Dead Bacterial Cells Research Articles

Investigation of Formation of Bacterial Biofilm upon Dead Siblings.

Biocides can effectively kill bacteria; however, whether the dead bacterial cells left on the surface influence the later growth of biofilm is unknown. In this study, we have cultured Pseudomonas aeruginosa (PAO1) biofilm on their dead siblings and have investigated their evolution by using magnetic force modulation atomic force microscopy (MF-AFM). The time dependence of the biofilm thickness indicates that the deposited dead siblings can slow down the growth of PAO1 biofilm. The biofilm growing on dead bacteria layers is softer in comparison with those upon alive siblings, as reflected by the static elastic modulus ( E) and dynamic stiffness ( kd) scaled to the disturbing frequency ( f) as kd = kd,0 fγ, where kd,0 is the scaling factor and γ is the power-law exponent. We reveal that the smaller population instead of the variation of extracellular polymeric substances (EPS) within the biofilm upon the dead siblings is responsible for the softer biofilm. The present study provides a better understanding of the biofilm formation, thus, making it significant for designing antimicrobial medical materials and antifouling coatings.

Atmospheric air plasma induces increased cell aggregation during the formation of Escherichia coli biofilms

Atmospheric air plasma has previously been shown to be a novel and effective method for biofilm eradication. Here we study the effects of plasma on both microbial inactivation and induced structural modification for forming biofilms. New structures are created from aggregates of extracellular polysaccharides and dead bacterial cells, forming a protective and resilient matrix in which the remaining living cells grow and reproduce under proper growth conditions. The new colonies are found to be more resilient in this state, reducing the efficacy of subsequent plasma treatment. We verify that the observed effect is not caused by chemicals produced by plasma reactive species, but instead by the physical processes of drying and convection caused by the plasma discharge.

Detection of Clavibacter michiganensis subsp. michiganensis in viable but nonculturable state from tomato seed using improved qPCR.

Clavibacter michiganensis subsp. michiganensis (Cmm) is a seed-borne pathogen that causes bacterial canker disease of tomato. Cmm is typically detected in tomato seeds using quantitative real-time polymerase chain reaction (qPCR) combined with culture-based isolation. The viable but nonculturable (VBNC) state of Cmm may result in the underestimation or false negative detection of the pathogen. In the present study, propidium monoazide (PMA) and its improved structure PMAxx were used to pretreat Cmm prior to DNA extraction, followed by qPCR. Both PMA and PMAxx could bind to the chromosomal DNA of dead bacterial cells and therefore block DNA amplification by PCR. This effect, however, does not occur in living bacterial cells, as the chemicals cannot penetrate through the undamaged cell membrane. Both viable and dead Cmm cells were treated with PMA and PMAxx at various concentrations. With this treatment, the range of the cell population was determined for effective detection. PMAxx showed a better discrimination effect than PMA on the viable and dead cells of Cmm and was therefore used throughout the present study. VBNC cells of Cmm (108 CFU mL-1) was induced by 50 μM copper sulfate, which was detected at different sampling times up to a month by using both PMAxx-qPCR and flow cytometry assays. The optimal PMAxx concentration was 20 μM for detecting membrane-intact Cmm cells. High specificity and sensitivity were obtained at Cmm concentrations ranging from 103 to 107 CFU mL-1. The accurate and robust results of PMAxx-qPCR were confirmed by flow cytometry method to detect viable Cmm cells. Furthermore, the PMAxx-qPCR assay was successfully used in detecting VBNC Cmm cells in tomato seeds with as few as 10 seeds per set.

Development and evaluation of a culture-free microbiota profiling platform (MYcrobiota) for clinical diagnostics

Microbiota profiling has the potential to greatly impact on routine clinical diagnostics by detecting DNA derived from live, fastidious, and dead bacterial cells present within clinical samples. Such results could potentially be used to benefit patients by influencing antibiotic prescribing practices or to generate new classical-based diagnostic methods, e.g., culture or PCR. However, technical flaws in 16S rRNA gene next-generation sequencing (NGS) protocols, together with the requirement for access to bioinformatics, currently hinder the introduction of microbiota analysis into clinical diagnostics. Here, we report on the development and evaluation of an “end-to-end” microbiota profiling platform (MYcrobiota), which combines our previously validated micelle PCR/NGS (micPCR/NGS) methodology with an easy-to-use, dedicated bioinformatics pipeline. The newly designed bioinformatics pipeline processes micPCR/NGS data automatically and summarizes the results in interactive, but simple web reports. In order to explore the utility of MYcrobiota in clinical diagnostics, 47 clinical samples (40 “damaged skin” samples and 7 synovial fluids) were investigated using routine bacterial culture as comparator. MYcrobiota confirmed the presence of bacterial DNA in 37/37 culture-positive samples and detected bacterial taxa in 2/10 culture-negative samples. Moreover, 36/38 potentially relevant aerobic bacterial taxa and 3/3 mixtures of anaerobic bacteria were identified using culture and MYcrobiota, with the sensitivity and specificity being 95%. Interestingly, the majority of the 448 bacterial taxa identified using MYcrobiota were not identified using culture, which could potentially have an impact on clinical decision-making. Taken together, the development of MYcrobiota is a promising step towards the introduction of microbiota analysis into clinical diagnostic laboratories.

Mapping bacteria on filter membranes, an innovative SERS approach

The existence of pathogenic bacteria in drinking water has been a threat to the safety of human well-being. Traditional methods to detect bacteria are standard plate counts or rapid methods such as ELISA and PCR. However, those methods can be time-consuming or require pre-enrichment when detecting low concentrations of bacteria. Filter membrane has been used for bacteria collection and culturing when monitoring environmental water samples. In this study, we applied surface enhanced Raman spectroscopy (SERS) to rapidly screen bacteria cells on a filter membrane based on the unique signal from an indicator molecule, 4-mercaptophenylboronic acid (4-mpba), that can specifically bind to the surface of bacteria through diol group in its structure and give off consistent SERS signal. With a pore size of 0.22 μm, the filter membrane functions as both a concentrating device and a separation mechanism that eliminates molecules smaller than the pore size. With SERS mapping, 4-mpba gives characteristic signal, indicating the presence of bacteria, whereas no 4-mpba signal was observed in the absence of bacteria. The developed method can detect Escherichia coli, Salmonella enterica, and Listeria monocytogenes on a filter membrane non-selectively in 80 min. Application of the method in pond water was demonstrated. However, this method may not be able to discriminate between live and dead bacterial cells and further development is needed.

Reduction of Aflatoxin M1 Levels during Ethiopian Traditional Fermented Milk (Ergo) Production

In this study, the reduction of aflatoxin M1 (AFM1) levels during lab-scale ergo production was investigated through determination of the residual levels of AFM1 using Enzyme Linked Immunosorbent Assay. The results showed gradual and incubation time dependent reduction of AFM1 level in the raw milk samples being fermented to ergo. The maximum reductions of 57.33 and 54.04% were recorded in AFM1 in natural and LAB inoculums initiated fermentations, respectively, in 5 days of incubation. Although a significant difference (P=0.05) in the AFM1 decrease in the two types of fermentations was recorded, such findings could vary with milk samples depending on initial load of the microorganisms as determined by hygienic conditions. However, the level of AFM1 in control (sterilized) samples showed only a 5.5% decrease during the entire period of incubation. Microbiological investigation showed increasing LAB counts with incubation time. A gradual decrease in pH of the milk samples was observed during fermentation. Considering the fact that both viable and dead bacterial cells could remove AFM1 during ergo production, the mechanism is proposed as predominantly involving noncovalent binding of the toxin with the chemical components of the bacterial cell wall.

Microbiological and Histopathological studies on cholesterol reducing effect of lactobacillus acidophilus

A total of 50 albino rats were used to carry out an in vivo study on the cholesterol lowering impact of isolated L. acidophilus isolated from diatery supplements. Blood serum of the experimental rats as well as their liver, heart and aorta tissues were analyzed and examined .The attained results revealed a great decrease in serum cholesterol due to the use of live or dead bacterial cells. The histopathological results collected from examination of liver revealed that in live lactobacillus treated group display significant amelioration compare to non treated group, while no significant improvement detected in dead lactobacillus treated group. The corresponding results of the heart in showed that some pathological lesions seen in cardiac tissue in high cholesterol feeding group, still observed. This is besides atrophy of many cardiomyocytes and apoptosis. Signe of improvement was noticed in dead lactobacillus treated hypercholesterolemic group, where the severity and incidence of the pathological lesions observed in hypercholesterolemia animals was significantly reduced. Concerning the aorta, the attained results demonstrated that in live lactobacillus treated group, early plague formation was observed in few areas, however fibrin covered wide area of the endothelial layer. Significant amelioration in aorta tissues of dead lactobacillus treated hypercholesterolemia group was detected.

Antibacterial Effect of Potassium Tetraborate Tetrahydrate against Soft Rot Disease Agent Pectobacterium carotovorum in Tomato.

Soft rot caused by Pectobacterium carotovorum is one of most common bacterial diseases occurring in fruits and vegetables worldwide, yet consumer-acceptable options for post-harvest disease management are still insufficient. We evaluated the effect of potassium tetraborate tetrahydrate (B4K2O7.4H2O) (PTB) on the growth of P. carotovorum using strain BA17 as a representative of high virulence. Complete inhibition of bacterial growth was achieved by treatment with PTB at 100 mM both at pH 9.2 and after adjustment to pH 7.0. Bactericidal activity was quantified and validated by counting fluorescently labeled live and dead bacterial cells using flow cytometry, and reconfirmed using qPCR with high-affinity photoreactive DNA binding dye propidium monoazide (PMA). The results of flow cytometry, qPCR, and culturing confirmed that bacterial cells were killed following exposure to PTB at 100 mM. Bacterial cell membranes were damaged following a 5-min treatment and extrusion of cytoplasmic material from bacterial cells was observed using electronic transmission microscopy. Soft rot incidence on inoculated tomato fruit was significantly reduced by dipping infected fruits in PTB at 100 mM for 5 min and no lesions developed following a 10-min treatment. PTB does not pose a hazard to human health and is an effective alternative to other bactericides and antibiotics for controlling soft rot disease of tomato caused by P. carotovorum.

New Insights into Antibiofilm Effect of a Nanosized ZnO Coating against the Pathogenic Methicillin Resistant Staphylococcus aureus.

ZnO nanoparticles (NPs) are arising as promising novel antibiotics toward device-related infections. The surface functionalization of Zn, a novel resorbable biomaterial, with ZnO NPs could present an effective solution to overcome such a threat. In this sense, the antibacterial and antibiofilm activity of nano- and microsized ZnO coatings was studied against clinically relevant bacteria, methicillin resistant Staphylococcus aureus (MRSA). The bacterial viability of planktonic and biofilm cells together with the corresponding biofilm structures revealed that only the nanosized ZnO coating had an antibiofilm effect. To elucidate this effect, a novel approach was taken: preconditioning of bacteria with this ZnO coating followed by exposure to subinhibitory concentrations of antibiotics with well-known modes of actions. This approached revealed (i) a decreased biofilm formation in combination with gentamycin, targeting protein synthesis, and (ii) an increased biofilm formation in the presence of rifampicin and vancomycin, acting on RNA and cell wall biosynthesis, respectively. The increased bacteria resistance to these two antibiotics gave new insights into the antibiofilm effect of this nanosized ZnO coating. The synergistic effect observed for gentamycin opened new perspectives for the design of effective solutions against implant-related infections. During the in vitro degradation of this nanosized ZnO-coated Zn, a specific corrosion product, hopeite [Zn3(PO4)2], was depicted. Interestingly, the increased deposition of hopeite-derived compounds on MRSA cells surface seemed to be related to unhealthy and dead bacterial cells. This observation suggested that hopeite may well play a key role in this antibiofilm activity. The results obtained herein shed light on the possible antibacterial effect of a nanosized ZnO coating, and strengthened its antimicrobial (antibacterial and antifungal) potential, therefore providing a potentially effective material to overcome the growing trend of implant-related infections.

Anti-biofouling enhancement of a polycarbonate membrane with functionalized poly(vinyl alcohol) electrospun nanofibers: Permeation flux, biofilm formation, contact, and regeneration tests

In this study, benzyl triethylammonium chloride (BTEAC)-functionalized poly(vinyl alcohol) (PVA) nanofibers were fabricated via electrospinning and deposited on a commercial polycarbonate (PC) membrane (f-PVA/PC membrane) in order to enhance the anti-biofouling activity of the membrane. Permeation flux, biofilm formation, contact, and regeneration tests were performed to evaluate the anti-biofouling potential of the f-PVA/PC membrane against bacteria, including Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. The permeation flux test showed that the normalized flux of the f-PVA/PC membrane was retained at 1.0 after filtration of 500mL of the bacterial solution (K. pneumoniae), whereas the fluxes of the PC and PVA/PC membranes decreased to 0.57 and 0.84, respectively. In the biofilm formation test, the number of biofilm cells on the f-PVA nanofibers (P. aeruginosa = 4.21–4.98log colony-forming unit (CFU), S. aureus = 3.74–4.39log CFU) was less than those on the PVA nanofibers (P. aeruginosa = 5.68–6.89log CFU, S. aureus = 4.72–5.82log CFU). The contact test demonstrated that mortality rates (contact time = 60min) on the f-PVA/PC membrane (K. pneumoniae = 94.08%, S. aureus = 99.99%, E. coli = 92.30%) were greater than those on the PC membrane (K. pneumoniae = 73.75%, S. aureus = 62.41%, E. coli = 76.80%). Fluorescence microscopy images illustrated that the population of red (dead) bacterial cells on the f-PVA/PC membranes was greater than that on the PVA/PC membrane. The regeneration test indicated that the f-PVA/PC membrane retained its anti-biofouling activity during regeneration and reuse over six cycles with mortality rates of 93.82–96.29% (K. pneumoniae), 74.27–85.15% (S. aureus), 91.68–95.19% (E. coli), and 94.94–96.90% (mixed-strain bacteria). The results demonstrated that the f-PVA nanofibers could enhance the anti-biofouling potential of the membrane through both anti-adhesive and anti-bacterial surface modifications.

Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex.

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

Novel amphiphilic poly(dimethylsiloxane) based polyurethane networks tethered with carboxybetaine and their combined antibacterial and anti-adhesive property

The traditional nonfouling materials are powerless against bacterial cells attachment, while the hydrophobic bactericidal surfaces always suffer from nonspecific protein adsorption and dead bacterial cells accumulation. Here, amphiphilic polyurethane (PU) networks modified with poly(dimethylsiloxane) (PDMS) and cationic carboxybetaine diol through simple crosslinking reaction were developed, which had an antibacterial efficiency of 97.7%. Thereafter, the hydrolysis of carboxybetaine ester into zwitterionic groups brought about anti-adhesive properties against bacteria and proteins. The surface chemical composition and wettability performance of the PU network surfaces were investigated by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and contact angle analysis. The surface distribution of PDMS and zwitterionic segments produced an obvious amphiphilic heterogeneous surface, which was demonstrated by atomic force microscopy (AFM). Enzyme-linked immunosorbent assays (ELISA) were used to test the nonspecific protein adsorption behaviors. With the advantages of the transition from excellent bactericidal performance to anti-adhesion and the combination of fouling resistance and fouling release property, the designed PDMS-based amphiphilic PU network shows great application potential in biomedical devices and marine facilities.

Dynamics of Huanglongbing-associated Bacterium Candidatus Liberibacter asiaticus in Citrus aurantifolia Swingle (Mexican Lime).

The bacterial disease citrus huanglongbing (HLB), associated with "Candidatus Liberibacter asiaticus" (C.Las) has severely impacted the citrus industry, causing a significant reduction in production and fruit quality. In the present study, it was monitored the C.Las population dynamics in symptomatic, HLB-positive Mexican lime trees (Citrus aurantifolia Swingle) in a tropical, citrus-producing area of Mexico. The objective of this study was to identify the dynamics of the population of huanglongbing-associated bacterium Candidatus Liberibacter asiaticus and its insect vector in Citrus aurantifolia Swingle (Mexican lime). Leaf samples were collected every 2 months over a period of 26 months for quantification of bacterial titers and young and mature leaves were collected in each season to determine preferential sites of bacterial accumulation. The proportion of living and dead bacterial cells could be determined through the use of quantitative real-time PCR in the presence of ethidium monoazide (EMA-qPCR). It was observed a lower bacterial titer at high temperatures in the infected trees relative to titers in mild weather, despite a higher accumulation of the insect vector Diaphorina citri in these conditions. This study also revealed seasonal fluctuations in the titers of bacteria in mature leaves when compared to young leaves. No statistically significant correlation between any meteorological variable, C.Las concentration and D. citri population could be drawn. Although, HLB management strategies have focused on vector control, host tree phenology may be important. The evaluation of citrus phenology, C.Las concentration, ACP population and environmental conditions provides insights into the cyclical, seasonal variations of both the HLB pathogen and its vector. These findings should help in the design of integrative HLB control strategies that take into account the accumulation of the pathogen and the presence of its vector.

Population Dynamics of Antimicrobial Peptide's Activity is Governed by their Retention in Dead Bacterial Cells

Antimicrobial peptides (AMPs) are broad spectrum antibiotics that utilize electrostatics to selectively target bacteria. Like every antibiotic, AMPs need a minimum concentration (known as MIC) to effectively inhibit growth of a bacterial population. Despite our molecular knowledge on AMPs, we still lack a comprehensive understanding of the AMP's dynamics at the cellular and the population level. In this talk, I present our recent findings that MIC of AMPs is strongly dependent on the cell density, even in dilute cultures where there is no direct cell-to-cell interactions. This suggests that individual cells may adsorb a large number of AMPs that considerably reduces their concentration in the solution. To further investigate this, we used a live single-cell imaging platform to track fluorescently tagged AMPs and the time evolution of their translocation into bacteria. Our single-cell analysis show that bacteria not only adsorb a large fraction of AMPs from the culture, but dead cells also retain them almost permanently which sequesters AMPs availability for attacking more cells.

Decay kinetics of microbial source tracking (MST) markers and human adenovirus under the effects of sunlight and salinity

Artificial seawater and freshwater microcosms inoculated with raw sewage were set up to compare the persistence of microbial source tracking (MST) markers (i.e. Bacteroides thetaiotaomicron (B. theta), Methanobrevibacter smithii (M. smithii), human polyomaviruses JC and BK (HPyVs)) and human adenoviruses under different sunlight intensity and salinity. PMA pretreatment successfully eliminated the false-positive detection of dead bacterial cells in the model-development experiment. The results were then validated using real environmental matrices in microcosms inoculated with raw sewage. The genome concentrations of the targets followed a first-order decay pattern with 90% reduction of the initial amounts in <5days for both artificial and natural surface waters. Decay rate constant (k1) were developed microorganisms in artificial water matrices. Due to the different water environment conditions, improved decay rates (k2) incorporated with sunlight, TSS and TOC adjustment coefficients were used for validation of the natural water matrices. Based on the predictive squared correlation coefficient (Q2F) and root-mean-square error (RMSE) validation criteria, the improved k2 were able to provide better prediction on the survival of target microorganisms in environmental surface waters (Q2F>0.6 and RMSE ranged from 0.05 to 1.81). For microbial source tracking purposes, HPyVs are suggested to be better MST markers in freshwater, while B. theta is recommended for seawater based on the decay models developed in this study. The targeted DNA of M. smithii should only be used to indicate recent human faecal pollution in surface waters due to their faster decay than human adenoviruses.

Structural damage of Bacillus subtilis biofilms using pulsed laser interaction with gold thin films.

There is a huge interest in developing strategies to effectively eliminate biofilms due to their negative impact in both industrial and clinical settings. In this study, structural damage was induced on two day-old B. subtilis biofilms using the interaction of 532 nm pulsed laser with gold thin films. Radiant exposure of 225mJ/cm2 induced distinct changes on the surface structure and overall morphology of the matured biofilms after laser irradiation. Moreover, at the radiant exposure used, changes in the colour and viscosity of the biofilm were observed which may indicate a compromised extracellular matrix. Irradiated biofilms in the presence of gold film also showed strong propidium iodide signal which implies an increase in the number of dead bacterial cells after laser treatment. Thus, this laser-based technique is a promising approach in targeting and eradicating matured biofilms attached on surfaces such as medical implants.

The use of propidium monoazide in conjunction with qPCR and Illumina sequencing to identify and quantify live yeasts and bacteria

Culture-independent methods of microbial identification have been developed, which allow for DNA extraction directly from environmental samples without subjecting microbes to growth on nutrient media. These methods often involve next generation DNA sequencing (NGS) for identifying microbes and qPCR for quantifying them. Despite the benefits of extracting all DNA from the sample, results may be compromised by amplifying DNA from dead cells. To address this short-coming, the use of propidium monoazide (PMA) has been used to deactivate DNA in non-viable cells. Nevertheless, its optimization has not been fully explored under a variety of conditions. In this study, we optimized the PMA method for both yeasts and bacteria. Specifically, we explored the effect different PMA concentrations and different cell densities had on DNA amplification (as part of next generation DNA sequencing) from both dead and viable bacterial and yeast cells. We found PMA was effective in eliminating DNA that was associated with dead yeast and bacterial cells for all cell concentrations. Nevertheless, DNA (extracted from viable yeast and bacterial cells) amplified most abundantly when PMA concentration was at 6μM and when yeast densities ranged between 106 to 107CFU/mL and bacterial densities were approximately 108CFU/mL.

The lipopeptide 6-2 produced by Bacillus amyloliquefaciens anti-CA has potent activity against the biofilm-forming organisms

Both the whole cells and protoplasts of Pseudomonas aeruginosa PAO1 and Bacillus cereus, two biofilm-forming bacteria, were disrupted by the lipopeptide 6-2 produced by Bacillus amyloliquefaciens anti-CA. The lipopeptide 6-2 could also effectively inhibit the formation of biofilms and disperse pre-formed biofilms. Live/dead staining of the biofilms grown in the absence or presence of the lipopeptide 6-2 showed that more dead bacterial cells in the presence of the lipopeptide than those in the absence of the lipopeptide and biofilm formation was greatly reduced by the lipopeptide 6-2. Expression of the PslC gene related to exopolysaccharides in P. aeruginosa PAO1 was also inhibited. All these results demonstrated that the lipopeptide 6-2 produced by B. amyloliquefaciens anti-CA had a high activity against biofilm-forming bacteria. The lipopeptide 6-2 also killed the larvae of Balanus amphitrite and inhibit the germination of Laminaria japonica spore and growth of protozoa, all of which were the fouling organisms in marine environments.

Dynamics of particulate phosphorus in a shallow eutrophic lake

We tested the hypothesis that in shallow, eutrophic Lake Kasumigaura, the concentration of particulate phosphorus (PP) is controlled by biogenic P (P in living or dead phytoplankton and bacterial cells), rather than by resuspension of inorganic P in sediment. Increases in wind velocity and turbidity were associated with bottom shear stress exceeding the critical value for the lake (τc=0.15Nm−2); this increased turbidity was due to sediment resuspension. However, concentrations of PP; HCl-extractable, reactive P in PP (P-rP); and HCl-extractable, non-reactive P in PP (P-nrP) were not correlated with wind velocity (PP vs. wind velocity: r=0.40, p>0.05). Rather, the P-nrP concentration accounted for approximately 79% of PP, and the concentrations of PP, P-rP, and P-nrP were correlated with the particulate organic carbon (POC) concentration (POC vs. PP: r=0.90, p<0.01; POC vs. P-rP: r=0.82, p<0.01; POC vs. P-nrP: r=0.86, p<0.01). In our 31P nuclear magnetic resonance spectroscopy results, mononucleotides accounted for the largest proportion among the detected P compound classes. In addition, concentrations of mononucleotides, orthophosphate, and pyrophosphate were significantly higher in samples with high POC concentrations, whereas the DNA-P concentration was not. These results suggest that biogenic P affects PP concentrations more strongly than does sediment resuspension, and the production of biogenic P creates a pool of mononucleotides, a class of easily degradable P, even in shallow, eutrophic Lake Kasumigaura.

Regeneration of the power performance of cathodes affected by biofouling

Air cathode microbial fuel cells (MFCs) were used in a cascade-system, to treat neat human urine as the fuel. Their long-term operation caused biodeterioration and biofouling of the cathodes. The cathodes were made from two graphite-painted layers, separated by a current collector. The initial performance of the MFCs was reaching average values of 105.5±32.2μW and current of 1164.5±120.2μA. After 3months of operation the power performance decreased to 9.8±3.5μW, whilst current decreased to 461.2±137.5μA. Polarisation studies revealed significant transport losses accompanied by a biofilm formation on the cathodes. The alkaline lysis procedure was established to remove the biomass and chemical compounds adsorbed on the cathode’s surface. As a result, the current increased from 378.6±108.3μA to 503.8±95.6μA. The additional step of replacing the outer layer of the cathode resulted in a further increase of current to 698.1±130μA. Similarly, the power performance of the MFCs was recovered to the original level reaching 105.3±16.3μW, which corresponds to 100% recovery. Monitoring bacterial cell number on the cathode’s surface showed that biofilm formed during operation was successfully removed and composed mainly of dead bacterial cells after treatment. To the best of the authors’ knowledge, this is the first time that the performance of deteriorating cathodes, has been successfully recovered for MFCs in-situ. Through this easy, fast and inexpensive procedure, designing multilayer cathodes may help enhance the range of operating conditions, if a biofilm forms on their surface.