Obtainment, selection and characterization of a mutant strain of Kluyveromyces marxianus that displays improved production of 2-phenylethanol and enhanced DAHP synthase activity.

AIMS: Yeasts produce 2-phenylethanol (2-PE) from sugars via de novo synthesis; however, its synthesis is limited due to feedback inhibition on the isofunctional 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) synthases (Aro3p and Aro4p). This work aimed to select Kluyveromyces marxianus mutant strains with improved capacity to produce 2-PE from sugars. METHODS AND RESULTS: Kluyveromyces marxianus CCT 7735 mutant strains were selected from UV irradiation coupled with screening of p-fluoro-dl-phenylalanine (PFP) tolerant strains on culture medium without l-Phe addition. Most of them produced 2-PE titres higher than the parental strain and the Km_PFP41 mutant strain stood out for displaying the highest 2-PE specific production rate. Moreover it showed higher activity of DAHP synthase than the parental strain. We sequenced both ARO3 and ARO4 genes of Km_PFP41 mutant and identified mutations in ARO4 which caused changes in both size and conformation of the Aro4p. These changes seem to be associated with the enhanced activity of DAHP synthase and improved production of 2-PE exhibited by that mutant strain. CONCLUSIONS: The Km_PFP41 mutant strain presented improved 2-PE production via de novo synthesis and enhanced DAHP synthase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The mutant strain obtained in this work may be exploited as a yeast cell factory for high-level synthesis of 2-PE.

Bacterial degradation of dissolved organic matter released by Planktothrix agardhii (Cyanobacteria).

Although Planktothrix agardhii often produces toxic blooms in eutrophic water bodies around the world, little is known about the fate of the organic matter released by these abundant Cyanobacteria. Thus, this study focused in estimating the bacterial consumption of the DOC and DON (dissolved organic carbon and dissolved organic nitrogen, respectively) produced by axenic P. agardhii cultures and identifying some of the bacterial OTUs (operational taxonomic units) involved in the process. Both P. agardhii and bacterial inocula were sampled from the eutrophic Barra Bonita Reservoir (SP, Brazil). Two distinct carbon degradation phases were observed: during the first three days, higher degradation coefficients were calculated, which were followed by a slower degradation phase. The maximum value observed for particulate bacterial carbon (POC) was 11.9 mg L-1, which consisted of 62.5% of the total available DOC, and its mineralization coefficient was 0.477 day-1 (t½ = 1.45 days). A similar pattern of degradation was observed for DON, although the coefficients were slightly different. Changes in the OTUs patterns were observed during the different steps of the degradation. The main OTUs were related to the classes Alphaproteobacteria (8 OTUs), Betaproteobacteria (2 OTUs) and Gammaproteobacteria (3 OTUs). The genus Acinetobacter was the only identified organism that occurred during the whole process. Bacterial richness was higher at the slower degradation phase, which could be related to the small amounts of DOM (dissolved organic matter) available, particularly carbon. The kinetics of the bacterial degradation of P. agardhii-originated DOM suggests minimal loss of DOM from the Barra Bonita reservoir.

Influence of the residue from an iron mining dam in the growth of two macrophyte species.

On November 5th, 2015 the worst environmental disaster in Brazil spilled 60 million m3 of iron mining residue into Gualaxo do Norte River (Minas Gerais State), an affluent of the highest River Basin of the Brazilian Southeast (Doce River Basin), reaching the Atlantic Ocean. To assess the impact of the iron residue on the aquatic plant metabolism, we performed macrophyte growth experiments under controlled light and temperature conditions using two species (Egeria densa and Chara sp.). The plants' growth data were fitted in a kinetic model to obtain the biomass yields (K) and growth rates (µ). Turbidity and electrical conductivity of the water were measured over time. Both plants showed the highest growth rates in the contaminated condition (0.056 d-1 for E. densa and 0.45 d-1 for Chara sp.) and the biomass increased in the short-term (≈20 days). The control condition (i.e. no impacted water) supported the biomass increasing over time and the development of vegetative buddings with high daily rates (1.75 cm d-1 for E. densa and 0.13 cm d-1 for Chara sp). Turbidity showed a sharp decrease in 48 h and had no effects in the plants growth in the contaminated condition. The contamination affected the plants' yields in the long-term affecting the biomass development. This study provides preliminary information about the ecological consequences of a mining dam rupture aiming to collaborate with monitoring and risk assessments.

Chemical constraints on new man-made lakes.

The formation of reservoirs often affects water quality strongly, with the changes in the physicochemical properties being ascribed to decomposition of remaining organic matter arising from leaching and (biological and chemical) breakdown processes. In this study, experiments under laboratory conditions were performed to show that the nature of the course particulate organic matter (CPOM; i.e., leaves, branches, barks, and litter) determines the decomposition kinetics in new reservoirs. Effects on the water quality can be of short-, mid-, and long-term duration for all types of CPOM, as indicated in the mathematical modeling of the decomposition kinetics. Leaves and litter displayed the shortest half-life times (51 and 40 days, respectively) and the highest potential of leaching/oxidation of labile compounds (19 and 21%, respectively). On the other hand, decomposition of branches and barks generated the lowest oxygen consumption (74 and 44 mg oxygen/g dry mass (DM), respectively). During formation of the reservoir, the incorporation and decomposition of organic matter prevailed over material exportation. Therefore, in addition to a decrease in oxygen availability the concentration of biochemical oxygen demand (BOD) and nutrients increased. After the filling stage, there was significant loss of organic matter via oxidation, sedimentation, biological assimilation, and export, thus causing the BOD concentration and the fertility of the water to decrease.

Oxygen uptake from aquatic macrophyte decomposition from Piraju Reservoir (Piraju, SP, Brazil).

The kinetics of oxygen consumption related to mineralisation of 18 taxa of aquatic macrophytes (Cyperus sp, Azolla caroliniana, Echinodorus macrophyllus, Eichhornia azurea, Eichhornia crassipes, Eleocharis sp1, Eleocharis sp2, Hetereanthera multiflora, Hydrocotyle raniculoides, Ludwigia sp, Myriophyllum aquaticum, Nymphaea elegans, Oxycaryum cubense, Ricciocarpus natans, Rynchospora corymbosa, Salvinia auriculata, Typha domingensis and Utricularia foliosa) from the reservoir of Piraju Hydroelectric Power Plant (São Paulo state, Brazil) were described. For each species, two incubations were prepared with ca. 300.0 mg of plant (DW) and 1.0 L of reservoir water sample. The incubations were maintained in the dark and at 20 ºC. Periodically the dissolved oxygen (DO) concentrations were measured; the accumulated DO values were fitted to 1st order kinetic model and the results showed that: i) high oxygen consumption was observed for Ludwigia sp (533 mg g-1 DW), while the lowest was registered for Eleocharis sp1 (205 mg g-1 DW) mineralisation; ii) the higher deoxygenation rate constants were verified in the mineralisation of A. caroliniana (0.052 day-1), H. raniculoides (0.050 day-1) and U. foliosa (0.049 day-1). The oxygen consumption rate constants of Ludwigia sp and Eleocharis sp2 mineralisation (0.027 day-1) were the lowest. The half-time of oxygen consumption varied from 9 to 26 days. In the short term, the detritus of E. macrophyllus, H. raniculoides, Ludwigia sp, N. elegans and U. foliosa were the critical resources to the reservoir oxygen demand; while in the long term, A. caroliniana, H. multiflora and T. domingensis were the resources that can potentially contribute to the benthic oxygen demand of this reservoir.

Decomposition of dissolved organic matter released by an isolate of Microcystis aeruginosa and morphological profile of the associated bacterial community.

This study concerns the kinetics of bacterial degradation of two fractions (molecular mass) of dissolved organic matter (DOM) released by Microcystis aeruginosa. Barra Bonita Reservoir (SP, Brazil) conditions were simulated in the laboratory using the associated local bacterial community. The extent of degradation was quantified as the amount of organic carbon transferred from each DOM fraction (< 3 kDa and 3-30 kDa) to bacteria. The variation of bacteria morphotypes associated with the decomposition of each fraction was observed. To find the degradation rate constants (kT), the time profiles of the total, dissolved and particulate organic carbon concentrations were fitted to a first-order kinetic model. These rate constants were higher for the 3-30 kDa fraction than for the lighter fraction. Only in the latter fraction the formation of refractory dissolved organic carbon (DOCR) compounds could be detected and its rate of mass loss was low. The higher bacterial density was reached at 24 and 48 hours for small and higher fractions, respectively. In the first 48 hours of decomposition of both fractions, there was an early predominance of bacillus, succeeded by coccobacillus, vibrios and coccus, and from day 5 to 27, the bacterial density declined and there was greater evenness among the morphotypes. Both fractions of DOM were consumed rapidly, corroborating the hypothesis that DOM is readily available in the environment. This also suggests that the bacterial community in the inocula readily uses the labile part of the DOM, until this community is able to metabolise efficiently the remaining of DOM not degraded in the first moment. Given that M. aeruginosa blooms recur throughout the year in some eutrophic reservoirs, there is a constant supply of the same DOM which could maintain a consortium of bacterial morphotypes adapted to consuming this substrate.

Cellulase and xylanase activity during the decomposition of three aquatic macrophytes in a tropical oxbow lagoon.

Due to the connection between enzymatic activity and degradation of different fractions of organic matter, enzyme assays can be used to estimate degradation rates of particulate and dissolved organic carbon in freshwater systems. The aim of this study was to quantify and model the enzymatic degradation involving the decomposition of macrophytes, describing temporal activity of cellulases (EC 3.2.1.4 and EC 3.2.1.91) and xylanase (EC 3.2.1.8) during in situ decomposition of three aquatic macrophytes (Salvinia sp., Eichhornia azurea and Cyperus giganteus) on the surface and water-sediment interface (w-s interface) of an oxbow lagoon (Óleo lagoon) within a natural Brazilian Savanna Reserve. Overall, the enzymatic degradation of aquatic macrophytes in Óleo lagoon occurred during the whole year and was initiated together with leaching. Xylanase production was ca. 5 times higher than cellulase values due to easy access to this compound by cellulolytic microorganisms. Enzymatic production and detritus mass decay were similar on the surface and w-s interface. Salvinia sp. was the most recalcitrant detritus, with low mass decay and enzymatic activity. E. azurea and C. giganteus decomposition rates and enzymatic production were high and similar. Due to the physicochemical homogeneity observed in the Óleo lagoon, the differences between the decay rates of each species are mostly related with detritus chemical quality.

Aerobic and anaerobic decomposition of Pistia stratiotes leachates from a tropical eutrophic reservoir (Barra Bonita, SP, Brazil).

The present study discussed the kinetic aspects of leachate decomposition from an aquatic macrophyte, Pistia stratiotes L (water lettuce). This species was collected from Barra Bonita Reservoir located in the State of São Paulo (Brazil). Decomposition chambers were prepared with high molecular weight (HMW), low molecular weight (LMW) and integral (INT = HMW + LMW) dissolved organic matter (DOM) diluted with reservoir water. The samples were incubated at 20 degrees C, in darkness and under aerobic or anaerobic conditions. For 79 days, the concentrations of dissolved oxygen (DO) and organic carbon (OC) were measured. For calculating the deoxygenation coefficients (k d) and maximum oxygen consumption (COmax) the concentration of DO was integrated and fitted to a first-order kinetics model, which also applied to the depletion of OC concentrations. The COmax of INT incubations were 4% higher than the sum of HMW and LMW fractions. The deoxygenation coefficients, k d, had the same order of magnitude for all treatments. In relation to carbon decay, regardless of the availability of oxygen, the INT DOM also showed higher mineralisation. These results suggest that the leachate mineralisations are short-term processes; when the fractionation of the leachates occurs, the LMW had organic compounds with more accessibility for heterotrophic metabolism. On the other hand, when compared to INT DOM, the HMW and LMW were less consumed suggesting an interaction of the reactivity of the leachate. Our data suggest that in the Barra Bonita Reservoir the mineralisation of P. stratiotes leachates occurs through two competitive pathways (i.e. mineralisation of the labile compounds and formation of recalcitrant organic resources and their mineralisation) in which the oxygen availability and the molecular mass of DOM can interfere in the rates of reactions.

Oxygen demand during mineralization of aquatic macrophytes from an oxbow lake.

This study presents a kinetic model of oxygen consumption during aerobic decomposition of detritus from seven species of aquatic macrophytes: Cabomba furcata, Cyperus giganteus, Egeria najas, Eichhornia azurea, Salvinia auriculata, Oxycaryum cubense and Utricularia breviscapa. The aquatic macrophytes were collected from Oleo Lagoon situated in the Mogi-Guaçu river floodplain (SP, Brazil). Mineralization experiments were performed using the closed bottles method. Incubations made with lake water and macrophytes detritus (500 mL and 200 mg.L(-1) (DM), respectively) were maintained during 45 to 80 days at 20 degrees C under aerobic conditions and darkness. Carbon content of leachates from aquatic macrophytes detritus and dissolved oxygen concentrations were analyzed. From the results we concluded that: i) the decomposition constants differ among macrophytes; these differences being dependent primarily on molecular and elemental composition of detritus and ii) in the short term, most of the oxygen demand seems to depend upon the demineralization of the dissolved carbon fraction.

Oxygen uptake during mineralization of photosynthesized carbon from phytoplankton of the Barra Bonita reservoir: a mesocosm study.

This study aimed to discuss and describe the oxygen consumption during aerobic mineralization of organic products (cells and excretion products) from five unialgal cultures: Cryptomonas sp., Microcystis aeruginosa, Anabaena spiroides, Thalassiosira sp. and Aulacoseira granulata. These species were isolated from Barra Bonita reservoir (22 degrees 29' S and 48 degrees 34' W) and cultivated in the laboratory. From each culture, two decomposition chambers were prepared; each chamber contained about 130 mg.L(-1) of carbon from water samples of the reservoir. The chambers were aerated and incubated in the dark at 20.0 degrees C. The concentration of dissolved oxygen, pH values and electrical conductivity of the solutions were determined during a period of 10 days. The results indicated increases in oxygen consumption for all the solutions studied and also for electrical conductivity. The pH values presented a decreasing tendency throughout the experiment. Oxygen consumption varied from 43 (Aulacoseira granulata chamber) to 345 mg O2 g(-1) C (Anabaena spiroides chamber). Decrease in pH values was probably due to increase in CO2 concentration from microbial respiration. Increase in electrical conductivity might be due to the liberation of ions during decomposition. The results demonstrate the potentiality of the studied genera in influencing oxygen availability followed by a die-off event. It also indicates the possibility of changing of the electrical conductivity and pH values in the water column due the aerobic algae mineralization.

Hydrologic cycle and dynamics of aquatic macrophytes in two intermittent rivers of the semi-arid region of Brazil.

The dynamics of aquatic macrophytes in intermittent rivers is generally related to the characteristics of the resistance and resilience of plants to hydrologic disturbances of flood and drought. In the semi-arid region of Brazil, intermittent rivers and streams are affected by disturbances with variable intensity, frequency, and duration throughout their hydrologic cycles. The aim of the present study is to determine the occurrence and variation of biomass of aquatic macrophyte species in two intermittent rivers of distinct hydrologic regimes. Their dynamics were determined with respect to resistance and resilience responses of macrophytes to flood and drought events by estimating the variation of biomass and productivity throughout two hydrologic cycles. Twenty-one visits were undertaken in the rewetting, drying, and drought phases in a permanent puddle in the Avelós stream and two temporary puddles in the Taperoá river, state of Paraíba, Northeast Brazil. The sampling was carried out by using the square method. Floods of different magnitudes occurred during the present study in the river and in the stream. The results showed that floods and droughts are determining factors in the occurrence of macrophytes and in the structure of their aquatic communities. The species richness of the aquatic macrophyte communities was lower in the puddles of the river and stream subject to flood events, when compared to areas where the run-off water is retained. At the beginning of the recolonization process, the intensity of the floods was decisive in the productivity and biomass of the aquatic macrophytes in the Taperoá river and the Avelós stream. In intermediate levels of disturbance, the largest values of productivity and biomass and the shortest time for starting the recolonization process occurred.

The effect of the size of particles on mineralization of Oxycaryum cubense (Poepp. & Kunth) Lye.

Assays were carried out to evaluate effects of detritus size on the mineralization of an aquatic macrophyte, the Oxycaryum cubense. Samples of plant and water were collected from an oxbow lake, the Infernão lagoon (21 degrees 35' S and 47 degrees 51' W) located at Mogi Guaçu river floodplain. The plants were taken to the laboratory, washed under tap water, dried (50 degrees C) and fractioned into six groups according to their size, viz. 100, 10, 1.13, 0.78, 0.61 and 0.25 mm. Decomposition chambers were prepared by adding 1.0 g of plant fragments to 4.1 L of water lagoon. In sequence, the incubations were aerated and the concentrations of dissolved oxygen, the pH, the electric conductivity and the temperature were monitored for 120 days. The occurrence of anaerobic processes was avoided by reoxygenating the solutions. The experimental results were fitted to a first order kinetic model and the consumption of dissolved oxygen from mineralization processes was obtained. The physical process of fragmentation of O. cubense detritus is unlikely to promote the consumption of higher quantities of dissolved oxygen in mineralization processes meaning that fragmentation should not interfere in the balance of DO in this aquatic system, however fragmentation processes favored the acidification and increased the liberation of dissolved ions from the Infernão lagoon.

Oxygen uptake during mineralization of humic substances from Infernão Lagoon (São Paulo, Brazil).

Assays were carried out to evaluate the dissolved oxygen uptake resulting from mineralization of humic substances (fulvic acid (FA) and humic acid (HA)) from different sources: sediment, dissolved organic matter (DOM) of 120-day decomposed aquatic macrophyte (Scirpus cubensis and Cabomba piauhyensis), and lagoon DOM. The experiments were also aimed at estimating the oxygen uptake coefficient of the mineralization. About 20-30 mg of substrate were added to 1.1 liters of water from Infernão Lagoon (21 degrees 33' to 21 degrees 37'S; 47 degrees 45' to 47 degrees 51'W). The solutions were aerated and the dissolved oxygen (DO) was monitored during 40 days. Dissolved organic carbon (DOC) and particulate organic carbon (POC) were estimated after 80 days of the experiment. Anaerobic processes were avoided by aerating the solutions. The results were fitted to a first-order kinetics model, from which the uptake of oxygen parameters was obtained. Oxygen consumption (OC) ranged from 4.24 mg L(-1) (HA--S. cubensis) to 33.76 mg L(-1) (FA--sediment). The highest deoxygenation coefficient (kD) was observed during mineralization of FA--DOM (0.299 day(-1)), followed in decreasing order by FA--S. cubensis, HA--sediment, HA--S. cubensis, FA --sediment, and FA--C. piauhyensis (0.282; 0.255; 0.178; 0.130, and 0.123 day(-1), respectively). The carbon analyses indicated that the FA and HA samples at the end of the experiment presented a decay that varied from 15.23% to 42.35% and that the FA and HA conversions into POC were relatively low (from 0.76% to 3.94%).