Ferredoxin5 Deletion Affects Metabolism of Algae during the Different Phases of Sulfur Deprivation.

Ferredoxin5 (FDX5), a minor ferredoxin protein in the alga Chlamydomonas (Chlamydomonas reinhardtii), helps maintain thylakoid membrane integrity in the dark. Sulfur (S) deprivation has been used to achieve prolonged hydrogen production in green algae. Here, we propose that FDX5 is involved in algal responses to S-deprivation as well as to the dark. Specifically, we tested the role of FDX5 in both the initial aerobic and subsequent anaerobic phases of S-deprivation. Under S-deprived conditions, absence of FDX5 causes a distinct delay in achieving anoxia by affecting photosynthetic O2 evolution, accompanied by reduced acetate uptake, lower starch accumulation, and delayed/lower fermentative metabolite production, including photohydrogen. We attribute these differences to transcriptional and/or posttranslational regulation of acetyl-CoA synthetase and ADP-Glc pyrophosphorylase, and increased stability of the PSII D1 protein. Interestingly, increased levels of FDX2 and FDX1 were observed in the mutant under oxic, S-replete conditions, strengthening our previously proposed hypothesis that other ferredoxins compensate in response to a lack of FDX5. Taken together, the results of our omics and pull-down experiments confirmed biochemical and physiological results, suggesting that FDX5 may have other effects on Chlamydomonas metabolism through its interaction with multiple redox partners.

The role of microorganisms in a full-scale sequencing batch reactor under low aeration and different cycle times.

This study describes the role of microorganisms in a full-scale step-feed sequencing batch reactor (SBR) system for urban wastewater treatment. Chemical profiles for three different cycle times were measured under low aeration conditions with a high carbon-to-nitrogen ratio. The applied organic load was above 1.0 g chemical oxygen demand (COD)/L x d. The removal efficiencies were higher than 81%, 93%, and 76% for soluble COD, N-NH4+, and total Kjeldahl nitrogen, respectively. The ratio of volatile suspended solids (VSS) to total suspended solids was 78%, and the food-to-microorganism ratio was an average of 1.41 g COD/g VSS x d. The active biomass was comprised of 87.8% heterotrophic and 12.2% autotrophic organisms. Nitrifying organisms were found with a low amount of ammonia-oxidizing bacteria (5%) and a much higher amount of nitrite-oxidizing bacteria. Polyphosphate-accumulating organisms (PAOs) were found at high amounts (25%) compared to glycogen-accumulating organisms, even in a system with a high carbon to phosphorus ratio. The activity of denitrifying PAOs was 72%.

Dynamics of planktonic prokaryotes and dissolved carbon in a subtropical coastal lake.

To understand the dynamics of planktonic prokaryotes in a subtropical lake and its relationship with carbon, we conducted water sampling through four 48-h periods in Peri Lake for 1 year. Planktonic prokaryotes were characterized by the abundance and biomass of heterotrophic bacteria (HB) and of cyanobacteria (coccoid and filamentous cells). During all samplings, we measured wind speed, water temperature (WT), pH, dissolved oxygen (DO), precipitation, dissolved organic carbon (DOC), dissolved inorganic carbon (DIC), and carbon dioxide (CO2). DOC was higher in the summer (average = 465 µM - WT = 27°C) and lower in the winter (average = 235 µM - WT = 17°C), with no significant variability throughout the daily cycles. CO2 concentrations presented a different pattern, with a minimum in the warm waters of the summer period (8.31 µM) and a maximum in the spring (37.13 µM). Daily trends were observed for pH, DO, WT, and CO2. At an annual scale, both biological and physical-chemical controls were important regulators of CO2. HB abundance and biomass were higher in the winter sampling (5.60 × 10(9) cells L(-1) and 20.83 µmol C L(-1)) and lower in the summer (1.87 × 10(9) cells L(-1) and 3.95 µmol C L(-1)). Filamentous cyanobacteria (0.23 × 10(8)-0.68 × 10(8) filaments L(-1)) produced up to 167.16 µmol C L(-1) as biomass (during the warmer period), whereas coccoid cyanobacteria contributed only 0.38 µmol C L(-1). Precipitation, temperature, and the biomass of HB were the main regulators of CO2 concentrations. Temperature had a negative effect on the concentration of CO2, which may be indirectly attributed to high heterotroph activity in the autumn and winter periods. DOC was positively correlated with the abundance of total cyanobacteria and negatively with HB. Thus, planktonic prokaryotes have played an important role in the dynamics of both dissolved inorganic and organic carbon in the lake.

Full-scale sequencing batch reactor (SBR) for domestic wastewater: performance and diversity of microbial communities.

This work describes the performance and microbial diversity in a sequencing batch reactor of a decentralized full-scale system for urban wastewater treatment under limited aeration. The removal efficiency was: 83% for soluble chemical oxygen demand (SCOD), 60% for N-NH4(+), 70% for total suspended solids (TSS) and 80% for volatile suspended solids (VSS). The biomass concentration had a maximum value around 8.7gVSSL(-1) for organic load rate of 0.6gCODL(-1)d(-1). The food/microorganism ratios showed average of 0.2gCOD/gVSSd. The sludge bacterial flocs were formed an irregular arrangement with organisms attached such as Euglypha sp. and pedunculate ciliates. It was observed the presence of Bacteria domains including Nitrosomonas spp., Nitrobacter spp., Nitrospira and C. "Accumulibacter" cluster. The DPAO activity was 70%. Denaturing gradient gel electrophoresis showed changes in ribotype number over biological treatment time among the groups observed being some are linked to nutrient removal. The reactor showed viability to treat domestic wastewater.

Microbial and chemical profile of a ponds system for the treatment of landfill leachate.

The present study describes the behavior of spatio-temporal variation of parameters and microbial profile of a pilot stabilization ponds system, consisted of three serial ponds, for the treatment of landfill leachate. Bacterial diversity was determined through molecular techniques (FISH, PCR and phylogenic analysis), while the phytoplankton community was evaluated through optical microscopy and quantified by the Sedgewick-Rafter chamber. Physicochemical parameters were also evaluated. The ponds system presented the following removal efficiency: 56% for TCOD; 83% for SBOD5 and 82% for N-NH4(+). Moreover, the analysis of chlorophyll a and DO showed stratification in the mass of water by the vertical profile. The analysis of the phytoplankton community showed a low number of species, with a predominance of Chlamydomonas sp. and presence of Cryptomonas sp. in lower density. The bacterial diversity analysis showed the presence of Planctomycetales, Verrucomicrobiales, some Desulfovibionaceae sulfate-reducing bacteria and Pseudomonas sp.

Bioconversion of cassava starch by-product into bacillus and related bacteria polyhydroxyalkanoates

Background: Unlike petroleum-based synthetic plastics, biodegradable biopolymer generation from industrial residue is a key strategy to reduce costs in the production process, as well as in the waste management, since efficient industrial wastewater treatment could be costly. In this context, the present work describes the prospection and use of bacterial strains capable to bioconvert cassava starch by-product into biodegradable polyhydroxyalkanoates (PHAs). Results: The first step of this study was the bacterial competence screening which was conducted with 72 strains covering 21 Bacillus and related species. The microorganism growth in a medium with a starch substrate was measured by an innovative MTT assay, while the ability of the bacteria to secrete amylase and produce PHA was evaluated by the Nile Red Dye method. Based on growth and potential for PHA production, four isolates were selected and identified as Bacillus megaterium by 16S rRNA sequencing. When cultivated in hydrolyzed cassava starch by-product, maximum production reached 4.97 g dry biomass/L with 29.7 percent of Poly-(3-hydroxybutyrate) (characterized by FTIR). Conclusions: MTT assay proved to be a reliable methodology for monitoring bacterial growth in insoluble media. Selected amylolytic strains could be used as an alternative industrial process for biodegradable plastics production from starchy residues, reducing costs for biodegradable biopolymer production and wastewater treatment operations.

Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum.

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.

Antioxidant properties of violacein: possible relation on its biological function.

Violacein, a violet pigment produced by Chromobacterium violaceum, has attracted much attention in recent literature due to its pharmacological properties. In this work, the antioxidant properties of violacein were investigated. The reactivity with oxygen and nitrogen reactive species and 1,1-diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, was evaluated. EPR studies were carried out to evaluate the reactivity with the hydroxyl radical. The action of violacein against lipid peroxidation in three models of lipid membranes, including rat liver microsomes, Egg and Soy bean phosphathidylcholine liposomes were also evaluated. The compound reacted with DPPH (IC(50)=30microM), nitric oxide (IC(50)=21microM), superoxide radicals (IC(50)=125microM) and decreased the hydroxyl radical EPR signal. The compound protected the studied membranes against peroxidation induced by reactive species in the micromolar range. The reconstitution of violacein into the membranes increased its antioxidant effect. These results indicate that the compound has strong antioxidant potential. Based on these results we suggest violacein plays an important role with the microorganism membrane in defense against oxidative stress.

In vitro testing for genotoxicity of violacein assessed by Comet and Micronucleus assays.

Chromobacterium violaceum is a Gram (-) bacteria found in water samples and soils from tropical and subtropical regions of the world. Violacein, the major pigment produced by these bacteria, has been shown to have antibiotic, antitumoral and trypanocidal activities. In the present work, the genotoxicity of violacein was investigated in four different cell lines by using the alkaline Comet assay and in VERO cells using the Micronucleus test. In the alkaline Comet assay, violacein, when tested at concentrations ranging from 0.19 to 1.5 microM, did not induce a significant increase in DNA damage in HEp-2 and MA104 cells. However, violacein was positive for DNA damage in FRhK-4 cells and for both DNA damage and micronuclei in VERO cells, in a concentration-response relationship. The results of this study indicated that violacein is genotoxic in VERO and FRhK-4 cells. These findings contribute to a comprehensive evaluation of the pharmacological potential of violacein.

Energetic metabolism of Chromobacterium violaceum.

Chromobacterium violaceum is a free-living microorganism, normally exposed to diverse environmental conditions; it has a versatile energy-generating metabolism. This bacterium is capable of exploiting a wide range of energy resources by using appropriate oxidases and reductases. This allows C. violaceum to live in both aerobic and anaerobic conditions. In aerobic conditions, C. violaceum is able to grow in a minimal medium with simple sugars, such as glucose, fructose, galactose, and ribose; both Embden-Meyerhoff, tricarboxylic acid and glyoxylate cycles are used. The respiratory chain supplies energy, as well as substrates for other metabolic pathways. Under anaerobic conditions, C. violaceum metabolizes glucose, producing acetic and formic acid, but not lactic acid or ethanol. C. violaceum is also able to use amino acids and lipids as an energy supply.

Molecular characterization of the poly(3-hydroxybutyrate) (PHB) synthase from Ralstonia eutropha: in vitro evolution, site-specific mutagenesis and development of a PHB synthase protein model.

A threading model of the Ralstonia eutropha polyhydroxyalkanoate (PHA) synthase was developed based on the homology to the Burkholderia glumae lipase, whose structure has been resolved by X-ray analysis. The lid-like structure in the model was discussed. In this study, various R. eutropha PHA synthase mutants were generated employing random as well as site-specific mutagenesis. Four permissive mutants (double and triple mutations) were obtained from single gene shuffling, which showed reduced activity and whose mutation sites mapped at variable surface-exposed positions. Six site-specific mutations were generated in order to identify amino acid residues which might be involved in substrate specificity. Replacement of residues T323 (I/S) and C438 (G), respectively, which are located in the core structure of the PHA synthase model, abolished PHA synthase activity. Replacement of the two amino acid residues Y445 (F) and L446 (K), respectively, which are located at the surface of the protein model and adjacent to W425, resulted in reduced activity without changing substrate specificity and indicating a functional role of these residues. The E267K mutant exhibited only slightly reduced activity with a surface-exposed mutation site. Four site-specific deletions were generated to evaluate the role of the C-terminus and variant amino acid sequence regions, which link highly conserved regions. Deleted regions were D281-D290, A372-C382, E578-A589 and V585-A589 and the respective PHA synthases showed no detectable activity, indicating an essential role of the variable C-terminus and the linking regions between conserved blocks 2 and 3 as well as 3 and 4. Moreover, the N-terminal part of the class II PHA synthase (PhaC(Pa)) from Pseudomonas aeruginosa and the C-terminal part of the class I PHA synthase (PhaC(Re)) from R. eutropha were fused, respectively, resulting in three fusion proteins with no detectable in vivo activity. However, the fusion protein F1 (PhaC(Pa)-1-265-PhaC(Re)-289-589) showed 13% of wild type in vitro activity with the fusion point located at a surface-exposed loop region.