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Chemiluminescence (CL) reactions are widely used for the detection and quantification of many types of analytes. Laccase has previously been proposed in CL reactions; however, its light emission behaviour has not been characterized. This study was conducted to characterize the laccase-luminol system, determine its kinetic parameters, and analyze the effects of protein and OH- concentration on the CL signal. Laccase from Coriolopsis gallica was combined with different concentrations of luminol (125 nM to 4 mM), and the enzyme kinetics were evaluated using diverse kinetic models. The laccase-luminol system was able to produce CL without an intermediate molecule, but it exhibited substrate-inhibition behaviour. A two-site random model was used and suggested that when the first luminol molecule was bound to the active site, laccase affinity for the second luminol molecule was increased. This inhibition effect could be avoided using a low luminol concentration. At 5 μM luminol concentration, 1 mg/ml (0.13 U) laccase is needed to achieve nearly 90% of the maximum CL signal, suggesting that the available luminol could not bind to all active sites. Furthermore, the concentration of NaOH negatively affected the CL signal. The laccase-luminol system represents an alternative to existing CL systems, with potential uses in molecular detection and quantification., Translational Omics and Bioengineering and Regenerative Medicine strategic focus groups of Tecnologico de Monterrey; CONACyT, Grant/Award Number: 492276, With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).

Bacillus amyloliquefaciens strain 83 is a bacterium isolated from mango phyllosphere effective to control anthracnose in various tropical crops. The antifungal activity of bacillomycin D homologues, produced by this strain, against Colletotrichum gloeosporioides was characterized in this study. Minimum inhibitory concentration (MIC100) of bacillomycin D homologues revealed that slight changes in the fatty acid chain length (hydrophobicity) of bacillomycin D cause significant differences of its antifungal activity against spores but no significant differences were found against mycelium. These lipopeptides inhibit both spore germination and mycelial growth. Mycelium was more susceptible to bacillomycin D homologues than spores. Propidium iodide staining of spores and mycelium showed that bacillomycin D causes damage to the cell membranes which directly correlates with its viability. The present work explores the relationship between the chemical structure and biological activity of bacillomycin homologues. As synergism between lipopeptides produced by B. amyloliquefaciens was observed, the biosynthesis of diverse bacillomycin D homologues should play a major role in the biological control of fungal pathogens by this strain. These findings will contribute to the design of better antimicrobial lipopeptides with high selectivity and potency.

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

Background Wild-type yeasts have been successfully used to obtain food products, yet their full potential as fermenting microorganisms for large-scale ethanol fuel production has to be determined. In this study, wild-type ethanologenic yeasts isolated from a secondary effluent were assessed for their capability to ferment saccharified microalgae sugars. Results Yeast species in wastewater were identified sequencing the Internal Transcribed Spacers 1 and 2 regions of the ribosomal cluster. Concurrently, microalgae biomass sugars were saccharified via acid hydrolysis, producing 5.0 ± 0.3 g L−1 of fermentable sugars. Glucose consumption and ethanol production of yeasts in hydrolyzed-microalgae liquor were tested at different initial sugar concentrations and fermentation time. The predominant ethanologenic yeast species was identified as Candida sp., and glucose consumption for this strain and S. cerevisiae achieved 75% and 87% of the initial concentration at optimal conditions, respectively. Relatively similar ethanol yields were determined for both species, achieving 0.45 ± 0.05 (S. cerevisiae) and 0.46 ± 0.05 g ethanol per g glucose (Candida sp.). Conclusion Overall, the results provide a first insight of the fermentation capacities of specific wild-type Candida species, and their potential role in ethanol industries seeking to improve their cost-efficiency.

In Pseudomonas spp. PsrA, a transcriptional activator of therpoS gene, regulates fatty acid catabolism by repressing the fadBA5 β-oxidation operon. In Azotobacter vinelandii, a soil bacterium closely related to Pseudomonas species, PsrA is also an activator of rpoS expression, although its participation in the regulation of lipid metabolism has not been analyzed. In this work we found that inactivation of psrA had no effect on the expression of β-oxidation genes in this bacterium, but instead decreased expression of the unsaturated fatty acid biosynthetic operon fabAB (3-hydroxydecanoyl-ACP dehydratase/isomerase and 3-ketoacyl-ACP synthase I). This inactivation also reduced the unsaturated fatty acid content, as revealed by the thin-layer chromatographic analysis, and confirmed by gas chromatography; notably, there was also a lower content of cyclopropane fatty acids, which are synthesized from unsaturated fatty acids. The absence of PsrA has no effect on the growth rate, but showed loss of cell viability during long-term growth, in accordance with the role of these unsaturated and cyclopropane fatty acids in the protection of membranes. Finally, an electrophoretic mobility shift assay revealed specific binding of PsrA to the fabA promoter region, where a putative binding site for this regulator was located. Taken together, our data show that PsrA plays an important role in the regulation of unsaturated fatty acids metabolism in A. vinelandii by positively regulating fabAB.

Additional file 1. Figure S1. Design matrix with coded levels and real values (in parenthesis) for a three-level full factorial design. Figure S2. Calibration curve for tested parameters in HPLC using laboratory grade reagents. Figure S3. Calibration curve for ethanol quantification in GC using laboratory grade reagents. Table S1. High-Performance Liquid Chromatographer instruments and chromatographic conditions. Table S2. Gas Chromatographer instruments and chromatographic conditions.

In this study, the exact contribution of T. versicolor fungal biomass and laccase in the removal of the Orange II dye from liquid culture was determined. Biomass and laccase were produced with three different carbon sources [bran flakes (BF), wheat bran (WB) and wheat flour (WF)]. The contribution of the biomass and the laccase enzyme in the removal of the Orange II dye was assessed as follows: (A) in vivo treatment with fungal biomass; in vivo treatment with fungal biomass and inhibited laccase (using 0.6 mM sodium azide); and (B) in vitro treatment with crude laccase. The results of fungal biomass production were similar for all the carbon sources evaluated, while laccase volumetric activities were different. The highest enzyme production was obtained with WB, followed by BF and WF. In the in vivo treatment with fungal biomass–laccase, dye removal was over 84% for all the carbon sources. Dye adsorption by fungal biomass varied from 1.5–2%, presenting enzymatic activities ranging from 62 to 163 U L(−1). In the in vivo treatment with fungal biomass-inhibited laccase, the removal of the dye varied from 30 to 72%. In this case, the percentage of dye adsorption by fungal biomass was significantly increased and ranged from 18 to 53%. In the in vitro treatment with laccase, the removal ranged from 80 to 84%. The best treatment for dye removal involved the use of both fungal biomass and laccase. The carbon source for biomass and laccase production had an impact on dye removal.

In this study the biosurfactant (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG-5 strain) was improved and the tenso-active agents were purified and characterized. A 23 factorial design was used to evaluate the effect of nitrogen and carbon sources on the surface tension (ST) reduction and emulsion index (EI24) of the produced Bs. Optimum Bs production by SM3 was achieved at high concentrations of carbon and nitrogen, reducing ST to 26.5 ± 0.28 dynes/cm, with an EI24 of 79.9 ± 0.2 %. Meanwhile, the best results for SMRG-5 were obtained at low concentrations, reducing the ST to 25.2 ± 0.2 dynes/cm, with an EI24 of 89.7 ± 0.28 %. The optimal conditions for Bs production were scaled up in a 2-L reactor, yielding 4.8 and 5.2 g/L for SM3 and SMRG-5, respectively. GC-MS analysis revealed the presence of two different lipopeptides (octadecanoic and hexadecanoic acid for SM3 and SMRG5, respectively). Both strains were capable of benzo [a] pyrene removal (59 % after 72 h of culture). This article is protected by copyright. All rights reserved

A novel poly-3-hydroxybutyrate depolymerase was identified in Azotobacter vinelandii. This enzyme, now designated PhbZ1, is associated to the poly-3-hydroxybutyrate (PHB) granules and when expressed in Escherichia coli, it showed in vitro PHB depolymerizing activity on native or artificial PHB granules, but not on crystalline PHB. Native PHB (nPHB) granules isolated from a PhbZ1 mutant had a diminished endogenous in vitro hydrolysis of the polyester, when compared to the granules of the wild-type strain. This in vitro degradation was also tested in the presence of free coenzyme A. Thiolytic degradation of the polymer was observed in the nPHB granules of the wild type, resulting in the formation of 3-hydroxybutyryl-CoA, but was absent in the granules of the mutant. It was previously reported that cultures of A. vinelandii OP grown in a bioreactor showed a decrease in the weight average molecular weight (Mw) of the PHB after 20 h of culture, with an increase in the fraction of polymers of lower molecular weight. This decrease was correlated with an increase in the PHB depolymerase activity during the culture. Here, we show that in the phbZ1 mutant, neither the decrease in the Mw nor the appearance of a low molecular weight polymers occurred. In addition, a higher PHB accumulation was observed in the cultures of the phbZ1 mutant. These results suggest that PhbZ1 has a role in the degradation of PHB in cultures in bioreactors and its inactivation allows the production of a polymer of a uniform high molecular weight.

Aspergillus sydowii is a moderate halophile fungus extensively studied for its biotechnological potential and halophile responses, which has also been reported as a coral reef pathogen. In a recent publication, the transcriptomic analysis of this fungus, when growing on wheat straw, showed that genes related to cell wall modification and cation transporters were upregulated under hypersaline conditions but not under 0.5 M NaCl, the optimal salinity for growth in this strain. This led us to study osmolyte accumulation as a mechanism to withstand moderate salinity. In this work, we show that A. sydowii accumulates trehalose, arabitol, mannitol, and glycerol with different temporal dynamics, which depend on whether the fungus is exposed to hypo- or hyperosmotic stress. The transcripts coding for enzymes responsible for polyalcohol synthesis were regulated in a stress-dependent manner. Interestingly, A. sydowii contains three homologs (Hog1, Hog2 and MpkC) of the Hog1 MAPK, the master regulator of hyperosmotic stress response in S. cerevisiae and other fungi. We show a differential regulation of these MAPKs under different salinity conditions, including sustained basal Hog1/Hog2 phosphorylation levels in the absence of NaCl or in the presence of 2.0 M NaCl, in contrast to what is observed in S. cerevisiae. These findings indicate that halophilic fungi such as A. sydowii utilize different osmoadaptation mechanisms to hypersaline conditions.

BACKGROUND: Multiple aqueous two‐phase systems (ATPS) were evaluated for the partial purification of laccase from a crude aqueous extract. The laccase extract was obtained from residual compost, generated from the production of the edible mushroom Pleurotus ostreatus. A total of 59 ATPS were evaluated; 16 systems using poly (ethylene glycol) (PEG) and phosphates, 20 systems using UCON (ethylene oxide/propylene oxide copolymer) and different salts, and 23 polymer–polymer systems (Ficoll, Dextran and PEG). RESULTS: Protein partitioning was not achieved with the PEG–phosphate systems. ATPS composed of UCON and salts presented great potential for laccase recovery, with up to 95.12 ± 5.27, 96.05 ± 4.99 and 98.31 ± 3.58% recovery, and purification factors of 5.02, 4.14 and 9.97 for the UCON‐NaH₂PO₄ , UCON‐NaSO₄, and UCON‐(NH₄)₂SO₄ systems, respectively. The PEG 10 000/Dextran 10 000 and the PEG 10 000/Dextran 100 000 polymer–polymer systems proved to have potential, presenting purification factors as high as 5.78 and 6.56, respectively. CONCLUSION: The laccase analyzed in this study had a molecular weight of approximately 30 kDa, and thus, is the smallest laccase yet to be reported for P. ostreatus. ATPS represent an attractive alternative for the efficient recovery of laccase from residual compost generated after P. ostreatus production. © 2016 Society of Chemical Industry

Many fungi produce several laccase isoenzymes endowed with different catalytic properties, however, the physiological significance of this multiplicity is still unclear. Multiple laccase isoforms produced by the same organism imply a greater flexibility and/or adaptation to constantly changing environments. In this study, we explore and discuss the laccase isoform diversity of Trametes versicolor under submerged culture conditions. Oak sawdust was used as a natural, abundant and cheap source of laccase inducers. Two laccase bands (lcc1 and lcc2) were detected by SDS-PAGE zymography. Lcc2 exhibited an apparent molecular mass of 100 kDa, and was purified to its characteristic blue color for biochemical characterization. Lcc2 displayed high affinities toward all the substrates used in this study (0.01 ± 0.0043, 0.5 ± 0.053, 2.23 ± 0.53 and 3.77 ± 0.85 mM, for ABTS, DMP, GUA and SYR, respectively). Isoelectric focusing analysis of this band revealed the presence of 5 distinct laccase isoforms. The suggested existence of different laccase heterodimers in lcc2 may have had an influence on its catalyzing capacity. Laccase isoform diversity was corroborated by molecular characterization of 5 allelic mRNA sequences. Multiple alignment analysis, 3-dimensional mapping and theoretical sequence-structure-function assumptions suggests that the newly characterized alleles would most likely display diversity in their biochemical behavior if functionally expressed.

Bacillus amyloliquefaciens spores have been used as the principal ingredient of biocontrol products. However, during the process of spore production, wild-type strains produce poly-γ-glutamic acid (γ-PGA), an undesirable byproduct that increases broth viscosity and hinders recovery and drying. This work examined the influence of specific glucose uptake rates (qGluc) in glucose-controlled overflow metabolism. Diverse scenarios, from glucose limitation to glucose sufficiency, were evaluated in continuous cultures to control qGluc. Cell yields of glucose were higher at low qGluc, while the opposing trend was found for γ-PGA and other overflow metabolic byproducts yields. However, γ-PGA production was still detectable in cultures with the highest glucose limitation (D = 0.06 h−1), even though high sporulation incidence was observed in these cultures. Indeed, in such conditions, nonsporulating vegetative cells seem to maintain glucose overflow metabolism, allowing limited γ-PGA production. These findings can be used to establish fed-batch culture strategies for high cell density Bacillus amyloliquefaciens cultures where γ-PGA production (and apparent viscosity) is significantly reduced. This is the first time that the dependence of qGluc on growth, sporulation and carbon overflow metabolism of a spore and biofilm producer, Bacillus amyloliquefaciens strain, has been reported.

Laccases are currently employed in several biotechnological applications such as pharmaceutical, environmental, textile and petrochemical. Their production in submerged bioreactors has been extensively reported using basidiomycetes, however, this approach exhibits a major drawback: the concomitant production of proteases, which reduces laccase activity in broths. In this work, aqueous two-phase systems (ATPS) composed by polyethylene glycol (PEG)-salt, UCON-salt and PEG-dextran were tested to separate laccase from proteases in culture media. Results showed that PEG of low molecular weight (400 g mol-1), volume ratio of 0.33 and tie line length of 45% (w/w) in a PEG-salt system allowed the separation of both enzymes. The extraction efficiency decreased when sodium chloride was added into the system, but increased with a second ATPS stage. A PEG 4600-dextran 500 000 system was also suitable to separate laccase from proteases with an enzymatic activity recovery of 95%. The UCON-salt systems tested were not adequate for the separation of both enzymes since they partitioned to the same phase. Therefore, two options for laccase separation from proteases are proposed: a two step PEG 400-salt system for laccase separation after the fermentation process and PEG 4600-dextran 500 000 for in situ recovery of laccase due to its lower salt content which is suitable for fungal growth. To our knowledge, this is the first time that conditions for the separation of laccase and proteases on ATPS different phases are reported. These findings open the opportunity to establish optimum process conditions for the efficient production and recovery of laccases produced by Pleurotus ostreatus.

Butanol is considered a superior liquid fuel that can replace gasoline in internal combustion engines. It is produced by acetone-butanol-ethanol (ABE) fermentation using various species of solventogenic clostridia. Performance of ABE fermentation process is severely limited mostly by high cost of substrate, substrate inhibition and low solvent tolerance; leading to low product concentrations, low productivity, low yield, and difficulty in controlling culture metabolism. In order to decrease the cost per substrate and exploit a waste generated by dairy industry, this study proposes using cheese whey as substrate for ABE fermentation. It was observed that the addition of an iron source was strictly necessary for the cheese whey to be a viable substrate because this metal is needed to produce ferredoxin, a key protein in the fermentative metabolism of Clostridium acetobutylicum serving as a temporary electron acceptor. Lack of iron in the cheese whey impedes ferredoxin synthesis and therefore, restricts pyruvate-ferredoxin oxidoreductase activity leading to the production of lactic acid instead of acetone, butanol and ethanol. Moreover, the addition of FeSO4 notably improved ABE production performance by increasing butanol content (7.13 ± 1.53 g/L) by 65% compared to that of FeCl3 (4.32 ± 0.94 g/L) under the same fermentation conditions.

In this study, the biosurfactants (Bs) production of two Serratia marcescens strains (SM3 and its isogenic SMRG-5 strain) was improved and the tenso-active agents were purified and characterized. A 2

The effects of nitrogen limitation on the specific growth rate (μ) and the accumulation of reserve metabolites in Neochloris oleoabundans during heterotrophic fed-batch cultivation were studied in a system consisting of three stages with an excess of glucose in a 40-L bioreactor. During the first stage, which was a batch cultivation with no carbon or nitrogen limitation, the cells grew at a μ of 0.045 h− 1, and the major cellular component was protein (44%, w/w) on a dry cell weight (DCW) basis. In the second, which was a fed-batch stage, two different exponential nitrate-feeding profiles were employed at μ values of 0.042 and 0.035 h− 1. The results show that a nitrogen limitation that causes a small reduction in the μ (only 20%) has a marked effect on the biochemical composition of the microalgal cells, promoting greater accumulation of lipids and carbohydrates. During the third stage, which was a post fed-batch stage, the cells were maintained under nitrogen-starvation and excess glucose conditions. When the glucose was depleted, the cells from the fed-batch culture at 0.042 h− 1 contained 53.8% lipids, reaching a cell mass of 20.9 gDCW L− 1, and the biomass, lipid and carbohydrate productivities were 1900, 1020 and 500 mg L− 1 day− 1, respectively. Thus far, these are the highest productivity values reported for N. oleoabundans cultures under phototrophic, mixotrophic or heterotrophic growth.

Mycelial growth and laccase production by Pleurotus ostreatus CP50 cultured in a 10-L mechanically agitated bioreactor were assessed through a 2(3) factorial experimental design. The main effects and interactions of three factors (agitation, aeration and copper induction) over five responses (μ, αLacc, βLacc, maximal volumetric laccase activity and maximal biomass concentration) were analyzed. P. ostreatus growth was significantly improved when culturing was conducted with high agitation (5.9kW/m(3)s) and aeration flow (0.5vvm) rates. Under the experimental conditions evaluated, no evidence of hydrodynamic stress affecting fungal growth was observed. However, the high agitation and aeration conditions were detrimental for the growth-associated laccase production constant (αLacc), leading to a very complex optimization of the process. The maximal laccase volumetric activity (1.2 and 3.8U/ml for non-induced and copper-induced cultures, respectively) was observed when the culturing was performed at a low agitation rate (0.9kW/m(3)s) and a high aeration flow rate (0.5vvm). Laccase proteolysis may explain the complex interactions observed between agitation and aeration and the effects of these factors on the laccase volumetric activity observed in the cultures.

Background Avian coccidiosis is a disease caused worldwide by several species of parasite Eimeria that causes significant economic losses. This disease affects chickens development and production, that most of times is controlled with anticoccidial drugs. Although efforts have been made to address this disease, they have been made to control Eimeria sporozoites, although enteric stages are often vulnerable, however; the parasite oocyst remains a problem that must be controlled, as it has a resistant structure that facilitates dispersion. Despite some commercial products based on chemical compounds have been developed as disinfectants that destroy oocysts, the solution of the problem remains to be solved. Results In this work, we assessed in vitro anticoccidial activity of a compound(s) secreted by yeast isolated in oocysts suspension from infected chickens. The yeast was molecularly identified as Meyerozyma guilliermondii, and its anticoccidial activity against Eimeria tenella oocysts was assessed. Here, we report the damage to oocysts walls caused by M. guilliermondii culture, supernatant, supernatant extract and intracellular proteins. In all cases, a significant decreased of oocysts was observed. Conclusions The yeast Meyerozyma guilliermondii secretes a compound with anticoccidial activity and also has a compound of protein nature that damages the resistant structure of oocyst, showing the potential of this yeast and its products as a feasible method of coccidiosis control. Electronic supplementary material The online version of this article (doi:10.1186/s12917-015-0589-0) contains supplementary material, which is available to authorized users.

The independent effects of hydrodynamic stress (assessed as the Energy Dissipation/Circulation Function, EDCF) and dissolved oxygen tension (DOT) on the growth, morphology and laccase production by Pleurotus ostreatus CP50 were studied using a 3(2) factorial design in a 10L reactor. A bell-shape function for fungus growth between 8 and 22% DOT was observed, as well as a significant negative effect on laccase production and the expression of poxc, the gene encoding for the most abundant laccase produced by P. ostreatus CP50. Increasing EDCF from 1 to 21 kW/m(3)s, had a positive effect on fungus growth, whereas no effect on poxc gene expression was observed. However, the increase in EDCF favored the specific laccase production due to the generation of smaller pellets with less diffusional limitations and increased metabolically active biomass. The results show, for the first time, that hydrodynamic effects on growth and laccase production are mainly physical and diffusional, while the influence of the dissolved oxygen is at transcriptional level.

International audience; Ethylene is a key molecule in organic synthesis currently produced by steam cracking of fossil hydrocarbons. In nature, ethylene is produced in higher plants by 1-aminocyclopropane-1-carboxylic acid oxidase (ACCO). Biocatalytic alternatives for ethylene production are still far from being competitive with traditional production plants. Furthermore, data dispersion shown in the literature adds uncertainty to the introduction of ACCO as a biocatalyst, especially when larger numbers of isoforms or mutants are to be compared. Here we propose a new method for measuring ACCO activity based on cyanide detection. Data provided here indicate that cyanide detection is more precise, more responsive, and much more stable than any other method tested for ACCO activity estimation so far. Briefly, enzymatically produced cyanide can be detected by its derivatization with naphthalene-2,3-dicarboxyaldehide (NDA) to generate 1-cyanobenz[f]isoindole (CBI), which is further detected by high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. Cyanide can be detected in the range between 0.99 and 60.17 pmol, which is three orders of magnitude more sensitive than the currently used ethylene estimation method.

Organophosphate pesticides are of great interest for research because they are currently the most commonly used pesticides. In this study, a bacterial strain capable of completely degrading methyl parathion (MP) was isolated from agricultural soils in central Mexico. This strain was designated strain S5‐2 and was identified as Burkholderia cenocepacia. To increase degradation yields, cells were immobilized on three different supports: powdered zeolite and Opuntia sp. and Agave sp. fibers. The results indicated a significant increase in MP hydrolysis and p‐nitrophenol (PNP) degradation with immobilized cells compared to free cell cultures. Furthermore, immobilized cells were capable of withstanding and degrading higher concentrations of PNP compared to cell suspension cultures. The cell viability in the free cell cultures, as well as PNP degradation, was affected at concentrations greater than 25 mg/L. In contrast, cells immobilized on Opuntia sp. and Agave sp. fibers completely degraded PNP at concentrations of 100 mg/L. To verify that MP solution toxicity was decreased by B. cenocepacia strain S5‐2 via pesticide degradation, we measured the acetylcholinesterase activity, both before and after treatment with bacteria. The results demonstrate that the activity of acetylcholinesterase was unaffected after MP degradation by bacteria.

Background In comparison with phototrophic growth, heterotrophic conditions can significantly increase growth rates, final cell number and cell mass in microalgae cultures. Neochloris oleoabundans is a microalga of biotechnological interest that accumulates lipids under phototrophic and nitrogen-limited conditions. Heterotrophic flask culture experiments were conducted to identify carbon sources that can be metabolized by N. oleoabundans, and bioreactor batch and fed-batch (nitrate pulse additions) cultures supplemented with glucose were performed to study the cellular composition of the microalgae under balanced and high C/N ratios (glucose/nitrate). Results N. oleoabundans was able to grow using glucose and cellobiose as sole carbon sources under strict heterotrophic conditions. Under a balanced C/N ratio of 17 and using bioreactor batch cultures containing 3 g/L glucose, a maximal cell mass of 1.72 g/L was found, with protein being the major cell component (44% w/w). A maximal cell mass of 9.2 g/L was obtained using batch cultures at a C/N ratio of 278. Under these conditions, lipid accumulation was promoted (up to 52% w/w) through N-limitation, resulting in high lipid productivity (528.5 mg/L/day). Fed-batch cultures were performed at a C/N ratio of 278 and with nitrate pulse additions. This condition allowed a maximal cell mass of 14.2 g/L to be achieved and switched the metabolism to carbohydrate synthesis (up to 54% of dry weight), mainly in the form of starch. It was found that transmembrane transport under these conditions was dependent on a proton-motive force, indicating that glucose is transported by a symporter. Conclusions N. oleoabundans was able to grow under strict heterotrophic culture conditions with glucose or cellobiose as the only carbon source. The glucose used is transported by a symporter system. Batch cultures with a balanced C/N ratio accumulate proteins as the major cellular component; a high C/N ratio significantly increased the dry cell mass and resulted in a high lipid content, and a high cell density was achieved using fed-batch cultures promoting carbohydrate accumulation. These results suggest heterotrophic batch cultures of N. oleoabundans as an alternative for the production of proteins or lipids with simple culture strategies and minimal-mineral media supplemented with glucose.

Microbial enzymes that can hydrolyze organophosphorus compounds have been isolated, identified and characterized from different microbial species in order to use them in biodegradation of organophosphorus compounds. We isolated a bacterial strain Cons002 from an agricultural soil bacterial consortium, which can hydrolyze methyl-parathion (MP) and other organophosphate pesticides. HPLC analysis showed that strain Cons002 is capable of degrading pesticides MP, parathion and phorate. Pulsed-field gel electrophoresis and 16S rRNA amplification were performed for strain characterization and identification, respectively, showing that the strain Cons002 is related to the genus Enterobacter sp. which has a single chromosome of 4.6 Mb and has no plasmids. Genomic library was constructed from DNA of Enterobacter sp. Cons002. A gene called opdE (Organophosphate Degradation from E nterobacter) consists of 753 bp and encodes a protein of 25 kDa, which was isolated using activity methods. This gene opdE had no similarity to any genes reported to degrade organophosphates. When kanamycin-resistance cassette was placed in the gene opdE, hydrolase activity was suppressed and Enterobacter sp. Cons002 had no growth with MP as a nutrients source.