Effect of mechanical stress on biofilms challenged by different chemicals

In this study a methodology was applied in order to ascertain the mechanical stability of biofilms, by using a stainless-steel (SS) rotating device immersed in a biological reactor where biofilms formed by Pseudomonas fluorescens were allowed to grow for 7 days at a Reynolds number of agitation of 2400. The biofilms developed with this system were characterised in terms of amount of total, extracellular and intracellular proteins and polysaccharides, amount of mass, metabolic activity and mechanical stability, showing that the biofilms were active, had a high content of extracellular constituents and an inherent mechanical stability. In order to assess the role of chemical agents on the mechanical stability, the biofilms were exposed to chemical agents followed by mechanical treatments by submission to increase Reynolds number of agitation. Seven different chemical agents were tested (two non-oxidising biocides, three surfactants and two oxidising biocides) and their effects on the biofilm mechanical stability were evaluated. The increase in the Reynolds number increased the biofilm removal, but total biofilm removal was not found for all the conditions tested. For the experiment without chemical addition (only mechanical treatment), the biofilm remaining on the surface was about 76%. The chemical treatment followed by the subsequent mechanical treatment did not remove all the biofilms from the surface. The biofilm remaining on the SS cylinder ranged from 3% to 62%, depending on the chemical treatment, showing that the chemical treatment is far from being a cause that induces massive biofilm detachment and even the synergistic chemical and mechanical treatments did not promote biofilm removal. Some chemical agents promoted an increase in the biofilm mechanical stability such as glutaraldehyde (GTA), benzalkonium chloride (BC), except for the lower concentration tested, and sodium dodecyl sulphate (SDS), except for the higher concentration tested. Treatments that promoted biofilm removal, to an extent similar to the control experiment (without chemical treatment), were BC, for the lower and the higher concentration of SDS. Cetyltrimethyl ammonium bromide (CTAB), ortho-phthalaldehyde (OPA), sodium hydroxide (NaOH) and sodium hypochlorite (SHC) promoted the weakening of the biofilm mechanical stability.     

Dynamics of lead immobilization in sulfate reducing biofilms

We have evaluated the effects of selected minerals present in subsoil environment on the efficiency of lead removal from contaminated groundwaters using biofilms composed of sulfate-reducing microorganisms, and examined the stability of metal deposits after the biofilms had been temporarily exposed to the air. To quantify the studied effects, lead was immobilized in biofilms of Desulfovibrio desulfuricans grown anaerobically in two flat-plate flow reactors, one filled with hematite and the other with quartz. While the biofilms in both reactors were heterogeneous and consisted of voids and channels, the biofilms grown on hematite were denser, thicker, and more porous than those grown on quartz. The average H₂S concentrations, measured using microelectrodes, were higher in the biofilms grown on quartz than those measured in the biofilms grown on hematite. During 18 weeks of operation, iron was continuously released from the hematite. Lead was immobilized more efficiently in the biofilms grown on quartz than it was in the biofilms grown on hematite. Lead deposits were partially reoxidized, especially in biofilms grown on hematite, and the biofilms in both reactors responded to the presence of oxygen by lowering their density and increasing the H₂S production rate.     

A simple optode based method for imaging O2 distribution and dynamics in tap water biofilms

A ratiometric luminescence intensity imaging approach is presented, which enables spatial O₂ measurements in biofilm reactors with transparent planar O₂ optodes. Optodes consist of an O₂ sensitive luminescent dye immobilized in a 1-10 μm thick polymeric layer on a transparent carrier, e.g. a glass window. The method is based on sequential imaging of the O₂ dependent luminescence intensity, which are subsequently normalized with luminescent intensity images recorded under anoxic conditions. We present 2-dimensional O₂ distribution images at the base of a tap water biofilm measured with the new ratiometric method and compare the results with O₂ distribution images obtained in the same biofilm reactor with luminescence lifetime imaging. Using conventional digital cameras, such simple normalized luminescence intensity imaging can yield images of 2-dimensional O₂ distributions with a high signal-to-noise ratio and spatial resolution comparable or even surpassing those obtained with expensive and complex luminescence lifetime imaging systems. The method can be applied to biofilm growth incubators allowing intermittent experimental shifts to anoxic conditions or in systems, in which the O₂ concentration is depleted during incubation.     

Kinetics and stoichiometry of sulfide oxidation by sewer biofilms

Oxidation of sulfide under aerobic conditions by biofilms grown on municipal wastewater in 6 identical pipe reactors was investigated. The biofilms were grown at pH 7.6 and temperatures of 20 and 25 degrees C under aerobic-anaerobic transient conditions with pulse dosing of sulfide in the bulk water. The pulse dosing of sulfide served to simulate conditions in a gravity sewer located downstream of a pressure main. During growth of the biofilms, sulfide was pulse dosed in concentrations of 0, 0.5, 2.0 and 5.0 g Sm(-3) with a frequency of 1h(-1). Based on a series of batch experiments, kinetics and stoichiometry of sulfide oxidation by the sewer biofilms was investigated and a rate equation and a stoichiometric constant proposed. Sulfide oxidation kinetics was significantly faster for biofilms grown at sulfide loadings of 0.5, 2.0 and 5.0 g Sm(-3)h(-1) than for biofilms grown in the absence of sulfide. However, the kinetics of sulfide oxidation was relatively constant for biofilms grown at sulfide loadings above 0.5 g Sm(-3)h(-1). Mass balance calculations of dissolved oxygen and sulfur compounds suggested the oxidation product to be elemental sulfur. Further oxidation of elemental sulfur could not be documented.     

Effects of initial molecular weight on removal rate of dextran in biofilms

Degradation kinetics of different size dextrans in a biofilm reactor were evaluated. Degradation rates of dextran standards, measured as time series of oxygen utilisation rates, decreased with increasing initial molecular weight. Removal of bulk phase total organic carbon with time was highly correlated (R2>0.99) and could be modelled with variable half-order degradation rate expressions. A power correlation between initial molecular weight and the variable half-order degradation rate coefficient was found for polymers in the range 6-500 kDa. Degradation of dextran in the colloid size range (MW>1 Mda) did not follow the same kinetics. Reductions in the observed removal rate with polymer size can be explained by the effect of reduced diffusivities of the substrate, without assuming reaction rate effects.     

Nitrate stimulation of indigenous nitrate-reducing, sulfide-oxidising bacterial community in wastewater anaerobic biofilms

The role of the nitrate-reducing, sulfide-oxidising bacteria (NR-SOB) in the nitrate-mediated inhibition of sulfide net production by anaerobic wastewater biofilms was analyzed in two experimental bioreactors, continuously fed with the primary effluent of a wastewater treatment plant, one used as control (BRC) and the other one supplemented with nitrate (BRN). This study integrated information from H(2)S and pH microelectrodes, RNA-based molecular techniques, and the time course of biofilm growth and bioreactors water phase. Biofilms were a net source of sulfide for the water phase (2.01 micromol S(2-)(tot)m(-2)s(-1)) in the absence of nitrate dosing. Nitrate addition effectively led to the cessation of sulfide release from biofilms despite which a low rate of net sulfate reduction activity (0.26 micromol S(2-)(tot)m(-2)s(-1)) persisted at a deep layer within the biofilm. Indigenous NR-SOB including Thiomicrospira denitrificans, Arcobacter sp., and Thiobacillus denitrificans were stimulated by nitrate addition resulting in the elimination of most sulfide from the biofilms. Active sulfate reducing bacteria (SRB) represented comparable fractions of total metabolically active bacteria in the libraries obtained from BRN and BRC. However, we detected changes in the taxonomic composition of the SRB community suggesting its adaptation to a higher level of NR-SOB activity in the presence of nitrate.     

Microscale geochemical gradients in Hanford 300 Area sediment biofilms and influence of uranium

The presence and importance of microenvironments in the subsurface at contaminated sites were suggested by previous geochemical studies. However, no direct quantitative characterization of the geochemical microenvironments had been reported. We quantitatively characterized microscale geochemical gradients (dissolved oxygen (DO), H₂, pH, and redox potential) in Hanford 300A subsurface sediment biofilms. Our results revealed significant differences in geochemical parameters across the sediment biofilm/water interface in the presence and absence of U(VI) under oxic and anoxic conditions. While the pH was relatively constant within the sediment biofilm, the redox potential and the DO and H₂ concentrations were heterogeneous at the microscale (<500-1000 μm). We found microenvironments with high DO levels (DO hotspots) when the sediment biofilm was exposed to U(VI). On the other hand, we found hotspots (high concentrations) of H₂ under anoxic conditions both in the presence and in the absence of U(VI). The presence of anoxic microenvironments inside the sediment biofilms suggests that U(VI) reduction proceeds under bulk oxic conditions. To test this, we operated our biofilm reactor under air-saturated conditions in the presence of U(VI) and characterized U speciation in the sediment biofilm. U L_III-edge X-ray absorption spectroscopy (XANES and EXAFS) showed that 80-85% of the U was in the U(IV) valence state.     

Disinfectant efficacy of chlorite and chlorine dioxide in drinking water biofilms

The drinking water industry is closely examining options to maintain disinfection in distribution systems. In particular this research compared the relative efficiency of the chlorite ion (ClO2-) to chlorine dioxide (ClO2) for biofilm control. Chlorite levels were selected for monitoring since they are typically observed in the distribution system as a by-product whenever chlorine dioxide is applied for primary or secondary disinfection. Previous research has reported the chlorite ion to be effective in mitigating nitrification in distribution systems. Annular reactors (ARs) containing polycarbonate and cast iron coupons were used to simulate water quality conditions in a distribution system. Following a 4 week acclimation period, individual ARs operated in parallel were dosed with high (0.25mg/l) and low (0.1mg/l) chlorite concentrations and with high (0.5 mg/l) and low (0.25mg/l) chlorine dioxide concentrations, as measured in the effluent of the AR. Another set of ARs that contained cast iron and polycarbonate coupons served as controls and did not receive any disinfection. The data presented herein show that the presence of chlorite at low concentration levels was not effective at reducing heterotrophic bacteria. Log reductions of attached heterotrophic bacteria for low and high chlorite ranged between 0.20 and 0.34. Chlorine dioxide had greater log reductions for attached heterotrophic bacteria ranging from 0.52 to 1.36 at the higher dose. The greatest log reduction in suspended heterotrophic bacteria was for high dose of ClO2 on either cast iron or polycarbonate coupons (1.77 and 1.55). These data indicate that it would be necessary to maintain a chlorine dioxide residual concentration in distribution systems for control of microbiological regrowth.     

Comparison of potentially pathogenic free-living amoeba hosts by Legionella spp. in substrate-associated biofilms and floating biofilms from spring environments

This study compares five genera of free-living amoebae (FLA) hosts by Legionella spp. in the fixed and floating biofilm samples from spring environments. Detection rate of Legionella spp. was 26.9% for the floating biofilms and 3.1% for the fixed biofilms. Acanthamoeba spp., Hartmanella vermiformis, and Naegleria spp. were more frequently detected in floating biofilm than in fixed biofilm samples. The percentage of pathogenic Acanthamoeba spp. among all the genus Acanthamoeba detected positive samples was 19.6%. The potential pathogenic Naegleria spp. (for example, Naegleria australiensis, Naegleria philippinensis, and Naegleria italica) was 54.2% to all the Naegleria detected positive samples. In the study, 12 serotypes of possible pneumonia causing Legionella spp. were detected, and their percentage in all the Legionella containing samples was 42.4%. The FLA parasitized by Legionella included unnamed Acanthamoeba genotype, Acanthamoeba griffini, Acanthamoeba jacobsi, H. vermiformis, and N. australiensis. Significant differences were also observed between the presence/absence of H. vermiformis and Legionella parasitism in FLA. Comparisons between the culture-confirmed method and the PCR-based detection method for detecting FLA and Legionella in biofilms showed great variation. Therefore, using these analysis methods together to detect FLA and Legionella is recommended.     

Enhancing the formation and shear resistance of nitrifying biofilms on membranes by surface modification

Polypropylene (PP) membranes and polyethylene (PE) surfaces were modified to enhance formation and shear resistance of nitrifying biofilms for wastewater treatment applications. A combination of plasma polymerization and wet chemistry was employed to ultimately introduce poly(ethyleneglycol) (PEG) chains with two different functional groups (-PEG-NH₂ and -PEG-CH₃). Biofilm growth experiments using a mixed nitrifying bacterial culture revealed that the specific combination of PEG chains with amino groups resulted in most biofilm formation on both PP and PE samples. Detachment experiments showed similar trends: biofilms on -PEG-NH₂ modified surfaces were much stronger compared to the other modifications and the unmodified reference surfaces. Electrostatic interactions between the protonated amino group and negatively charged bacteria as well as PEG chain density which can affect the surface structure might be possible explanations of the superiority of the -PEG-NH₂ modification. The success of the-PEG-NH₂ modification was independent of the original surface and might, therefore, be used in wastewater treatment bioreactors to improve reactor performance by making biofilm formation more stable and predictable.     

Temporal and spatial inhibitory effects of zinc and copper on wastewater biofilms from oxygen concentration profiles determined by microelectrodes

To understand the temporal and spatial toxic effect of heavy metals on the microbial activities of biofilms, microelectrodes were used to measure the inhibitory oxygen (O₂) concentration profiles resulted from the effects of zinc (Zn²⁺) and copper (Cu²⁺). Using the O₂ microprofiles as bases, the spatial distributions of net specific O₂ respiration were determined in biofilms with and without treatment of 5 mg/L Zn²⁺ or 1 mg/L Cu²⁺. Results show that microbial activities were inhibited only in the outer layer (∼400 μm) of the biofilms and bacteria present in the deeper sections of the biofilms became even more active. The inhibition caused by the heavy metals was evaluated by two methods. One was derived from the oxygen influx at the interface and the other was based on the integral of the oxygen consumption calculated from the entire O₂ profile. The two methods yielded significantly different results. We argue that the integral method results in more accurate assessment of toxicity than the surface flux determination.     

A review of mathematical modelling of biofilm processes: advances in modelling of selected biofilm processes

The paper reviews the developments in the mathematical modelling of important aerobic biofilm processes‐nitrification, combined carbon oxidation and nitrification, simultaneous nitrification and denitrification (SND) and simultaneous adsorption and biodegradation — that have been made during the past two decades. This article coniders only a selection of those publications that have created a significant impact on the trends and techniques of modeling of these processes. The recent advances in the areas of simultaneous nitrification and denitrification as well as bioregeneration of the activated carbon have also been summarized.     

Effects of temperature and biodegradable organic matter on control of biofilms by free chlorine in a model drinking water distribution system

This study used annular reactors (AR) to investigate, under controlled laboratory conditions, the effects of temperature and biodegradable organic matter (BOM) on the free chlorine residual needed to control biofilm accumulation, as measured by heterotrophic plate count (HPC) bacteria. Biofilm was grown on PVC coupons, initially in the absence of chlorine, at 6, 12, and 18 degrees C, in the presence and absence of a BOM supplement (250 microg C/L) added as acetate. During the early stages of chlorine addition, when no measurable free chlorine residual was present, a reduction in biofilm HPC numbers was observed. Subsequently, once sufficient chlorine was added to establish a residual, the biofilm HPC numbers expressed as log CFU/cm2 fell exponentially with the increase in free chlorine residual. Temperature appeared to have an important effect on both the chlorine demand of the system and the free chlorine residual required to control the biofilm HPC numbers to the detection limit (3.2 Log CFU/cm2). For the water supplemented with BOM, a strong linear correlation was found between the temperature and the free chlorine residual required to control the biofilm. At 6 degrees C, the presence of a BOM supplement appeared to substantially increase the level of free chlorine residual required to control the biofilm. The results of these laboratory experiments provide qualitative indications of effects that could be expected in full-scale systems, rather than to make quantitative predictions.     

Investigation of the microbial degradation of phenazone-type drugs and their metabolites by natural biofilms derived from river water using liquid chromatography/tandem mass spectrometry (LC-MS/MS)

The degradation of the pharmaceuticals phenazone and metamizole, two pyrazolone-derivates in widespread use, using biofilms created by natural organisms from the national park Unteres Odertal, Germany, were investigated. An analytical method based on LC-MS/MS was optimised to determine the substances phenazone and methylaminoantipyrine (MAA), the hydrolysis product of metamizole (also known as dipyrone), as well as their metabolites 1,5-dimethyl-1,2-dehydro-3-pyrazolone (DP), acetaminoantipyrine (AAA), formylaminoantipyrine (FAA) and 4-aminoantipyrine (AA). Performance characteristics of the method were evaluated in terms of recovery, standard deviation, coefficient of variation, method detection limits (MDL) and method quantification limits (MQL). Degradation studies of phenazone and MAA were conducted using a laboratory-scale continuous flow biofilm reactor fed with different nutrient media and with variable hydraulic retention times of 24 and 32 h. MAA was degraded rapidly to FAA and AA, while phenazone was not degraded under the prevailing conditions even after 32 h. By operating the bioreactor in batch mode to study the phenazone degradation potential of the biofilm under limiting nutrient conditions, an elimination rate of 85% phenazone was observed, but because of the slow elimination rate and aerobic conditions, the metabolite DP was not detected. In additional batch experiments using bacterial isolates from the natural biofilm to decompose phenazone, some bacterial strains were able to form DP from phenazone in marginal concentrations over the sampling period of eight weeks. Obviously, the microorganisms need a reasonably long time to adapt their metabolisms to enable the removal of phenazone from water samples.     

应用水力层析法刻画含水层非均质性

为了研究含水层多孔介质参数非均质性,利用一系列连续的交叉孔水力试验获取含水层对于外界干扰的反应信息,应用序贯连续线性估计方法（Sequential Successive Linear Estimator）对多系列水头信息进行随机参数估计,即水力层析法。应用该方法对随机产生参数的一维含水层进行了渗透系数K和储水率Ss的估算,并对估计值和真实值进行了对比,其中使用单井数据计算就可使K值的平均相对误差控制在2.67％,证明了该方法的有效性。在对某地浸采铀厂区长、宽为150m×150m的含水层进行了实例研究,使用29口井孔进行层析试验,对于该二维水平分布含水层的非均质特征进行了刻画,其中K值为0.5-1.4m／d,Ss为0.00005／m—0.0002／m。该数据在后期的地下水污染溶质运移模拟中取得了很好的应用。作为研究地下水参数的新方法,水力层析法能够对三维分布的含水层特征进行高精度刻画,这种层析的概念和反演方法可广泛应用于环境和地球科学领域的研究。     

The effects of changing water flow velocity on the formation of biofilms and water quality in pilot distribution system consisting of copper or polyethylene pipes

We studied the effects of flow velocity on the formation of biofilms and the concentration of bacteria in water in copper and plastic (polyethylene, PE) pipes. The formation of biofilms increased with the flow velocity of water. The increase in microbial numbers and contents of ATP was clearer in the PE pipes than in the copper pipes. This was also seen as increased consumption of microbial nutrients in the pipeline system. This indicates that the mass transfer of nutrients is in major role in the growth of biofilms. However, the increased biomass of biofilms did not affect microbial numbers in the water. Rapid changes in water flow rate resuspended biofilms and sediments which increased the concentrations of bacteria and copper in water. The disturbance caused by the changing water flow was also seen as an increase in the particle counts and water turbidity recorded with online instrumentation.     

Bacterially mediated mineralisation processes lead to biodeterioration of artworks in Maltese catacombs

Mineral structures formed by bacterial and microalgal biofilms growing on the archaeological surface in Maltese hypogea were studied using Energy Dispersive X-Ray Spectroscopy (EDS) coupled to Environmental Scanning Electron Microscopy (ESEM), X-ray micro-diffraction (XRD) and X-ray fluorescence (XRF). These techniques have shown that mineral structures having different morphologies and chemical composition were associated with the microorganisms in the subaerophytic biofilm. Salt efflorescences and mineral deposits on the archaeological surface were often formed from gypsum (CaSO₄? 2H₂O), halite (NaCl) and calcite (CaCO₃). Biogenic carbonates produced by microbial activities were a common occurrence. These assumed different forms, such as the production of mineral coats around cyanobacterial sheaths and the occurrence of calcite fibres with different morphologies on the surface of the biofilms. Moreover, vaterite (CaCO₃) spherulites which appeared hollow in cross-section were observed. The presence of struvite was recorded from one catacomb site. These investigations have facilitated the study of the neoformation of metastable minerals by microbially mediated processes, which potentially contribute to a better understanding of the biodeterioration of artworks in Maltese palaeo-Christian catacombs.     

方差体技术在识别断层及陷落柱中的应用

从引入方差体技术的必要性出发 ,介绍了方差体技术的原理。通过该技术在寺河煤矿的实际应用 ,体现了方差体技术对小断层、陷落柱等地质异常体良好的自动识别效果。     

The influence of biofilms on the migration of uranium in acid mine drainage (AMD) waters

The uranium mine in Königstein (Germany) is currently in the process of being flooded. Huge mass of Ferrovum myxofaciens dominated biofilms are growing in the acid mine drainage (AMD) water as macroscopic streamers and as stalactite-like snottites hanging from the ceiling of the galleries. Microsensor measurements were performed in the AMD water as well as in the biofilms from the drainage channel on-site and in the laboratory. The analytical data of the AMD water was used for the thermodynamic calculation of the predominance fields of the aquatic uranium sulfate (UO₂SO₄) and UO₂⁺⁺ speciation as well as of the solid uranium species Uranophane [Ca(UO₂)₂(SiO₃OH)₂?5H₂O] and Coffinite [U(SiO₄)_1 − x(OH)_4x], which are defined in the stability field of pH > 4.8 and Eh < 960 mV and pH > 0 and Eh < 300 mV, respectively. The plotting of the measured redox potential and pH of the AMD water and the biofilm into the calculated pH-Eh diagram showed that an aqueous uranium(VI) sulfate complex exists under the ambient conditions. According to thermodynamic calculations a retention of uranium from the AMD water by forming solid uranium(VI) or uranium(IV) species will be inhibited until the pH will increase to > 4.8. Even analysis by Energy-filtered Transmission Electron Microscopy (EF-TEM) and electron energy loss spectroscopy (EELS) within the biofilms did not provide any microscopic or spectroscopic evidence for the presence of uranium immobilization. In laboratory experiments the first phase of the flooding process was simulated by increasing the pH of the AMD water. The results of the experiments indicated that the F. myxofaciens dominated biofilms may have a substantial impact on the migration of uranium. The AMD water remained acid although it was permanently neutralized with the consequence that the retention of uranium from the aqueous solution by the formation of solid uranium species will be inhibited.     

Predation influences the structure of biofilm developed on ultrafiltration membranes

This study investigates the impact of predation by eukaryotes on the development of specific biofilm structures in gravity-driven dead-end ultrafiltration systems. Filtration systems were operated under ultra-low pressure conditions (65 mbar) without the control of biofilm formation. Three different levels of predation were evaluated: (1) inhibition of eukaryotic organisms, (2) addition of cultured protozoa (Tetrahymena pyriformis), and (3) no modification of microbial community as a control. The system performance was evaluated based on permeate flux and structures of the biofilm. It was found that predation had a significant influence on both the total amount and also the structure of the biofilm. An open and heterogeneous structure developed in systems with predation whereas a flat, compact, and thick structure that homogeneously covered the membrane surface developed in absence of predation. Permeate flux was correlated with the structure of the biofilm with increased fluxes for smaller membrane coverage. Permeate fluxes in the presence or absence of the predators was 10 and 5 L m⁻² h⁻¹, respectively. It was concluded that eukaryotic predation is a key factor influencing the performance of gravity-driven ultrafiltration systems.