A genomics approach to improve the analysis and design of strain selections

Strain engineering has been traditionally centered on the use of mutation, selection, and screening to develop improved strains. Although mutational and screening methods are well-characterized, selection remains poorly understood. We hypothesized that we could use a genome-wide method for assessing laboratory selections to design selections with enhanced sensitivity (true positives) and specificity (true negatives) towards a single desired phenotype. To test this hypothesis, we first applied multi-SCale Analysis of Library Enrichments (SCALEs) to identify genes conferring increased fitness in continuous flow selections with increasing levels of 3-hydroxypropionic acid (3-HP). We found that this selection not only enriched for 3-HP tolerance phenotypes but also for wall adherence phenotypes (41% false positives). Using this genome-wide data, we designed a serial-batch selection with a decreasing 3-HP gradient. Further examination by ROC analysis confirmed that the serial-batch approach resulted in significantly increased sensitivity (46%) and specificity (10%) for our desired phenotype (3-HP tolerance).     

Aldehyde dehydrogenase activity is important to the production of 3-hydroxypropionic acid from glycerol by recombinant Klebsiella pneumoniae

3-Hydroxypropionic acid (3-HP) can be produced from glycerol via two enzymatic reactions catalyzed by a coenzyme B₁₂-dependent glycerol dehydratase (GDHt) and aldehyde dehydrogenase (ALDH) in Klebsiella pneumoniae. As the intracellular GDHt activity in K. pneumoniae is high, the overall rate of 3-HP production is controlled by the ALDH activity. To examine the effect of different ALDH activity on 3-HP production, three different ALDHs, AldH from Escherichia coli (EaldH), PuuC from K. pneumoniae (PuuC) and KGSADH from Azospirillum brasilense (KGSADH), were overexpressed and compared in various recombinant K. pneumoniae strains. In addition, the genes encoding DhaT and YqhD, which are responsible for the conversion of 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PDO), were disrupted individually from K. pneumoniae to enhance the carbon flux from 3-HPA to 3-HP. When the ALDH activity was measured in various recombinant K. pneumoniae, KGSADH showed the highest crude cell activity of 8.0 U/mg protein, which was 2 and 4 times higher than that of PuuC and EaldH, respectively. The different ALDH activities had a significant effect on 3-HP production in a flask culture containing 100 mM glycerol, and K. pneumoniae ΔdhaT (KGSADH) resulted in the highest titer (64 mM) among the nine recombinant strains (three ALDH × three host strains; one wild type and two mutants). In glycerol fed-batch bioreactor cultivation, K. pneumoniae ΔdhaT (KGSADH) exhibited 3-HP production at >16 g/L in 48 h with a glycerol carbon yield of >40%. In comparison, K. pneumoniae ΔdhaT (PuuC) produced only 11 g/L 3-HP in 48 h with a yield of >23%. This study demonstrates that a high ALDH activity is essential for the effective production of 3-HP from glycerol with recombinant K. pneumoniae.     

Metabolic engineering of Corynebacterium glutamicum for the production of 3-hydroxypropionic acid from glucose and xylose

3-Hydroxypropionic acid (3-HP) is a promising platform chemical which can be used for the production of various value-added chemicals. In this study,Corynebacterium glutamicum was metabolically engineered to efficiently produce 3-HP from glucose and xylose via the glycerol pathway. A functional 3-HP synthesis pathway was engineered through a combination of genes involved in glycerol synthesis (fusion of gpd and gpp from Saccharomyces cerevisiae) and 3-HP production (pduCDEGH from Klebsiella pneumoniae and aldehyde dehydrogenases from various resources). High 3-HP yield was achieved by screening of active aldehyde dehydrogenases and by minimizing byproduct synthesis (gapA^A1GΔldhAΔpta-ackAΔpoxBΔglpK). Substitution of phosphoenolpyruvate-dependent glucose uptake system (PTS) by inositol permeases (iolT1) and glucokinase (glk) further increased 3-HP production to 38.6 g/L, with the yield of 0.48 g/g glucose. To broaden its substrate spectrum, the engineered strain was modified to incorporate the pentose transport gene araE and xylose catabolic gene xylAB, allowing for the simultaneous utilization of glucose and xylose. Combination of these genetic manipulations resulted in an engineered C. glutamicum strain capable of producing 62.6 g/L 3-HP at a yield of 0.51 g/g glucose in fed-batch fermentation. To the best of our knowledge, this is the highest titer and yield of 3-HP from sugar. This is also the first report for the production of 3-HP from xylose, opening the way toward 3-HP production from abundant lignocellulosic feedstocks.     

Biosynthesis of platform chemical 3-hydroxypropionic acid (3-HP) directly from CO2 in cyanobacterium Synechocystis sp. PCC 6803

3-hydroxypropionic acid (3-HP) is an important platform chemical with a wide range of applications. So far large-scale production of 3-HP has been mainly through petroleum-based chemical processes, whose sustainability and environmental issues have attracted widespread attention. With the ability to fix CO₂ directly, cyanobacteria have been engineered as an autotrophic microbial cell factory to produce fuels and chemicals. In this study, we constructed the biosynthetic pathway of 3-HP in cyanobacterium Synechocystis sp. PCC 6803, and then optimized the system through the following approaches: i) increasing expression of malonyl-CoA reductase (MCR) gene using different promoters and cultivation conditions; ii) enhancing supply of the precursor malonyl-CoA by overexpressing acetyl-CoA carboxylase and biotinilase; iii) improving NADPH supply by overexpressing the NAD(P) transhydrogenase gene; iv) directing more carbon flux into 3-HP by inactivating the competing pathways of PHA and acetate biosynthesis. Together, the efforts led to a production of 837.18 mg L⁻¹ (348.8 mg/g dry cell weight) 3-HP directly from CO₂ in Synechocystis after 6 days cultivation, demonstrating the feasibility photosynthetic production of 3-HP directly from sunlight and CO₂ in cyanobacteria. In addition, the results showed that overexpression of the ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) gene from Anabaena sp. PCC 7120 and Synechococcus sp. PCC 7942 led to no increase of 3-HP production, suggesting CO₂ fixation may not be a rate-limiting step for 3-HP biosynthesis in Synechocystis.     

Coupled incremental precursor and co-factor supply improves 3-hydroxypropionic acid production in Saccharomyces cerevisiae

3-Hydroxypropionic acid (3-HP) is an attractive platform chemical, which can be used to produce a variety of commodity chemicals, such as acrylic acid and acrylamide. For enabling a sustainable alternative to petrochemicals as the feedstock for these commercially important chemicals, fermentative production of 3-HP is widely investigated and is centered on bacterial systems in most cases. However, bacteria present certain drawbacks for large-scale organic acid production. In this study, we have evaluated the production of 3-HP in the budding yeast Saccharomyces cerevisiae through a route from malonyl-CoA, because this allows performing the fermentation at low pH thus making the overall process cheaper. We have further engineered the host strain by increasing availability of the precursor malonyl-CoA and by coupling the production with increased NADPH supply we were able to substantially improve 3-HP production by five-fold, up to a final titer of 463 mg l⁻¹. Our work thus led to a demonstration of 3-HP production in yeast via the malonyl-CoA pathway, and this opens for the use of yeast as a cell factory for production of bio-based 3-HP and derived acrylates in the future.     

Effect of puuC overexpression and nitrate addition on glycerol metabolism and anaerobic 3-hydroxypropionic acid production in recombinant Klebsiella pneumoniae ΔglpKΔdhaT

3-Hydroxypropionic acid (3-HP), an industrially important platform chemical, is used as a precursor during the production of many commercially important chemicals. Recently, recombinant strains of K. pneumoniae overexpressing an NAD⁺-dependent γ-glutamyl-γ-aminobutyraldehyde dehydrogenase (PuuC) enzyme of K. pneumoniae DSM 2026 were shown to produce 3-HP from glycerol without the addition coenzyme B₁₂, which is expensive. However, 3-HP production in K. pneumoniae is accompanied with NADH generation, and this always results in large accumulation of 1,3-propanediol (1,3-PDO) and lactic acid. In this study, we investigated the potential use of nitrate as an electron acceptor both to regenerate NAD⁺ and to prevent the formation of byproducts during anaerobic production of 3-HP from glycerol. Nitrate addition could improve NAD⁺ regeneration, but decreased glycerol flux towards 3-HP production. To divert more glycerol towards 3-HP, a novel recombinant strain K. pneumoniae ΔglpKΔdhaT (puuC) was developed by disrupting the glpK gene, which encodes glycerol kinase, and the dhaT gene, which encodes 1,3-propanediol oxidoreductase. This strain showed improved cellular NAD⁺ concentrations and a high carbon flux towards 3-HP production. Through anaerobic cultivation in the presence of nitrate, this recombinant strain produced more than 40±3 mM 3-HP with more than 50% yield on glycerol in shake flasks and 250±10 mM 3-HP with approximately 30% yield on glycerol in a fed-batch bioreactor.     

Engineering improved ethanol production in Escherichia coli with a genome-wide approach

A key challenge to the commercial production of commodity chemical and fuels is the toxicity of such molecules to the microbial host. While a number of studies have attempted to engineer improved tolerance for such compounds, the majority of these studies have been performed in wild-type strains and culturing conditions that differ considerably from production conditions. Here we applied the multiscalar analysis of library enrichments (SCALEs) method and performed a growth selection in an ethanol production system to quantitatively map in parallel all genes in the genome onto ethanol tolerance and production. In order to perform the selection in an ethanol-producing system, we used a previously engineered Escherichia coli ethanol production strain (LW06; ATCC BAA-2466) (Woodruff et al., in press), as the host strain for the multiscalar genomic library analysis (>10⁶ clones for each library of 1, 2, or 4 kb overlapping genomic fragments). By testing individually selected clones, we confirmed that growth selections enriched for clones with both improved ethanol tolerance and production phenotypes. We performed combinatorial testing of the top genes identified (uspC, otsA, otsB) to investigate their ability to confer improved ethanol tolerance or ethanol production. We determined that overexpression of otsA was required for improved tolerance and productivity phenotypes, with the best performing strains showing up to 75% improvement relative to the parent production strain.     

Quantification of 2-hydrazinopyridine derivatized steroid hormones in fathead minnow (Pimephales promelas) blood plasma using LC-ESI+/MS/MS

Fathead minnows (Pimephales promelas) comprise a species-of-choice for the hazard assessments of various environmental contaminants, including compounds capable of disrupting endocrine function. Towards this end, the use of liquid chromatography coupled with mass spectrometry (LC–MS) and/or tandem mass spectrometry (MS/MS) is gaining common use for the quantification of steroid hormones as biomarkers of endocrine stress in small-fish toxicological studies. In this work, 2-hydrazinopyridine (2-HP) was used to derivatize and quantify the physiologically relevant steroid hormones of: 17α-hydroxypregnenolone, progesterone, 11-ketotestosterone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, in the blood plasma of male and female fathead minnows. Liquid chromatographic separation was achieved using a Waters? Sunfire C₁₈ column (2.1 mm × 50 mm with a 3.5 μm particle size) and Milli-Q water:methanol (both with 0.1% formic acid) mobile phase over a gradient of 15 min. All mass analyses were conducted using electrospray ionization in the positive mode with tandem mass spectrometry (ESI+/MS/MS). This is the first such application of 2-HP derivatization for the quantifications of the structurally and functionally diverse C19 androgen of 11-ketotestosterone; C21 progestogens of 17α-hydroxypregnenolone, progesterone and17α,20β-dihydroxypregnenone; and C21 corticosteroid of 11-deoxycortisol, in fathead minnow blood plasma. The limits of detection (LOD) were set to the lowest calibration standard that gave a signal-to-background response of ≥3, and were: 0.16 ng/ml for progesterone, 0.63 ng/ml for 17α-hydroxypregnenolone, 11-deoxycortisol and 17α,20β-dihydroxypregnenone, and 1.25 ng/ml for 11-ketotestosterone. This study demonstrates the application of 2-HP derivatization for the analysis of a variety of steroid hormones representative of endocrine function in a species of fish commonly used in toxicological studies.     

Effect of corticosterone on growth and welfare of broiler chickens showing long or short tonic immobility

Tonic immobility (TI) test is commonly used to assess fear. Animals showing different TI durations demonstrate distinct behavior and biochemical responses to stress. However, less is known about how TI phenotype affects growth and welfare of domestic fowl. In this study, broiler chickens (Gallus gallus) were classified into short and long TI duration (STI and LTI) phenotypes and treated chronically with vehicle (CON) or corticosterone (CORT). STI broilers demonstrated significantly higher growth rate with higher breast muscle yield (P < 0.05) and liver weight relative to BW tended to be lower (P = 0.053), which was accompanied by higher serum concentration of CORT (P < 0.05) and uric acid (P < 0.01), but lower serum level of T4 (P = 0.01). CORT severely reduced body weight, as well as the relative weight of muscle, bursa of Fabricius and spleen (P < 0.001), but relative liver weight was increased (P < 0.001). CORT-treated chickens had reduced serum CORT, elevated heterophile/lymphocyte ratio, and increased serum levels of total and free T3. STI broilers displayed more preening behavior (P < 0.05), yet CORT elicited more walking behavior (P < 0.05). No difference was observed in the welfare assessment scores between STI and LTI phenotypes under basal situation, while LTI chickens showed significantly increased incidence of pad dermatitis compared to STI under CORT exposure. The results suggest that STI broilers demonstrate better growth performance and higher adaptability to stress compared to LTI chickens.     

Does the white clover response to sulphur availability correspond to phenotypic or ontogenetic plasticity?

White clover (Trifolium repens L.) is a key species in grasslands. Its performance in grassland communities is strongly linked to nitrogen (N) availability. A decrease in soil sulphur (S) content has appeared in the last few decades in grasslands in Northern Europe and this could change the behaviour of white clover. S is essential for plants and particularly for legumes through its effect upon nitrogen fixation. The aim of this study was to determine the effects of S deficiency on white clover fitness, analysing its plasticity in a time course of growth.Three concentrations of SO₄^2?, “Low S” (0.009 mM), “Medium S” (0.384 mM) and “High S” (1.509 mM), were used to grow plants in a hydroponic system. S availability modified biomasses significantly only at the end of the experiment (11 weeks). Medium S appeared optimal while Low S induced a lower aboveground dry mass. An appropriate S availability (Medium S) not only increased S content but also increased N content by stimulating N₂ fixation. Plant growth analysis using growth fitted curves and the calculation of RGR revealed that S effects on biomass corresponded to the production of different phenotypes and not to a growth delay. This work shows that the acceleration growth phase (49–56 days) is a key period for the nutritional needs of white clover and should be the best period for a sulphur fertilisation regime that aims to enhance white clover fitness.     

GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

Inhibition kinetics of a commercial mixed culture by ammonium sulfate

The inhibitory effect of ammonium sulfate on a commercial mixed culture, used in biological waste-water treatment was studied under aerobic batch conditions. Several mathematical models of enzyme and growth kinetics including a death factor were analyzed through nonlinear regression to find the best fit to corresponding data of inhibition. The best fit model was found to be the generalized Monod type with a death factor having the biokinetic parameters; μ_max 0.681 h⁻¹, K_s 0.224 g dm⁻³, K_i 56240 g dm⁻³, K 0.055 g dm⁻³ and k_d 0.052 h⁻¹ to represent the experimental data accurately. The low saturation coefficient value along with high maximum specific growth rate and inhibition coefficient denotes the competitive characteristics of commercial mixed cultures in the biological treatment of high ammonium polluted waste waters.     

Biological and molecular responses of Chironomus riparius (Diptera,Chironomidae) to herbicide 2,4-D (2,4-dichlorophenoxyacetic acid)

2,4-Dichlorophenoxyacetic acid (2,4-D) is an agricultural contaminant found in rural ground water. It remains to be determined whether neither 2,4-D poses environmental risks, nor is the mechanism of toxicity known at the molecular level. To evaluate the potential ecological risk of 2,4-D, we assessed the biological parameters including the survival rate, adult sex ratio of emerged adults, and mouthpart deformities in Chironomus riparius after long-term exposure to 2,4-D. The larvae were treated with 0.1, 1 or, 10 μg L^? 1 of 2,4-D for short- and long-term exposure periods. The sex ratio was changed in C. riparius exposed to only 10 μg L^? 1 of 2,4-D, whereas mouthpart deformities were observed as significantly higher in C. riparius exposed to 0.1 μg L^? 1 of 2,4-D. Survival rates were not significantly affected by 2,4-D. Furthermore, we evaluated the molecular and biochemical responses of biomarker genes such as gene expression of heat shock proteins (HSPs), ferritins and glutathione S-transferases (GSTs) in C. riparius exposed to 2,4-D for 24 h. The expressions of HSP70, HSP40, HSP90 and GST levels in C. riparius were significantly increased after exposure to a 10 μg L^? 1 concentration of 2,4-D, whereas ferritin heavy and light chain gene expressions were significantly increased at all concentrations of 2,4-D exposure. Finally, these results may provide an important contribution to our understanding of the toxicology of 2,4-D herbicide in C. riparius. Moreover, the 2,4-D-mediated gene expressions may be generated by 2,4-D is the causative effects on most probable cause of the observed alterations. These biological, molecular and morphological parameters and the measured parameters can be used to monitor 2,4-D toxicity in an aquatic environment.     

Construction of recombinant Escherichia coli for enhanced bioconversion of colchicine into 3-demethylated colchicine at 70 l bioreactor level

A recombinant strain of Escherichia coli with CYP102A1 gene was developed for the demethylation of colchicine into their derivatives. The CYP102A1 gene responsible for demethylation was isolated from Bacillus megaterium ACBT03 and amplified using suitable primers. The amplified product was cloned into pET28a+ expression vector using host E. coli BL21(DE3) cells. The CYP3A4 (product of CYP102A1 gene) protein expression and other parameters like substrate toxicity, product toxicity and enzyme activity were optimized in shake flasks; and further scaled-up to 5 l bioreactor with 3 l working volume. In 5 l bioreactor, dissolved oxygen (DO) was optimized for maximum specific growth and enhanced 3-demethylated colchicine (3-DMC) production. The optimized conditions from shake flasks were scaled-up to 70 l bioreactor and resulted into ∼80% conversion of 20 mM colchicine in 48 h with a volumetric productivity of 6.62 mg l⁻¹ h⁻¹. Scale-up factors were measured as volumetric oxygen transfer coefficient (k_La) i.e., 56 h⁻¹ and impeller tip velocity (V_tip) i.e., 7.065 m s⁻¹, respectively. The kinetic parameters K_m, k_cat, and k_cat/K_m of the CYP3A4 enzyme using colchicine as the substrate were determined to be 271 ± 30 μM, 8533 ± 25 min⁻¹, and 31.49 μM min⁻¹, respectively, when IPTG induced recombinant E. coli culture was used.     

Cadmium detoxification in roots of Pisum sativum seedlings: relationship between toxicity levels,thiol pool alterations and growth

The evaluation of thiol metabolism in plant adaptation to relevant levels of cadmium stress is important for understanding the real importance of phytochelatins and related thiols in stress coping. The present work was designed to study the process of stress adaptation in roots of Pisum sativum L. plants during an exposure to different cadmium concentrations, ranging from more realistic exposures to those usually employed in PC studies. The balance between individual PCs and their homologous hPCs in constitutive thiol pools and root growth was also accessed. Roots of intact plants were submitted to 1, 3, 30, 60 or 120 μM Cd and harvested after 1, 3, 6 and 9 days after exposure. Growth parameters and root tissue cadmium accumulation were analysed. High-performance liquid chromatography (HPLC) with fluorescence detection was used due to its high sensitivity. Root growth was only affected in concentrations higher than 30 μM Cd, but the presence of low cadmium concentrations induced significant alterations in constitutive thiols and triggered the synthesis of PCs and hPCs, bearing two to four olygomeric repeats. Increasing Cd stress levels were generally associated with higher polythiol production; however, with the time-course of the experiments, higher degrees of toxicity were associated with a reduction in thiol production. This behaviour was attributed to the Cys and GSH depletion, which limited polythiol synthesis, as well as root growth. In tolerable concentrations, the rate of root length recovery matched the increase in PC and hPC synthesis. In higher concentrations (60 and 120 μM), the reduction in non-protein polythiol synthesis was associated with higher Cd toxicity, leading to a severe growth reduction. The synthesis of hPCs seemed to have a reduced importance in tolerance; however, their production was stimulated when the GSH deficit was higher. Our results suggest that the reductions in PC levels, observed in higher degrees of stress, were not related to the activation of other tolerance mechanisms but were instead associated with the high metabolic cost of this thiol-based tolerance mechanism.     

Use of tolerance values to diagnose water-quality stressors to aquatic biota in New England streams

Identification of stressors related to biological impairment is critical to biological assessments. We applied nationally derived tolerance indicator values for four water-quality variables to fish and benthic macroinvertebrate assemblages at 29 sites along an urban gradient in New England. Tolerance indicator values (TIVs), as biologically based predictors of water-quality variables, were determined for dissolved oxygen, nitrite plus nitrate (nitrate), total phosphorus, and water temperature for each site based on observed biological assemblages (TIV_O), and for expected assemblages (TIV_E). The quotient method, based on a ratio of the TIVs for observed and expected assemblages (tolerance units), was used to diagnose potential water-quality stressors. In addition, the ratio of measured water-quality values to water-quality criteria (water-quality units) was calculated for each water-quality variable to assess measured water-quality stressors. Results from a RIVPACS predictive model for benthic macroinvertebrates and Bray-Curtis dissimilarity for fish were used to classify sites into categories of good or impaired ecological condition. Significant differences were detected between good and impaired sites for all biological tolerance units (fish and benthic macroinvertebrate assemblages averaged) except for nitrate (P = 0.480), and for all water-quality units except for nitrate (P = 0.183). Diagnosis of water-quality stressors at selected sites was, in general, consistent with State-reported causes of impairment. Tolerance units for benthic macroinvertebrate and fish assemblages were significantly correlated for water temperature (P = 0.001, r = 0.63), dissolved oxygen (P = 0.001, r = 0.61), and total phosphorus (P = 0.001, r = 0.61), but not for nitrate (P = 0.059, r = −0.35). Differences between the two assemblages in site-specific diagnosis of water-quality stressors may be the result of differences in nitrate tolerance.     

Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli

An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 °C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use.     

Ecological effects of cow manure compost on soils contaminated by landfill leachate

The experiment was conducted to evaluate the effect of cow manure compost (CMC) application on leaching toxicity of leachate polluted soils by using Tetrahymena pyriformis (TP). Soils treated with various levels of leachate (0, 12.5 ml, 25 ml, 37.5 ml, and 50 ml leachate per 300 g soil) were amended with 0, 25 g and 50 g CMC, respectively. The results showed CMC application resulted in 7–18% lower leaching toxicity while excessive CMC has no significant benefit for decreasing leaching toxicity further. The alleviating effect of CMC on biotoxicity of soil extract was mainly attributed to either pH increase, high content of P and organic matter, or promotion on soil microbial metabolism and especially pH played an important role in alleviating effect. And the observations indicated that death rate (DR) of TP was more sensitive to leachate level respect to other biological parameters above and TP was effective as the test organism for leaching toxicity. Further studies are needed to unambiguously determine in-deep mechanism of toxicity impacts on TP posed by leachate pollutants.     

The toxicity and enzyme activity of a chlorine and sulfate containing aeruginosin isolated from a non-microcystin-producing Planktothrix strain

The toxicity of six different Planktothrix strains was examined in acute toxicity assays with the crustacean Thamnocephalus platyurus. The presence of toxicity in two strains could be explained by the occurrence of microcystins. The other four Planktothrix strains were not able to produce microcystins due to different mutations in the microcystin synthetase (mcy) gene cluster. In these strains, toxicity was attributed to the presence of chlorine and sulfate containing compounds. The main representative, called aeruginosin 828A, of such a compound in the Planktothrix strain 91/1 was isolated, and structure elucidation by 2D NMR and MS methods revealed the presence of phenyllactic acid (Pla), chloroleucine (Cleu), 2-carboxy-6-(4′-sulfo-xylosyl)-octahydroindole (Choi), and 3-aminoethyl-1-N-amidino-Δ-3-pyrroline (Aeap) residues. Aeruginosin 828A was found to be toxic for T. platyurus with a LC₅₀ value of 22.4 μM, which is only slightly higher than the toxicity found for microcystins. Additionally, very potent inhibition values for thrombin (IC₅₀ = 21.8 nM) and for trypsin (IC₅₀ = 112 nM) have been determined for aeruginosin 828A. These data support the hypothesis that aeruginosins containing chlorine and sulfate groups, which were found in microcystin-deficient Planktothrix strains, can be considered as another class of toxins.