The radish defensins RsAFP1 and RsAFP2 act synergistically with caspofungin against Candida albicans biofilms

The radish defensin RsAFP2 was previously characterized as a peptide with potent antifungal activity against several plant pathogenic fungi and human pathogens, including Candida albicans. RsAFP2 induces apoptosis and impairs the yeast-to-hypha transition in C. albicans. As the yeast-to-hypha transition is considered important for progression to mature biofilms, we analyzed the potential antibiofilm activity of recombinant (r)RsAFP2, heterologously expressed in Pichia pastoris, against C. albicans biofilms. We found that rRsAFP2 prevents C. albicans biofilm formation with a BIC-2 (i.e., the minimal rRsAFP2 concentration that inhibits biofilm formation by 50% as compared to control treatment) of 1.65 ± 0.40 mg/mL. Moreover, biofilm-specific synergistic effects were observed between rRsAFP2 doses as low as 2.5 μg/mL to 10 μg/mL and the antimycotics caspofungin and amphotericin B, pointing to the potential of RsAFP2 as a novel antibiofilm compound. In addition, we characterized the solution structure of rRsAFP2 and compared it to that of RsAFP1, another defensin present in radish seeds. These peptides have similar amino acid sequences, except for two amino acids, but rRsAFP2 is more potent than RsAFP1 against planktonic and biofilm cultures. Interestingly, as in case of rRsAFP2, also RsAFP1 acts synergistically with caspofungin against C. albicans biofilms in a comparable low dose range as rRsAFP2. A structural comparison of both defensins via NMR analysis revealed that also rRsAFP2 adopts the typical cysteine-stabilized αβ-motif of plant defensins, however, no structural differences were found between these peptides that might result in their differential antifungal/antibiofilm potency. This further suggests that the conserved structure of RsAFP1 and rRsAFP2 bears the potential to synergize with antimycotics against C. albicans biofilms.     

Effect of ferrocene-substituted porphyrin RL-91 on Candida albicans biofilm formation

Ferrocene-substituted porphyrin RL-91 exhibits antifungal activity against opportune human pathogen Candida albicans. RL-91 efficiently inhibits growth of both planktonic C. albicans cells and cells within biofilms without photoactivation. The minimal inhibitory concentration for plankton form (PMIC) was established to be 100 μg/mL and the same concentration killed 80% of sessile cells in the mature biofilm (SMIC80). Furthermore PMIC of RL-91 efficiently prevents C. albicans biofilm formation. RL-91 is cytotoxic for human fibroblasts in vitro in concentration of 10 μg/mL, however it does not cause hemolysis in concentrations of up to 50 μg/mL. These findings open possibility for application of RL-91 as an antifungal agent for external antibiofilm treatment of medical devices as well as a scaffold for further development of porphyrin based systemic antifungals.     

Antibiofilm activity and post antifungal effect of lemongrass oil on clinical Candida dubliniensis isolate

Candidal infections are often difficult to eradicate due to the resistance of biofilms to antifungal agents. This study aimed at determining the effects of lemongrass (Cymbopogon citratus DC) oil against Candida dubliniensis in both planktonic and biofilms form. The results from broth microdilution method revealed that the minimum inhibitory and minimum fungicidal concentration of lemongrass oil on C. dubliniensis were 0.43 and 0.86 mg/ml, respectively. Employing a formazan salt (XTT tetrazolium) reduction assay for biofilm study, the results showed that the average percentage (mean ± SD) inhibition of lemongrass oil (0.43 mg/ml) on biofilm formation was 91.57 ± 1.31%, while it exhibited more than 80% killing activity against C. dubliniensis in biofilm at concentrations of 1.7 mg/ml. In addition, a significant reduction (P = 0.03) of candidal adhesion to acrylic occurred after a 15 min exposure to 1.7 mg/ml of lemongrass oil. Moreover, limited exposure of yeasts to lemongrass oil at subcidal concentration can suppress growth for more than 24 h. Altogether, the results obtained indicate that lemongrass oil possessed antifungal and antibiofilm activities and could modulate candidal colonization. Therefore, the efficacy of lemongrass oil merits further development of this agent for the therapy of oral candidiasis.     

Inhibition of biofilm formation by conformationally constrained indole-based analogues of the marine alkaloid oroidin

Herein, we describe indole-based analogues of oroidin as a novel class of 2-aminoimidazole-based inhibitors of methicillin-resistant Staphylococcus aureus biofilm formation and, to the best of our knowledge, the first reported 2-aminoimidazole-based inhibitors of Streptococcus mutans biofilm formation. This study highlighted the indole moiety as a dibromopyrrole mimetic for obtaining inhibitors of S. aureus and S. mutans biofilm formation. The most potent compound in the series, 5-(trifluoromethoxy)indole-based analogue 4b (MBIC₅₀ = 20 μM), emerged as a promising hit for further optimisation of novel inhibitors of S. aureus and S. mutans biofilms.     

γ-Alkylidene-γ-lactones and isobutylpyrrol-2(5H)-ones analogues to rubrolides as inhibitors of biofilm formation by Gram-positive and Gram-negative bacteria

Several molecules have been discovered that interfere with formation of bacterial biofilms, opening a new strategy for the development of more efficient treatments in case of antibiotic resistant bacteria. Amongst the most active compounds are some natural brominated furanones from marine algae Delisea pulchra that have proven to be able to control pathogenic biofilms. We have recently reported that some rubrolide analogues are able to inhibit biofilm formation of Enterococcus faecalis. In the present Letter we describe results of the biological evaluation of a small library of 28 compounds including brominated furanones and the corresponding lactams against biofilm formation of Staphylococcus aureus, Pseudomonas aeruginosa, Staphylococcus epidermidis and Streptococcus mutans. Our results showed that in general these compounds were more active against biofilms of S. epidermidis and P. aeruginosa, with little or no inhibition of planktonic bacterial growth. In some cases they were able to prevent biofilm formation of P. aeruginosa at concentrations as low as 0.6 μg/mL (1.3 μM, compound 3d) and 0.7 μg/mL (1.3 μM, 3f). Results also indicate that, in general, lactams are more active against biofilms than their precursors, thus designating this class of molecules as good candidates for the development of a new generation of antimicrobial drugs targeted to biofilm inhibition.     

δ15N and δ13C diet–tissue discrimination factors for large sharks under semi-controlled conditions

Stable isotopes (δ¹⁵N and δ¹³C) are being widely applied in ecological research but there has been a call for ecologists to determine species- and tissue-specific diet discrimination factors (?¹³C and ?¹⁵N) for their study animals. For large sharks stable isotopes may provide an important tool to elucidate aspects of their ecological roles in marine systems, but laboratory based controlled feeding experiments are impractical. By utilizing commercial aquaria, we estimated ?¹⁵N and ?¹³C of muscle, liver, vertebral cartilage and a number of organs of three large sand tiger (Carcharias taurus) and one large lemon shark (Negaprion brevirostris) under a controlled feeding regime. For all sharks mean ± SD for ?¹⁵N and ?¹³C in lipid extracted muscle using lipid extracted prey data were 2.29‰ ± 0.22 and 0.90‰ ± 0.33, respectively. The use of non-lipid extracted muscle and prey resulted in very similar ?¹⁵N and ?¹³C values but mixing of lipid and non-lipid extracted data produced variable estimates. Values of ?¹⁵N and ?¹³C in lipid extracted liver and prey were 1.50‰ ± 0.54 and 0.22‰ ± 1.18, respectively. Non-lipid extracted diet discrimination factors in liver were highly influenced by lipid content and studies that examine stable isotopes in shark liver, and likely any high lipid tissue, should strive to remove lipid effects through standardising C:N ratios, prior to isotope analysis. Mean vertebral cartilage ?¹⁵N and ?¹³C values were 1.45‰ ± 0.61 and 3.75‰ ± 0.44, respectively. Organ ?¹⁵N and ?¹³C values were more variable among individual sharks but heart tissue was consistently enriched by ~ 1–2.5‰. Minimal variability in muscle and liver δ¹⁵N and δ¹³C sampled at different intervals along the length of individual sharks and between liver lobes suggests that stable isotope values are consistent within tissues of individual animals. To our knowledge, these are the first reported diet–tissue discrimination factors for large sharks under semi-controlled conditions, and are lower than those reported for teleost fish.     

Antimicrobial activity of the synthetic peptide Lys-a1 against oral streptococci

The peptide LYS-[TRP⁶]-Hy-A1 (Lys-a1) is a synthetic derivative of the peptide Hy-A1, initially isolated from the frog species Hypsiboas albopunctatus. According to previous research, it is a molecule with broad antimicrobial activity. The objective of this study was to evaluate the antimicrobial activity of the synthetic peptide Lys-a1 (KIFGAIWPLALGALKNLIK-NH₂) on the planktonic and biofilm growth of oral bacteria. The methods used to evaluate antimicrobial activity include the following: determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) in microtiter plates for growth in suspension and quantification of biomass by crystal violet staining and counting of colony forming units for biofilm growth. The microorganisms Streptococcus oralis, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus salivarius, Streptococcus mutans and Streptococcus sobrinus were grown in Brain Heart Infusion broth at 37 °C under atmospheric pressure with 10% CO₂. The peptide was solubilized in 0.1% acetic acid (v/v) at various concentrations (500–1.9 μg mL⁻¹). Chlorhexidine gluconate 0.12% was used as the positive control, and BHI culture medium was used as the negative control. The tested peptide demonstrated a remarkable antimicrobial effect, inhibiting the planktonic and biofilm growth of all strains tested, even at low concentrations. Thus, the peptide Lys-a1 is an important source for potential antimicrobial agents, especially for the control and prevention of microbial biofilms, which is one of the most important factors in cariogenic processes.     

Rapid methods to assess sanitizing efficacy of benzalkonium chloride to Listeria monocytogenes biofilms

Different methods were used to investigate biofilm growth including crystal violet staining, ATP bioluminescence and total viable count. Seven strains of Listeria monocytogenes and 8 of their derivative strains were screened for their capacity to form biofilms. Both adaptation to benzalkonium chloride (BC) and curing of plasmids did not significantly affect biofilm-forming ability. The strains of L. monocytogenes belonging to serotype 1 formed biofilms significantly better as compared to serotype 4 (P = 0.0003). To estimate the efficacy of BC for biofilm elimination the best and the poorest biofilm-formers were used (C719 and LJH 381). It was observed that, L. monocytogenes strain C719 in biofilms is at least 1000 times more resistant to BC than in planktonic form. Cells present in biofilms were shown to recover and grow after BC treatment thus providing a source of recontamination. It was shown that ATP bioluminescence provides good correlation with bacterial counts of L. monocytogenes in biofilms. Staining with crystal violet, on the contrary, did not correlate with bacterial growth in biofilms in the presence of high concentrations of BC but provided information on the concentration of bacterial cells, both live and dead, attached to the surface. ATP bioluminescence was found to be a reliable method for rapid estimation of the efficacy of sanitizers for biofilm disinfection. Crystal violet staining, on the other hand, was shown to be a suitable method to monitor removal of biofilms. Our investigation showed that for Listeria biofilms concentrations of BC higher then 10 mg/ml should be applied for at least 30 min to kill almost all the live cells in biofilms. However, this concentration was still not enough to remove biofilms from the surface of plastic.     

An in vitro urinary tract catheter system to investigate biofilm development in catheter-associated urinary tract infections

Biofilm development in urinary tract catheters is an often underestimated problem. However, this form of infection leads to high mortality rates and causes significant costs in health care. Therefore, it is important to analyze these biofilms and establish avoiding strategies. In this study a continuous flow-through system for the cultivation of biofilms under catheter-associated urinary tract infection conditions was established and validated. The in vitro urinary tract catheter system implies the composition of urine (artificial urine medium), the mean volume of urine of adults (1 mL min^–1), the frequently used silicone catheter (foley silicon catheter) as well as the infection with uropathogenic microorganisms like Pseudomonas aeruginosa. Three clinical isolates from urine of catheterized patients were chosen due to their ability to form biofilms, their mobility and their cell surface hydrophobicity. As reference strain P. aeruginosa PA14 has been used. Characteristic parameters as biofilm thickness, specific biofilm growth rate and substrate consumption were observed. Biofilm thicknesses varied from 105 ± 16 μm up to 246 ± 67 μm for the different isolates. The specific biofilm growth rate could be determined with a non invasive optical biomass sensor. This sensor allows online monitoring of the biofilm growth in the progress of the cultivation.     

Temporal–spatial variation and source analysis of carbon and nitrogen in a tidal wetland of Luoyuan Bay

Carbon and nitrogen are important elements in biogeochemical studies of tidal wetlands. Three wetland zones in Luoyuan Bay in the Fujian province were chosen for this study; the Spartina alterniflora flat zone with Spartina alterniflora growing, the silt zone with no Spartina alterniflora growing and the Spartina alterniflora-silt flat zone – a transition zone between the two. The spatial and seasonal variations of total organic carbon (TOC), total nitrogen (TN), stable isotopes of organic material (δ¹³C, δ¹⁵N), C/N ratio, average particle size and sediment composition in surface and vertical sediments of different ecological zones were analyzed. Carbon and nitrogen accumulation and particle size effects in the different ecological zones were discussed and the indicators of δ¹³C and C/N ratios were also compared. TOC, TN, δ¹³C contents, C/N ratios, and average particle size varied within the ranges of 0.611–1.133%, 0.053–0.090%, ?22.60 to ?18.92‰, 12.3–15.7, and 6.4–8.7 μm, respectively. Sediments were mainly silt-sized. Besides δ¹⁵N values, the other parameters, such as TOC, TN, δ¹³C contents, C/N ratios, and average particle size showed an obvious zonal distribution in surface sediments. The distribution of TOC and TN contents reflected the distribution of Spartina alterniflora within the bay. The profile and seasonal variations of these parameters in different ecological zones indicated that variations in the Spartina alterniflora flat and transition zones were complex because of the effect of Spartina alterniflora. Vertical and seasonal variations were sampled in the silt flat area. The profile and seasonal variations of TOC, TN and δ¹³C were similar in the transition zone and the Spartina alterniflora flat zone. Seasonal concentrations of TOC, TN and δ¹³C decreased from autumn > spring > winter > summer. The seasonal variation of carbon and nitrogen in the sediments may be influenced by temperature, particle size, plankton and benthos. The particle size effect was significant in the surface sediments and profile sediments of the transition zone. However, other factors had a greater effect on the distributions of TOC and TN in the Spartina alterniflora flat and silt flat zones. C/N ratios in sediments of the Spartina alterniflora flat, transition zone and silt flat were close to or > 12, indicating that the organic material source was dominated by terrestrial inputs. However, δ¹³C values decreased from the Spartina alterniflora flat zone > transition zone > silt flat zone indicating that the organic material source was predominantly from marine inputs. Thus the indications from C/N ratios and δ¹³C were different. There was no clear relationship between C/N ratios and δ¹³C values and a better relationship between δ¹³C values and TOC concentrations suggested that δ¹³C values provided a better indication of the organic source. Limited amounts of organic material came from Spartina alterniflora. This study has provided basic data for researching biogeochemical processes of biogenic elements in tidal wetlands and vegetation restoration, and has also provided a reference for assessing and protecting the environment and ecological systems in wetlands.     

Identification of aryl 2-aminoimidazoles as biofilm inhibitors in Gram-negative bacteria

The synthesis and biofilm inhibitory activity of a 30-member aryl amide 2-aminoimidazole library against the three biofilm forming Gram-negative bacteria Escherichia coli, Psuedomonas aeruginosa, and Acinetobacter baumannii is presented. The most active compound identified inhibits the formation of E. coli biofilms with an IC₅₀ of 5.2 μM and was observed to be non-toxic to planktonic growth, demonstrating that analogues based on an aryl framework are viable options as biofilm inhibitors within the 2-aminoimidazole family.     

Ensayo de formación y cuantificación de biopelículas mixtas de Candida albicans y Staphylococcus aureus

This study quantifies the production of single and mixed biofilms of Candida albicans and Staphylococcus aureus to determine if such mixed biofilms have synergistic effects. Assays were performed using polystyrene microtitre plates of 96 wells, metabolic activity was measured by the enzymatic reduction of a tetrazolium salt (XTT) and colorimetric changes were measured at 490 nm. Confocal scanning laser microscopy was used to visualise the biofilms of each microorganism and its growth kinetics. The highest levels of biofilm formation were observed in mixed biofilms, followed by those of Candida albicans only, with the lowest levels of biofilm formation being detected for Staphylococcus aureus; all together these results suggest a synergistic relationship between the tested microorganisms.     

Possible conformation of amphotericin B dimer in membrane-bound assembly as deduced from solid-state NMR

Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between ¹³C-labled AmB methyl ester and ¹⁹F-labled AmB. The dimer showed slightly weaker but significant biological activities against fungi and red blood cells compared with those of monomeric AmB. Then the dimer was subjected to ¹³C{¹⁹F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short ¹³C/¹⁹F distance (6.9 Å) accounting for 23% of the REDOR dephasing, and a longer one (14 Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel.     

Biofilm growth kinetics of a monomethylamine producing Alphaproteobacteria strain isolated from an anaerobic reactor

Industrial fishing effluents are characterized by high loads of protein and sulfate that stimulate the activity of proteolytic and sulfate reducing bacteria during anaerobic digestion. Their metabolic products (NH₃ and H₂S respectively) have a well-known detrimental effect on the activity of methanogens.Since methylamine is a carbon source used by methylaminotrophic methane producing archaea (mMPA) but not by sulfate reducing bacteria (SRB), enriched mMPA anaerobic biofilms have been developed on ceramics. We propose that methylated amines could be produced in the biofilm by using betaine, a known precursor of methylamine, as a carbon and energy source.We isolated an anaerobic betainotrophic methylaminogenic bacterial strain (bMB) from an anaerobic bioreactor, using betaine as the only carbon and energy source. This strain was identified by a standard biochemical test (API 20NE), cloning, and 16S rDNA sequencing.bMB biofilm structure and biofilm growth kinetic parameters were determined by means of scanning electron microscopy (SEM), and the Gompertz growth model, respectively. Monomethylamine production was determined by infrared spectroscopy and by high pressure liquid chromatography.The isolated bMB strain was determined as Stappia stellulata (Proteobacteria phylum). It was able to form biofilm on ceramics and its kinetic growth parameters resulted in: maximum biofilm bacterial count (A) of 6.25 × 10⁸ UFC/cm² and maximum specific growth rate (μ_m) of 0.022 1/h. Production of monomethylamine was about 4.027 atogram/cell/day (at/cell/day) after 15 days of incubation in biofilms.This study confirms the adhesion capacity of this bMB strain on ceramic supports, assuring that monomethylamine production in biofilms could be enriched with mMPA that use monomethylamine.     

Effect of tertiary sewage effluent additions on Prymnesium parvum cell toxicity and stable isotope ratios

We investigated the ability of the ichthyotoxic haptophyte Prymnesium parvum to use sewage-originated nutrients applying stable carbon (C) and nitrogen (N) isotope techniques. P. parvum was cultured under N and phosphorus (P) sufficient and deficient conditions in either sewage effluent-based medium or in a nitrate- and phosphate-based control. Cell densities and toxicities were monitored and stable carbon N isotopes signatures (δ¹³C and δ¹⁵N) of P. parvum and the sewage effluent analysed. Nitrogen and P sufficient cultures achieved the highest biomass followed by P and N deficient cultures, regardless of sewage effluent additions. The P deficient cultures with sewage effluent had higher toxicity, estimated as haemolytic activity (9.4 ± 0 × 10^?5 mg Saponin equiv. cell^?1) compared to the P deficient control and to all N deficient and NP sufficient cultures. Nutrient deficient conditions had no effect on the cell δ¹⁵N, but a decreasing effect on δ¹³C in the inorganic N deficient treatment. Growth in sewage-based media was followed by a substantial increase in the cell δ¹⁵N (10.4–16.1‰) compared to the control treatments (2.4–4.9‰), showing that P. parvum is capable of direct use of sewage-originated N, inorganic as well as organic. Uptake of terrestrial derived C in the sewage treatments was confirmed by a decrease in cell δ¹³C, implying that P. parvum is able to utilize organic nutrients in sewage effluent.     

Determination of food sources and trophic position in Malaysian tropical highland streams using carbon and nitrogen stable isotopes

Stable isotope analysis has been extensively used as an effective tool in determination of trophic relationship in ecosystems. In freshwater ecosystem, aquatic invertebrates represent main component of a river food web. This study was carried out to determine potential food sources of freshwater organism together with pattern of trophic position along the river food web. In this study, rivers of Belum-Temengor Forest Complex (BTFC) has been selected as sampling site as it is a pristine area that contains high diversity and abundance of organisms and can be a benchmark for other rivers in Malaysia. Stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N) were applied to estimate trophic position and food web paradigm. Analysis of stable isotopes based on organic material collected from the study area revealed that the highest δ¹³C value was reported from filamentous algae (? 22.68 ± 0.126⁰/₀₀) and the lowest δ¹³C was in allocthonous leaf packs (? 31.58 ± 0.187⁰/₀₀). Meanwhile the highest δ¹⁵N value was in fish (8.45 ± 0.177⁰/₀₀) and the lowest value of δ¹⁵N was in autochthonous aquatic macrophyte (2.00 ± 1.234⁰/₀₀). Based on the δ¹⁵N results, there are three trophic levels in the study river and it is suggested that the trophic chain begins with organic matter followed by group of insects and ends with fish (organic matter < insects < fish).     

Soil and tree ring chemistry of Pinus banksiana and Populus tremuloides stands as indicators of changes in atmospheric environments in the oil sands region of Alberta,Canada

The impact of chronic air pollution such as increased CO₂ and NO_x emissions on forest ecosystems in the Athabasca oil sands region in Alberta, Canada, was investigated in Pinus banksiana (jack pine) and Populus tremuloides (trembling aspen, aspen) stands in two watersheds (NE7 and SM8) located at different distances from the main emission sources of oil sands mining and upgrading facilities, using δ¹³C, δ¹⁵N, and Ca/Al of soil and tree ring samples as indicators. Watershed NE7 was exposed to greater amounts of acid deposition due to its closeness to the mining and upgrading area. The δ¹⁵N in the forest floor was lower (p < 0.05) in NE7 (ranged from −1.42 to −0.87‰) than in SM8 (−0.54 to 1.43‰), implying a greater amount of recent deposition of ¹⁵N-depleted N in NE7. Tree ring δ¹³C gradually decreased over time for both tree species/watersheds, indicating the influence of ¹³C-depleted CO₂ emitted from industrial sources. Tree ring N concentration and δ¹⁵N were not different between watersheds and did not significantly change with time. Interestingly, however, the difference between watersheds (NE7–SM8) that is expressed as Diff_N (for N) increased with concomitant decreases in Diff_δ¹⁵N over time, implying greater increases in ¹⁵N-depleted N input in NE7 than in SM8. Such trends were stronger in aspen stands (R² = 0.64 and p < 0.001 for Diff_N and R² = 0.44 and p < 0.01 for Diff_δ¹⁵N between 1964 and 2009) than in jack pine stands. We conclude that δ¹⁵N in the forest floor and differences in N and δ¹⁵N of tree rings between watersheds are useful indicators reflecting the impact of spatial variations of air pollution on forest stands in the Athabasca oil sands region in western Canada.     

Use of stable isotope ratios of fish larvae as indicators to assess diets and patterns of anthropogenic nitrogen pollution in estuarine ecosystems

A stable isotope study was carried out to investigate the feeding ecology of the common goby Pomatoschistus microps larvae (Krøyer, 1838), and to assess differences in the response of planktonic food web to nutrient enrichment, in two ecosystems from the Southern European coast with different levels of historical pollution (estuaries of the Minho and Lima Rivers). At each estuary and time (July 2012, November 2012, February 2013, and June 2013), the fish larvae of two size classes (class 0: 0–10 mm; class 1: 10–15 mm), particulate organic matter (POM), and pelagic zooplankton were collected. The stable isotope mixing model SIAR revealed that, despite temporal differences in the relative proportion of prey items ingested, in both estuaries P. microps larvae feed on both planktonic-hyperbenthic food sources, predating mainly on copepods (from 34% to 60%), Mysidacea (from 16% to 28%), and brachyuran zoea (from 14% to 29%). Fish larvae size classes did not differ significantly for δ¹⁵N, and exhibited a very narrow range of the δ¹³C signature. Enriched δ¹⁵N values of all biota in the Lima estuary throughout the study period, with a marked nitrogen enrichment in colder months, are indicative of higher anthropogenic inputs of nitrogen (e.g. sewage and industrial discharges, agriculture) into this system. The δ¹⁵N values of fish larvae and other planktonic groups can be a sensitive bioindicator, because they are highly correlated with the nitrogen content of water (ammonium), indicating that this element has transferred through the planktonic food web. Enriched carbon isotope ratios were observed in warmer months, in both estuaries, and the heavier δ¹³C values in Lima are best explained by differences in the degree of marine influence. This research emphasises the utility of stable isotopes in trophic interactions studies, highlighting the relevance of the stable nitrogen isotope of zooplanktonic communities as a reliable bioindicator to detect patterns of anthropogenic nitrogen contamination in estuarine ecosystems.     

Phenol and sulfide oxidation in a denitrifying biofilm reactor and its microbial community analysis

A microbial consortium attached onto a polyethylene support was used to evaluate the simultaneous oxidation of sulfide and phenol by denitrification. The phenol, sulfide and nitrate loading rates applied to an inverse fluidized bed reactor were up to 168 mg phenol–C/(l d), 37 mg S^2?/(l d) and 168 mg NO₃^?–N/(l d), respectively. Under steady state operation the consumption efficiencies of phenol, sulfide and nitrate were 100%. The N₂ yield (g N₂/g NO₃^?–N) was 0.89. The phenol was mineralized resulting in a yield of 0.82 g bicarbonate–C/g phenol–C and sulfide was completely oxidized to sulfate with a yield of 0.99 g SO₄^2?–S/g S^2?. 16S rRNA gene-based microbial community analysis of the denitrifying biofilm showed the presence of Thauera aromatica, Thiobacillus denitrificans, Thiobacillus sajanensis and Thiobacillus sp. This is the first work reporting the simultaneous oxidation of sulfide and phenol in a denitrifying biofilm reactor.     

Characterization of two quaternary ammonium chloride-resistant bacteria isolated from papermaking processing water and the biocidal effect on their biofilm formation

Biofilm formation and growth on equipment surfaces is detrimental to papermaking processes. However, a fundamental understanding leading to an optimal control strategy is yet to be found. Quaternary ammonium compounds (QAC) are being increasingly applied in the papermaking processes. Among them, the most frequently applied, N-alkyl-benzyl-dimethyl ammonium chloride, was employed in this study. To foster fundamental understanding of QAC efficacy towards biofilm control, two of the highest QAC-resistant strains of bacteria were isolated from the papermaking processing water and employed as model organisms. By the 16S rRNA gene sequencing technique, two Gram-negative rods with QAC resistance were identified as Morganella morganii (HB22) and the biofilm-forming Pseudomonas putida (HB45). The minimal inhibition concentration (MIC) values were 8 mg L⁻¹ for HB22 and 16 mg L⁻¹ for HB45, respectively, against QAC in basal medium (BM). However, both strains could grow under more than 150 mg L⁻¹ QAC in basal medium at neutral pH. As observed by crystal violet assay and fluorescent confocal microscopy, HB45 formed biofilm more slowly on stainless steel coupon which is the prime material of papermachine than on the surface of polystyrene, the most common material for food packaging and semi-finished/finished products. HB45 formed biofilm more slowly on stainless steel coupons than on polystyrene Petri dish surfaces, as observed by crystal violet assay and fluorescent confocal microscopy. For HB45, there was a marginal increase of inhibition of biofilm formation by increasing QAC concentration from 50 to 75 mg L⁻¹. By comparison of inhibition concentration in liquid state and in biofilm formation, the results implicate that the current practice in papermaking processes of adding biocide to qualitatively control planktonic bacterial communities does not ensure control of biofilm formation.