·d⁻¹ was attained. More than 27% of the oxidized nitrogen in the reactor was denitrified at the same time in most cases.     

Over expression of GroESL in Cupriavidus necator for heterotrophic and autotrophic isopropanol production

We previously reported a metabolic engineering strategy to develop an isopropanol producing strain of Cupriavidus necator leading to production of 3.4 g L⁻¹ isopropanol. In order to reach higher titers, isopropanol toxicity to the cells has to be considered. A toxic effect of isopropanol on the growth of C. necator has been indeed observed above a critical value of 15 g L⁻¹. GroESL chaperones were first searched and identified in the genome of C. necator. Native groEL and groES genes from C. necator were over-expressed in a strain deleted for PHA synthesis. We demonstrated that over-expressing groESL genes led to a better tolerance of the strain towards exogenous isopropanol. GroESL genes were then over-expressed within the best engineered isopropanol producing strain. A final isopropanol concentration of 9.8 g L⁻¹ was achieved in fed-batch culture on fructose as the sole carbon source (equivalent to 16 g L⁻¹ after taking into account evaporation). Cell viability was slightly improved by the chaperone over-expression, particularly at the end of the fermentation when the isopropanol concentration was the highest. Moreover, the strain over-expressing the chaperones showed higher enzyme activity levels of the 2 heterologous enzymes (acetoacetate carboxylase and alcohol dehydrogenase) of the isopropanol synthetic operon, translating to a higher specific production rate of isopropanol at the expense of the specific production rate of acetone. Over-expressing the native chaperones led to a 9–18% increase in the isopropanol yield on fructose.     

Temporal variability in egg production rates of Acartia tonsa Dana in Long Island Sound

The relationship between week-by-week variations in the in situ egg production rates of Acartia tonsa Dana and changes in chlorophyll concentration in several size fractions was investigated by incubating adult females in natural sea water for 24-h periods. Our results indicate that the egg production of A. tonsa in Long Island Sound was better related to the 10 μm chlorophyll size fraction than to the total chlorophyll concentration. The < 10 μm size fraction comprised the greatest percentage of the chlorophyll during July and August when the water column was stratified. Egg production rates were lowest (8.7 eggs · female⁻¹ · day⁻¹) in early August when less than 0.5 μg chlorophyll 1 ⁻¹ was observed in the 10 μm chlorophyll a size fraction. Following destratification in late August, the “fall” diatom bloom occurred and egg production rates increased to the maximum observed rate of 56.6 eggs · female⁻¹ · day⁻¹. At this time, the concentration of the 10μm chlorophyll size fraction was 5.5 μg 1⁻¹. Maximum egg production rates were observed at chlorophyll concentrations as low as 0.8 μg 1⁻¹ in the 10 μm size fraction.     

Reverse NaCa exchange requires internal Ca and/or ATP in squid axons

Classical NaCa exchange models are based on a symmetric carrier system where Na and Ca competing from the same site, can produce net movement of the other against its electrochemical gradient. We have explored this symmetric assumption by studying the Ca_o and Na_o-dependent Na efflux in dialyzed squid axons in which proper control of both external and internal medium was achieved. The results show: (1) In axons dialyzed without Ca_i and ATP, Ca_o-dependent Na efflux cannot be detected even in the absence of Na_o. Under these conditions, the level of Na efflux (1 pmol · cm⁻² · s⁻¹) is close to that predicted by an electrical ‘leak’. (2) In axons dialyzed with Ca_i (100 μM) and without ATP, Na efflux measured in 440 mM Na_o, is about 4–5 pmol · cm⁻² · s⁻¹ and rather insensitive to Ca_o between 0 and 10 mM. However, in the absence of Na_o, a Ca_o-dependent Na efflux is observed similar in magnitude to that found in the presence of external Na. (3) In the presence of both Ca_i and ATP, Na efflux into artificial sea-water (440 mM Na, 10 mM Ca) is 18 pmol · cm⁻² · s⁻¹. In the absence of Na_o the efflux of Na is 7.5 pmol · cm⁻² · s⁻¹. In the absence of both Na_o and Ca_o the efflux is close to ‘leak’. With full Na_o but no Ca_o, the Na efflux average 12.6 pmol · cm⁻² · s⁻¹. These results indicate a marked asymmetry in the modus operandi of the NaCa exchange system with respect to Ca_i and ATP. These two substrates are required from the cis side to promote Ca_o-dependent Na efflux (reversal NaCa exchange).