Treatment of gray water using anaerobic biofilms created on synthetic and natural fibers

Gray water treatment and reuse is an immediate option to counter the upcoming water shortages in various parts of world, especially urban areas. Anaerobic treatment of gray water in houses is an alternative low cost, low energy and low sludge generating option that can meet this challenge. Typical problems of fluctuating VFA, low pH and sludge washout at low loading rates with gray water feedstock was overcome in two chambered anaerobic biofilm reactors using natural fibers as the biofilm support. The long term performance of using natural fiber based biofilms at moderate and low organic loading rates (OLR) have been examined. Biofilms raised on natural fibers (coir, ridge-gourd) were similar to that of synthetic media (PVC, polyethylene) at lower OLR when operated in pulse fed mode without effluent recirculation and achieved 80-90% COD removal at HRT of 2 d showing a small variability during start-up. Confocal microscopy of the biofilms on natural fibers indicated thinner biofilms, dense cell architecture and low extra cellular polymeric substances (EPS) compared to synthetic supports and this is believed to be key factor in high performance at low OLR and low strength gray water. Natural fibers are thus shown to be an effective biofilm support that withstand fluctuating characteristic of domestic gray water. (C) 2013 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

DNA Assisted Self-Assembly of PAMAM Dendrimers

We report DNA assisted self-assembly of polyamidoamine (PAMAM) dendrimers using all atom Molecular Dynamics (MD) simulations and present a molecular level picture of a DNA-linked PAMAM dendrimer nanocluster, which was first experimentally reported by Choi et al. (Nano Lett., 2004, 4, 391-397). We have used single stranded DNA (ssDNA) to direct the self-assembly process. To explore the effect of pH on this mechanism, we have used both the protonated (low pH) and nonprotonated (high pH) dendrimers. In all cases studied here, we observe that the DNA strand on one dendrimer unit drives self-assembly as it binds to the complementary DNA strand present on the other dendrimer unit, leading to the formation of a DNA-linked dendrimer dimeric complex. However, this binding process strongly depends on the charge of the dendrimer and length of the ssDNA. We observe that the complex with a nonprotonated dendrimer can maintain a DNA length dependent inter-dendrimer distance. In contrast, for complexes with a protonated dendrimer, the inter-dendrimer distance is independent of the DNA length. We attribute this observation to the electrostatic complexation of a negatively charged DNA strand with the positively charged protonated dendrimer.

Dissecting Ubiquitin Folding Using the Self-Organized Polymer Model

Folding of Ubiquitin (Ub), a functionally important protein found in eukaryotic organisms, is investigated at low and neutral pH at different temperatures using simulations of the coarse-grained self-organized-polymer model with side chains (SOP-SC). The melting temperatures (T-m's), identified with the peaks in the heat capacity curves, decrease as pH decreases, in qualitative agreement with experiments. The calculated radius of gyration, showing dramatic variations with pH, is in excellent agreement with scattering experiments. At T-m Ub folds in a two-state manner at low and neutral pH. Clustering analysis of the conformations sampled in equilibrium folding trajectories at T-m with multiple transitions between the folded and unfolded states, shows a network of metastable states connecting the native and unfolded states. At low and neutral pH, Ub folds with high probability through a preferred set of conformations resulting in a pH-dependent dominant folding pathway. Folding kinetics reveal that Ub assembly at low pH occurs by multiple pathways involving a combination of nucleation-collapse and diffusion collision mechanism. The mechanism by which Ub folds is dictated by the stability of the key secondary structural elements responsible for establishing long-range contacts and collapse of Ub. Nucleation collapse mechanism holds if the stability of these elements are marginal, as would be the case at elevated temperatures. If the lifetimes associated with these structured microdomains are on the order of hundreds of microseconds, then Ub folding follows the diffusion collision mechanism with intermediates, many of which coincide with those found in equilibrium. Folding at neutral pH is a sequential process with a populated intermediate resembling that sampled at equilibrium. The transition state structures, obtained using a P-fold analysis, are homogeneous and globular with most of the secondary and tertiary structures being native-like. Many of our findings for both the thermodynamics and kinetics of folding are not only in agreement with experiments but also provide missing details not resolvable in standard experiments. The key prediction that folding mechanism varies dramatically with pH is amenable to experimental tests.

Sequence specific complexation of b DNA at sites containing g,c base pairs

A series of eight related analogs of distamycin A has been synthesized. Footprinting and affinity cleaving reveal that only two of the analogs, pyridine-2- car box amide-netropsin (2-Py N) and 1-methylimidazole-2-carboxamide-netrops in (2-ImN), bind to DNA with a specificity different from that of the parent compound. A new class of sites, represented by a TGACT sequence, is a strong site for 2-PyN binding, and the major recognition site for 2-ImN on DNA. Both compounds recognize the G•C bp specifically, although A's and T's in the site may be interchanged without penalty. Additional A•T bp outside the binding site increase the binding affinity. The compounds bind in the minor groove of the DNA sequence, but protect both grooves from dimethylsulfate. The binding evidence suggests that 2-PyN or 2-ImN binding induces a DNA conformational change.

In order to understand this sequence specific complexation better, the Ackers quantitative footprinting method for measuring individual site affinity constants has been extended to small molecules. MPE•Fe(II) cleavage reactions over a 10^5 range of free ligand concentrations are analyzed by gel electrophoresis. The decrease in cleavage is calculated by densitometry of a gel autoradiogram. The apparent fraction of DNA bound is then calculated from the amount of cleavage protection. The data is fitted to a theoretical curve using non-linear least squares techniques. Affinity constants at four individual sites are determined simultaneously. The distamycin A analog binds solely at A•T rich sites. Affinities range from 10^(6)- 10^(7)M^(-1) The data for parent compound D fit closely to a monomeric binding curve. 2-PyN binds both A•T sites and the TGTCA site with an apparent affinity constant of 10^(5) M^(-1). 2-ImN binds A•T sites with affinities less than 5 x 10^(4) M^(-1). The affinity of 2-ImN for the TGTCA site does not change significantly from the 2-PyN value. At the TGTCA site, the experimental data fit a dimeric binding curve better than a monomeric curve. Both 2-PyN and 2-ImN have substantially lower DNA affinities than closely related compounds.

In order to probe the requirements of this new binding site, fourteen other derivatives have been synthesized and tested. All compounds that recognize the TGTCA site have a heterocyclic aromatic nitrogen ortho to the N or C-terminal amide of the netropsin subunit. Specificity is strongly affected by the overall length of the small molecule. Only compounds that consist of at least three aromatic rings linked by amides exhibit TGTCA site binding. Specificity is only weakly altered by substitution on the pyridine ring, which correlates best with steric factors. A model is proposed for TGTCA site binding that has as its key feature hydrogen bonding to both G's by the small molecule. The specificity is determined by the sequence dependence of the distance between G's.

One derivative of 2-PyN exhibits pH dependent sequence specificity. At low pH, 4-dimethylaminopyridine-2-carboxamide-netropsin binds tightly to A•T sites. At high pH, 4-Me_(2)NPyN binds most tightly to the TGTCA site. In aqueous solution, this compound protonates at the pyridine nitrogen at pH 6. Thus presence of the protonated form correlates with A•T specificity.

The binding site of a class of eukaryotic transcriptional activators typified by yeast protein GCN4 and the mammalian oncogene Jun contains a strong 2-ImN binding site. Specificity requirements for the protein and small molecule are similar. GCN4 and 2-lmN bind simultaneously to the same binding site. GCN4 alters the cleavage pattern of 2-ImN-EDTA derivative at only one of its binding sites. The details of the interaction suggest that GCN4 alters the conformation of an AAAAAAA sequence adjacent to its binding site. The presence of a yeast counterpart to Jun partially blocks 2-lmN binding. The differences do not appear to be caused by direct interactions between 2-lmN and the proteins, but by induced conformational changes in the DNA protein complex. It is likely that the observed differences in complexation are involved in the varying sequence specificity of these proteins.

Some observations on the absence of bacteria from acid waters in northwest England

This brief report concentrates on the effect of low pH on the initial stages of decomposition and the conditioning of incoming particulate carbon or detritus by microbes, particularly certain genera of filamentous bacteria. Although many previous reports have concentrated on bacterial decomposition processes, little attention has been given to the composition of the bacterial community and the role of its component parts, particularly in nutrient-poor waters which are provided with sources of organic carbon and reducing power in the form of poor quality detritus.

A chemical survey of standing waters in south-east England, with reference to acidification and eutrophication

This study looks at the distribution and magnitude of acidification and eutrophication in south-east England where there are no natural lakes but a large number of shallow artificial ponds. The study area is defined as the region lying within a 100 km radius of central London but excluding the area within the M25 motorway. Water samples were taken from 120 sites between mid-January and the end of February 1990, with a subsequent monthly survey of a subset of 31 of these waters. Twelve chemical variables were measured in the laboratory using standard techniques. PH values for the full dataset ranged from 3.2 to 8.4, although the majority of sites had pH values in the range 7.0 to 8.5; only five sites had a pH of less than 6.0. The five low pH sites expectedly had low alkalinities and are the only sites with values below 0.1 meq per litre. Concentrations of calcium, sodium, potassium, magnesium, chloride, sulphate and nitrate had normal distributions. The majority of sites had total phosphorus concentrations in the range 25 to 200 mu g per litre, although 10 sites had concentrations above 400 mu g per litre. The low number of acid sites suggests that surface water acidity is not a widespread regional problem in south-east England. However the survey shows that a large number of standing waters in the region have high total phosphorus and nitrate concentrations, and 89% may be considered moderately to considerably eutrophic.

Chemical studies of viral entry mechanisms: I. Hydrophobic protein-lipid interactions during sendai virus membrane fusion. II. Kinetics of bacteriophage λ DNA injection.

Viruses possess very specific methods of targeting and entering cells. These methods would be extremely useful if they could also be applied to drug delivery, but little is known about the molecular mechanisms of the viral entry process. In order to gain further insight into mechanisms of viral entry, chemical and spectroscopic studies in two systems were conducted, examining hydrophobic protein-lipid interactions during Sendai virus membrane fusion, and the kinetics of bacteriophage λ DNA injection.

Sendai virus glycoprotein interactions with target membranes during the early stages of fusion were examined using time-resolved hydrophobic photoaffinity labeling with the lipid-soluble carbene generator3-(trifluoromethyl)-3-(m-^(125 )I] iodophenyl)diazirine (TID). The probe was incorporated in target membranes prior to virus addition and photolysis. During Sendai virus fusion with liposomes composed of cardiolipin (CL) or phosphatidylserine (PS), the viral fusion (F) protein is preferentially labeled at early time points, supporting the hypothesis that hydrophobic interaction of the fusion peptide at the N-terminus of the F_1 subunit with the target membrane is an initiating event in fusion. Correlation of the hydrophobic interactions with independently monitored fusion kinetics further supports this conclusion. Separation of proteins after labeling shows that the F_1 subunit, containing the putative hydrophobic fusion sequence, is exclusively labeled, and that the F_2 subunit does not participate in fusion. Labeling shows temperature and pH dependence consistent with a need for protein conformational mobility and fusion at neutral pH. Higher amounts of labeling during fusion with CL vesicles than during virus-PS vesicle fusion reflects membrane packing regulation of peptide insertion into target membranes. Labeling of the viral hemagglutinin/neuraminidase (HN) at low pH indicates that HN-mediated fusion is triggered by hydrophobic interactions, after titration of acidic amino acids. HN labeling under nonfusogenic conditions reveals that viral binding may involve hydrophobic as well as electrostatic interactions. Controls for diffusional labeling exclude a major contribution from this source. Labeling during reconstituted Sendai virus envelope-liposome fusion shows that functional reconstitution involves protein retention of the ability to undergo hydrophobic interactions.

Examination of Sendai virus fusion with erythrocyte membranes indicates that hydrophobic interactions also trigger fusion between biological membranes, and that HN binding may involve hydrophobic interactions as well. Labeling of the erythrocyte membranes revealed close membrane association of spectrin, which may play a role in regulating membrane fusion. The data show that hydrophobic fusion protein interaction with both artificial and biological membranes is a triggering event in fusion. Correlation of these results with earlier studies of membrane hydration and fusion kinetics provides a more detailed view of the mechanism of fusion.

The kinetics of DNA injection by bacteriophage λ. into liposomes bearing reconstituted receptors were measured using fluorescence spectroscopy. LamB, the bacteriophage receptor, was extracted from bacteria and reconstituted into liposomes by detergent removal dialysis. The DNA binding fluorophore ethidium bromide was encapsulated in the liposomes during dialysis. Enhanced fluorescence of ethidium bromide upon binding to injected DNA was monitored, and showed that injection is a rapid, one-step process. The bimolecular rate law, determined by the method of initial rates, revealed that injection occurs several times faster than indicated by earlier studies employing indirect assays.

It is hoped that these studies will increase the understanding of the mechanisms of virus entry into cells, and to facilitate the development of virus-mimetic drug delivery strategies.

Episodic variations in stream water chemistry associated with acid rainfall and run-off and the effect on aquatic ecosystem, with particular reference to fish populations in N.W. England

This is the episodic variations in stream water chemistry associated with acid rainfall and run-off and the effect on aquatic ecosystems, with particular reference to fish populations in North West England produced by the North West Water Authority in 1985. This report looks at the biological, physical and chemical information collected over a five year period from over 100 sites on upland streams in the North West Region of which drained rocks of low buffering capacity. In both Lake District and South Pennine sites striking differences were found between the composition of invertebrate communities inhabiting acid-stressed and less acid-stressed streams. Electric fishing surveys showed that acidic streams (geometric mean pH <5.5) generally had abnormally low densities of salmonids ( < 0 .2m2) and that 0+ fish were very few or absent. The latter indicates recruitment failure. Salmon were more sensitive than trout to low pH.

Impact of the invasive colonial tunicate Didemnum vexillum on the recruitment of the bay scallop (Argopecten irradians irradians) and implications for recruitment of the sea scallop (Placopecten magellanicus) on Georges Bank

The invasive colonial tunicate Didemnum vexillum has become widespread in New England waters, colonizing large areas of shell-gravel bottom on Georges Bank including commercial sea scallop (Placopecten magellanicus) grounds. Didemnum vexillum colonies are also fouling coastal shellfish aquaculture gear which increases maintenance costs and may affect shellfish growth rates. We hypothesized that D. vexillum will continue to spread and may affect shellfish larval settlement and survival. We conducted a laboratory experiment to assess interactions between larval bay scallops (Argopectin irradians irradians) and D. vexillum. We found that larval bay scallops avoid settling on D. vexillum colonies, possibly deterred by the low pH of the tunicate’s surface tissue. The results of this study suggest that widespread colonization of substrata by D. vexillum could affect scallop recruitment by reducing the area of quality habitats available for settlement. We propose that the bay scallop can serve as a surrogate for the sea scallop in estimating the negative impact D. vexillum could have on the recruitment of sea scallops on Georges Bank.

Macrobenthos of intertidal zone of Versova along the coast of Mumbai

To assess the biodiversity of macro benthos in the changing environment along the coast of Mumbai, the intertidal zone of Versova was identified. The water quality in this intertidal region was poor with low pH, salinity and dissolved oxygen, and high nitrite, nitrate, phosphate and ammonia. The substratum was sandy with 1.29% organic matter in it. Mean faunal density of 2257 no./m² was recorded during the study which was mainly contributed by polychaetes (83.5%) followed by amphipods (14.5%), while other groups represented were isopods, crabs, hermit crabs, unidentified decapods, pelecypods and gastropods. Average biomass of 34.83 g/m² (93.7%) was contributed by polychaetes. Shannon and Wiener Index (0.4107) indicated heavy pollution in the intertidal area of Versova.

Development of intermediate moisture marinated fish product and its storage characteristics

An attempt was made to prepare an intermediate moisture (around 44% moisture) marinated (pH around 4) fish product. Fillets from Sciaenid fish (each fish weighing 70-80 gm) were dipped in a solution containing 7% acetic acid, 20% common salt and 1% propionic acid for 2 hours. After soaking, the soaked fillets were partially dried to about 44% moisture. Three effective hurdles like low pH (by using 7% acetic add and 1% propionic acid), low water activity (by using 20% salt and partially drying the fillets) and preservative (1% propionic add), were used to prepare a shelf-stable product at room temperature. The dried product was sprayed with 0.0 5% BHA in 50% alcohol and further dried for 10 minutes to remove added water and alcohol, thereby another hurdle (preservative) against fat oxidation. The product was packed in 300 gauge polythene bags and stored in transparent screw cap plastic jars. Fortnightly samples were drawn and subjected to biochemical, bacteriological and organoleptic evaluation to study its storage characteristics. The product was in good acceptable form up to 4 months at ambient temperature. The product needed one hour soaking in water with two changes of water in between to make it free from excess salt and acid smell.

Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China

A previously unknown cyanophage, PaV-LD (Planktothrix agardhii Virus isolated from Lake Donghu), which causes lysis of the bloom-forming filamentous cyanobacterium P. agardhii, was isolated from Lake Donghu, Wuhan, China. PaV-LD only lysed P. agardhii strains isolated from Lake Donghu and not those isolated from other lakes. The PaV-LD particle has an icosahedral, non-tailed structure, ca. 70 to 85 nm (mean +/- SD = 76 +/- 6 nm) in diameter. PaV-LD was stable at freezing temperature, but lost its infectivity at temperatures >50 degrees C. Lysis of host cells was delayed about 3 d after the PaV-LD treatment with chloroform, and the virus was inactivated by exposure to low pH (<= 4). The latent period and burst size of the PaV-LD were estimated to be 48 to 72 h and about 340 infectious units per cell, respectively. The regrowth cultures of surviving host filaments were not lysed by the PaV-LD suspension. To our knowledge, this is the first isolation and cultivation of a virus infectious to the filamentous bloom-forming cyanobacterium Planktothrix from a freshwater lake.

Pressure-assisted field-amplified sample injection with reverse migrating micelles for analyzing trace steroids in MEKC

This paper describes a novel method that applies pressure-assisted field-amplified sample injection with reverse migrating micelles (PA-FASI-RMM) for the online concentration of neutral analytes in MEKC with a low-pH BGE. After injection of a plug of water into the separation capillary, negative voltage and positive pressure were simultaneously applied to initialize PA-FASI-RMM injection. The hydrodynamic flow generated by the positive pressure compensated the reverse EOF in the water plug and allowed the water plug to remain in the capillary during FASI with reverse migrating micelles (FASI-RMM) to obtain a much longer injection time than usual, which improved stacking efficiency greatly. Equations describing this injection mode were introduced and were supported by experimental results. For a 450-s online PA-FASI-RMM injection, three orders of magnitude sample enhancement in terms of peak area could be observed for the steroids and an achievement of detection limits was between 1 and 10 ng/mL.

Characterisation of a pathogenic virus isolated from marine threadfin fish (Eleutheronema tetradactylus) during a disease outbreak

An unknown virus was isolated from massive mortality of cultured threadfin (Eleutheronema tetradactylus) fingerlings. The virus replicated in BF-2 fish cell line and produced a plaque-like cytopathic effect. Electron micrographs revealed non-enveloped, icosahedral particles approximately 70-80 nm in diameter composed of a double capsid layer. Viroplasms and subviral particles approximately 30 run in diameter and complete particles of 70 nm in diameter were also observed in the infected BF-2 tissue culture cells. The virus was resistant upon pH 3 to 11 and ether treatment. It is also stable to heat treatment (3 h at 56 T). Replication was not inhibited by 5-iododeoxyuridine (5-IUdR). Acridine orange stain revealed typical reovirus-like cytoplasmic inclusion bodies. Electrophoresis of purified virus revealed 11 segments of double-stranded RNA and five major structural polypeptides of approximately 136, 132, 71, 41 and 33 kDa. Based on these findings, the virus isolated was identified to belong to the genus Aquareovirus and was designated as threadfin reovirus. This virus differed from a majority of other aquareovirus by its increase in virus infectivity upon exposure to various treatments such as high and low pH, heat (56 degreesC), ether and 5-IUdR. The RNA and virion protein banding pattern of the threadfin reovirus was shown to differ from another Asian isolate, the grass carp hemorrhage reovirus (GCV). Artificial injection of the threadfin reovirus into threadfin fingerlings resulted in complete mortality, whereas sea bass (Lates calcarifer) fingerlings infected via bath route showed severe mortality within a week after exposure. These results indicate that the threadfin virus is another pathogenic Asian aquareovirus isolate that could cross-infect into another marine fish, the sea bass. (C) 2002 Elsevier Science B.V. All rights reserved.