Aerobic Biofilm Research Articles

Start-up of aerobic granular biofilm at low temperature: Performance and microbial community dynamics

Low temperature is a great challenge for the biological treatment of wastewater. In this study, the rapid start-up of aerobic granular biofilm (AGF) reactor was realized by adding micro-sized polyurethane (PU) sponges as matrices at 10 °C. The results showed that the granulation process of AGF was different from that of traditional aerobic granular sludge and biofilms, which was formed by using the sludge intercepted in PU matrix instead of sponge skeletons as granulation carriers. During the 5-month operation period, stable pollutants removal performance was achieved within 70 days, besides, the corresponding ammonium, total nitrogen, and total phosphorus removal efficiencies were 98%, 70%, and 95%, respectively. The addition of PU matrices inhibited the growth of filamentous bacteria and provided support for high structural stability of AGF. With the operation of the reactor, the relative abundance of traditional denitrifying bacteria (genera Thauera and Acidovorax, etc.) decreased gradually, and the putative denitrifying phosphorus accumulating genus, Dechloromonas, occupied a dominant position in the system. This experiment showed that AGF system could be successfully started-up and operated with efficient pollutants removal performance under low temperature when using micro-sized PU sponges as matrices.

Efficient azo dye wastewater treatment in a hybrid anaerobic reactor with a built-in integrated bioelectrochemical system and an aerobic biofilm reactor: Evaluation of the combined forms and reflux ratio

A combined process that consisted of a hybrid anaerobic reactor (HAR) with an integral bioelectrochemical system and aerobic biofilm reactor (ABFR) was established for simulated azo dye wastewater treatment (domestic wastewater containing dye acid orange 7). The split combination form that separated HAR and ABFR into two individual reactors recorded a decolorization efficiency of 81.23 ± 0.12%, which was about 8% higher than that HAR and ABFR were stacked together into a single up-flow reactor. Implementation of reflux improved the decolorization and chemical oxygen demand (COD) removal in both the processes. Decolorization efficiency achieved 97.52 ± 0.66% in split process at a reflux ratio of 1 and the COD was 89 ± 2 mg/L in the final effluent. Further increasing the reflux ratio to 3 did not have any significance in treatment performance of the reactors. This study comprehensively revealed the influence of combination forms and reflux ratio on the performance of combined process.

Developmental dynamics of antibiotic resistome in aerobic biofilm microbiota treating wastewater under stepwise increasing tigecycline concentrations

This study aimed to investigate the impact of tigecycline, the third generation tetracycline, on the antibiotic resistance development in environmental microbiota. Two biological contact oxidation reactors containing aerobic biofilm microbiota were constructed, one of which was constantly fed with synthetic wastewater spiked with increasing concentrations of tigecycline (0 to 25 mg/L) under a hydrolytic retention time of 24 h. Over a period of 636 days, chemical oxygen demand removal over 90% and complete nitrification were achieved for both the control and tigecycline-exposed reactors, and effluent tigecycline concentrations in the tigecycline-exposed system were always <0.051 mg/L. Significant increases (p < 0.01) in resistome abundance and resistant bacteria ratio were detected at a tigecycline dose of 10 and 25 mg/L, respectively, revealed by metagenomic sequencing and culture-based method. The increase of resistome in the tigecycline system was mainly attributed to the enrichment of tetX, one cooperative tetracycline degrading gene. Partial canonical correspondence analysis showed that the change of resistome was mainly driven by bacterial community shift (vertical pathway). Network and genome binning analyses further suggested that the proliferation of Flavobacterium harboring tetX contributed to a relatively low community-wide resistance development in the aerobic biofilm microbiota under tigecycline selection by reducing the antibiotic concentration. This work provides scientific bases for the management and evaluation of the resistance risk induced by this novel antibiotic.

Enhanced hydrolyzed polyacrylamide removal from water by an aerobic biofilm reactor-ozone reactor-aerobic biofilm reactor hybrid treatment system: Performance, key enzymes and functional microorganisms

Degradation of hydrolyzed polyacrylamide-containing (HPAM-containing) wastewater was investigated in a lab-scale aerobic-ozonic-aerobic hybrid treatment system. When the HPAM concentration was 500 mg L−1 and the ozone dose was 25 g O3/g TOC, the HPAM removal rate reached 90.79%. Experimental results obtained from gel permeation chromatography (GPC) and rheometer indicated that the refractory HPAM was decomposed into small-molecule compounds. High performance liquid chromatography (HPLC) analysis showed that there was no acrylamide (AM) in the effluent of the system. Microbial communities in two aerobic biofilm reactors (ABRs) were analyzed by Illumina MiSeq Sequencing, which indicated that norank_f_Cytophagaceae, Meiothermus, Bacillus, etc. were keystone functional bacterial genera and Methanobacterium, norank_p_Bathyarchaeota, norank_c_Marine_Group_Ⅰ, etc. were dominant functional archaeal groups. To our knowledge, this is the first study to treat HPAM-containing wastewater using an aerobic-ozonic-aerobic hybrid process. Good removal efficiencies and presence of functional microorganisms demonstrated that the hybrid treatment system was practical for treating HPAM-containing wastewater.

Impacts of Continuous Inflow of Low Concentrations of Silver Nanoparticles on Biological Performance and Microbial Communities of Aerobic Heterotrophic Wastewater Biofilm.

Attached-growth wastewater processes are currently used in water resource recovery facilities (WRRFs) for required upgrades due to an increase in influent loading or to reach more stringent discharge criteria. Yet, the distribution and long-term inhibitory effects of silver nanoparticles (AgNPs) in attached-growth biological wastewater processes and their impact on involved microbial communities are poorly understood at relevant, low concentrations. Retention, distribution, and long-term inhibitory effect of polyvinylpyrrolidone (PVP)-coated AgNPs were evaluated in bench-scale moving bed biofilm reactors (MBBRs), achieving soluble organic matter removal, over a 64 day exposure to nominal concentrations of 10 and 100 μg/L. Distributions of continuously added AgNPs were characterized in the influent, bioreactor, and effluent of MBBRs using single particle inductively coupled plasma mass spectroscopy (spICP-MS). Aerobic heterotrophic biofilms in MBBRs demonstrated limited retention capacity for AgNPs over long-term exposure, with release of AgNPs, and Ag-rich biofilm sloughed from the carriers. Continuous exposure to both influent AgNP concentrations significantly decreased soluble chemical oxygen demand (SCOD) removal efficiency (11% to 31%) and reduced biofilm viability (8% to 30%). Specific activities of both intracellular dehydrogenase (DHA) and extracellular α-glucosidase (α-Glu) and protease (PRO) enzymes were significantly inhibited (8% to 39%) with an observed NP dose-dependent intracellular reactive oxygen species (ROS) production and shift in biofilm microbial community composition by day 64. Our results indicated that long-term exposure to AgNPs in biofilm processes at environmentally relevant concentrations can impact the treatment process stability and the quality of the discharged effluent.

Antibiofilm Efficacy of Nitric Oxide-Releasing Alginates against Cystic Fibrosis Bacterial Pathogens.

Colonization of the lungs by biofilm-forming pathogens is a major cause of mortality in cystic fibrosis (CF). In CF patients, these pathogens are difficult to treat due to the additional protection provided by both the biofilm exopolysaccharide matrix and thick, viscous mucus. The antibiofilm efficacy of nitric oxide (NO)-releasing alginates was evaluated against Pseudomonas aeruginosa, Burkholderia cepacia, Staphylococcus aureus, and methicillin-resistant S.aureus biofilms in both aerobic and anaerobic environments. Varying the amine precursor grafted onto alginate oligosaccharides imparted tunable NO storage (∼0.1-0.3 μmol/mg) and release kinetics (∼4-40 min half-lives) in the artificial sputum media used for biofilm testing. The NO-releasing alginates were highly antibacterial against the four CF-relevant pathogens, achieving a 5-log reduction in biofilm viability after 24 h of treatment, with biocidal efficacy dependent on NO-release kinetics. Aerobic biofilms required greater starting NO doses to achieve killing relative to the anaerobic biofilms. Relative to tobramycin (the minimum concentration of antibacterial agent required to achieve a 5-log reduction in viability after 24 h, MBEC24h, of ≥2000 μg/mL) and vancomycin (MBEC24h ≥ 1000 μg/mL), the NO-releasing alginates proved to be more effective (NO dose ≤ 520 μg/mL) regardless of growth conditions.

Design of Sewage Treatment Plants for High-Density Urban Reclamation Land

The success of any coastal reclamation project depends strongly on a sound planning and design practices. Due to the land use limitations, sewage treatment plant (STP) of the reclamation land should be fully studied to save land and reduce its adverse effects on the environment. This research focuses on the selection of biological treatment process for a novel STP, as various pollutants of the sewage can be effectively removed via the biological process. The paper presents a design of the STP implemented in Johor as an example, to achieve the goal of ‘zero discharge’ in the reclaimed wetland. The domestic sewage is treated, recycled and reused in the artificial wetland as an ecological green water to achieve Class IIB standard (effluent quality). The novel STP design comprised of the Immobilized Aerobic Biofilm (BioAX) and Mass Bio System (MBS) was compared against the conventional Sequencing Batch Reactor (SBR) process. Combination BioAX + MBS process is advantageous due to significantly less land needed, lower power consumption and lower sludge generation. The system was demonstrated as a viable process to treat the sewage wastewater in the reclamation land to meet Class IIB discharge at a lower environmental footprint, saving up to 50 % of the area.

Sensitivity to Oxygen in Microbial Electrochemical Systems Biofilms

SummaryThe formation and bioelectric performance of anode biofilms in microbial electrochemical systems (MESs) are sensitive to oxygen. Investigating the temporal-spatial structure of anode biofilms will help elucidate the interfaces between oxygen and bacteria, thereby facilitating the applications of MESs in wastewater treatment and energy recovery. Here, use of optical coherence tomography, frozen sections, and a microsensor revealed that the aerobic biofilms exhibited a multilayered sandwich structure with a sparse gap between the aerobe- and amphimicrobe-enriched outer layer and the dense exoelectrogen-enriched inner layer, whereas the anaerobic biofilm consisted of only a single dense layer. Our results showed that the inner layer of aerobic anode biofilms performed electricity generation, whereas the outer layer only consumed oxygen. In this case, electron donor diffusion through the outer layer became the limiting factor in electricity generation by the bioanode. Consequently, as the anode biofilms matured, current generation decreased.

Application of a microbial fuel cell-based biosensor for the energy-saving operation of macrophyte residues bioreactor with low concentration of dissolved organic carbon in effluents

The increasing application of plant residues bioreactor for aquatic environment remediation may release numerous dissolved organic carbon (DOC) into aquatic ecosystems. In this study, a microbial fuel cell (MFC) sensor was integrated with a macrophyte residues bioreactor (MRBR) to provide an energy-saving way for reduction of DOC concentrations in the effluent. Through re-utilization of macrophyte residues as solid carbon source, DOC concentrations in the effluent of MRBR increased to the maximum on day 5 and then dropped down rapidly to a low value, while the ratio of bioavailable DOC decreased gradually. Interestingly, it was found that there existed a linear relationship between DOC concentrations in initial residue leachate and the voltage from MFC biosensor (R2 = 0.9852). Accordingly, aerobic biofilm through aeration were applied in the upper part of MRBR to enhance the degradation of DOC prior to discharge to aquatic systems, and aeration rate was adjusted based on MFC sensor signal. Further experiments demonstrated that when voltage decreased from 0.18 V to 0.09 V, a half of aeration rate (7.5 L min−1) could still lead to a high DOC degradation efficiency (above 50%) and a low DOC concentration (∼10 mg L−1) in the reactor effluent. Thus, the integrated MFC signal could be used to regulate the aeration rate in order to obtain a low DOC concentration in effluents under an energy-saving way.

Diversity and abundance of denitrifying bacteria in a simultaneously nitrifying and denitrifying rotating biological contactor treating real wastewater at low temperatures

Abstract Nitrification and denitrification processes occur simultaneously in aerobic wastewater biofilms. Although wide regions of the world have average temperatures of less than 15 °C for a half year, few studies have investigated the nitrogen removal by nitrification and denitrification in a single-stage aerobic biofilm reactor used for treating real wastewater under low-temperature conditions. This study showed successful wastewater treatment in a high average nitrogen removal rate of 78% at low water temperatures by simultaneous nitrification and denitrification in a rotating biological contactor (RBC) biofilm. Batch operations using the RBC to evaluate the rates of ammonium decrease at low temperatures demonstrated that the rate of ammonium decrease at 8 °C was 76% of that at 20 °C. Daily monitoring of nitrification and denitrification rates suggested that the denitrification rate was highly correlated with the nitrification rate. Next-generation sequencing (NGS) analysis revealed the presence of diverse and abundant denitrifying bacteria and aerobic bacteria in the RBC biofilm more than those in the activated sludge samples, which probably enabled the achievement of the high nitrogen removal rates at such low temperatures. Furthermore, correlation with the colony counts showed that the NGS analysis had the quantification range of three orders of magnitude (from 0.001% to 1%).

Mixed aerobic-anaerobic incubation conditions induce proteolytic activity from in vitro salivary biofilms

ABSTRACTOral biofilms have not been studied using both metabolome and protein profiling concurrently. Bacteria produce proteases that lead to degradation of functional salivary proteins. The novel protocol described here allows for complete characterisation of in vitro oral biofilms, including proteolytic, metabolic, and microbiome analysis.Biofilms were grown on hydroxyapatite discs from whole mouth saliva, using sterilised saliva as a growth-medium, in different growth environments. Salivary protein degradation was assessed from spent saliva growth-medium using SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and metabolic activity by nuclear magnetic resonance (NMR). Discs were assessed for depth and coverage of biofilms by confocal laser scanning microscopy (CLSM), and biofilms were collected at the end of the experiment for 16S rRNA gene sequence analysis.There was a significant difference in biofilm viability, salivary protein degradation, and metabolites identified between biofilms grown aerobically and biofilms exposed to an anaerobic environment. Bacterial 16S rRNA gene sequencing showed the predominant genus in the 7-day aerobic biofilms was Streptococcus, in aerobic-anaerobic and anaerobic 7-day biofilms Porphyromonas, and in aerobic-anaerobic and anaerobic 13-day biofilms Fusobacterium.This data suggests new growth requirements and capabilities for analysing salivary biofilms in vitro, which can be used to benefit future research into oral bacterial biofilms.

A cascade of a denitrification bioreactor and an aerobic biofilm reactor for heavy oil refinery wastewater treatment

The performance of an efficient denitrification bioreactor–aerobic biofilm reactor cascade for heavy oil refinery wastewater treatment was investigated. Optimum operation parameters for denitrification were found as follows: (1) hydraulic retention time of 8 h; (2) C/NO3−–N molar ratio of 3.75 with acetate as the carbon source; (3) 20% (v/v) carrier filling ratio in the denitrification bioreactor. Under such optimal conditions, a volumetric removal of 0.82 kg N m−3 d−1 was obtained. As an alternative low-cost carbon source to acetate, secondary DAF effluent (COD/NO3−–N mass ratio of 5.4) was also detected and a stable activity of denitrification was achieved with adding 25% volume fraction of secondary DAF effluent. Effluent COD of the subsequent aerobic biofilm reactor further decreased satisfying the requirements of the current discharge standards. High-throughput sequencing results exhibited that Rhodocyclaceae and Comamonadaceae were the dominant denitrifiers in the denitrification reactor and Pseudomonas was the dominant microbe in the aerobic biofilm reactor.

Biohybrid Cathode in Single Chamber Microbial Fuel Cell

The aim of this work is to investigate the properties of biofilms, spontaneously grown on cathode electrodes of single-chamber microbial fuel cells, when used as catalysts for oxygen reduction reaction (ORR). To this purpose, a comparison between two sets of different carbon-based cathode electrodes is carried out. The first one (Pt-based biocathode) is based on the proliferation of the biofilm onto a Pt/C layer, leading thus to the creation of a biohybrid catalyst. The second set of electrodes (Pt-free biocathode) is based on a bare carbon-based material, on which biofilm grows and acts as the sole catalyst for ORR. Linear sweep voltammetry (LSV) characterization confirmed better performance when the biofilm is formed on both Pt-based and Pt-free cathodes, with respect to that obtained by biofilm-free cathodes. To analyze the properties of spontaneously grown cathodic biofilms on carbon-based electrodes, electrochemical impedance spectroscopy is employed. This study demonstrates that the highest power production is reached when aerobic biofilm acts as a catalyst for ORR in synergy with Pt in the biohybrid cathode.

A full-scale hybrid vertical anaerobic and aerobic biofilm wastewater treatment system: case study

Abstract The first full-scale Hybrid Vertical Anaerobic Aerobic Biofilm (HyVAB) reactor has been set up for treating wastewater from a vegetable processing industry in Grimstad, Norway. The novel HyVAB reactor integrates a bottom expanded granular sludge bed with a top aerobic biofilm stage, resulting in a small footprint and high treatment efficiency. The full scale holistic treatment plant consists of a pretreatment system of a sand trap and an equalization tank, a HyVAB reactor and an effluent sludge settlement tank. The HyVAB system has been operated continuously for 219 days with flow and chemical oxygen demand (COD) fluctuations corresponding to different product seasons. The reactor hydraulic retention time ranges from 32 to 10 hours, with the anaerobic organic loading rate (OLR) reaching a maximum 16 kg-COD/m3·d. The HyVAB removed on average of 90% of the total feed COD, at an operational temperature of 25 °C. Sludge production was low at 0.11 kg-volatile suspended solids/kg-COD removed. Odorless effluent from HyVAB can be discharged directly to a local municipal wastewater treatment plant without sludge handling. Over 82% of feed COD was converted to methane, leaving high methane content (84 ± 2%) biogas out of the reactor. Energy consumption of HyVAB was 0.5 kwh/ton wastewater. The cost of wastewater treatment is 1.5 NOK/kg COD removed (based on rates in Norway).

Dynamics of Biofilm Formation by Salmonella Typhimurium and Beef Processing Plant Bacteria in Mono- and Dual-Species Cultures.

This study aimed to determine the impact of bacteria from a beef plant conveyor belt on the biofilm formation of Salmonella in dual-species cultures. Beef plant isolates (50) including 18 Gram-negative aerobes (GNA), 8 Gram-positive aerobes (GPA), 5 lactic acid bacteria (LAB), 9 Enterobacteriaceae (EB), and 10 generic Escherichia coli (GEC) were included for developing biofilms in mono- and co-culture with S. Typhimurium at 15°C for 6days. Five selected cultures in planktonic form and in biofilms were tested for susceptibility to two commonly used sanitizers (i.e. E-San and Perox-EPlus). In mono-cultures, ≥ 80, 67, 61, 20, and 13% of GEC, EB, GNA, LAB, and GPA, respectively, developed measurable biofilms after 2days, while all co-culture pairings with S. Typhimurium achieved some level of biofilm production. The predominant effect of EB and only effect of GEC strains on the biofilm formation of S. Typhimuriumwas antagonistic, while that of Gram-positive bacteria was synergistic, with the effect being more prominent on day 6. The effect was highly variable for the GNA isolates. Six aerobic isolates that formed moderate/strong biofilms by day 2 greatly boosted the co-culture biofilm formation. Seven Gram-negative bacteria were antagonistic against the biofilm formation of the co-cultures. Both sanitizers completely inactivated the selected planktonic cultures, but were largely ineffective against biofilms. In conclusion, all beef plant isolates assessed formed biofilms when paired with S. Typhimurium. Aerobic biofilm formers may create a more favorable condition for Salmonella biofilm formation, while some beef plant isolates have potential as a biocontrol strategy for Salmonella biofilms.

Potential use of treated domestic sewage for cultivation of biofuel crops in Egypt

Sewage treatment in a biological compact unit and effluent reuse for cultivation of Jojoba, Jatropha and Castor beans was examined. The biological compact unit includes three zones: settling and anaerobic, aerobic biofilm and final sedimentation. Quality of raw sewage and treated effluents was monitored. Impact of water quality on growth rate of biofuel crops and soil properties was investigated. Reuse of raw sewage for irrigation of biofuel crops has major impact on microbial quality and heavy metals concentration of irrigated soil. Sequence of heavy metals accumulation in the soil was Zn > Cu > Cr > Pb. Castor beans irrigated with the final effluent has the highest growth rate and crop yield followed by Jatropha and Jojoba. Growing Castor beans using treated sewage is economically feasible since the plant produces harvestable seeds during the first year and thereafter. Castor beans yield was highest (1.78 ton/ha) with the final effluent. Estimated market price is $1241/ha.

Startup strategy for nitrogen removal via nitrite in a BAF system.

A biological aerated filter (BAF) pilot plant consisting of two reactors (aerobic and anoxic one) was used to determine a strategy to remove nitrogen via nitrite. RNA/DNA analysis was performed to assess microbial activity and support chemical results. In less than 13 days the pilot plant was able to remove COD and suspended solids. Nitrogen removal via nitrite pathway could not be observed until day 130 when the empty bed contact time (EBCT) was set at 0.71h. Nitrite was detected in the aerated BAF effluent but never nitrate. qPCR of amoA gene from RNA and DNA extracts of the aerobic biofilm confirmed that ammonia oxidizing bacteria (AOB) were present from the beginning of the operation but not active. AOB activity increased with time, reaching stability from operational day 124. The combination of both, low EBCT together with high OLR, has been demonstrated to be a feasible strategy to startup a BAF to achieve nitrogen removal via nitrite.

Nitrous oxide emissions from biofilm processes for wastewater treatment.

This paper discusses the microbial basis and the latest research on nitrous oxide (N2O) emissions from biofilms processes for wastewater treatment. Conditions that generally promote N2O formation in biofilms include (1) low DO values, or spatial DO transitions from high to low within the biofilm; (2) DO fluctuations within biofilm due to varying bulk DO concentrations or varying substrate concentrations; (3) conditions with high reaction rates, which lead to greater formation of intermediates, e.g., hydroxylamine (NH2OH) and nitrite (NO2-), that promote N2O formation; and (4) electron donor limitation for denitrification. Formation of N2O directly results from the activities of ammonia-oxidizing bacteria (AOB), ammonia-oxidizing archaea (AOA), and heterotrophic denitrifying bacteria. More research is needed on the roles of AOA, comammox, and specialized denitrifying microorganisms. In nitrifying biofilms, higher bulk ammonia (NH3) concentrations, higher nitrite (NO2-) concentrations, lower dissolved oxygen (DO), and greater biofilm thicknesses result in higher N2O emissions. In denitrifying biofilms, N2O accumulates at low levels as an intermediate and at higher levels at the oxic/anoxic transition regions of the biofilms and where COD becomes limiting. N2O formed in the outer regions can be consumed in the inner regions if COD penetrates sufficiently. In membrane-aerated biofilms, where nitrification takes place in the inner, aerobic biofilm region, the exterior anoxic biofilm can serve as a N2O sink. Reactors that include variable aeration or air scouring, such as denitrifying filters, trickling filters, or rotating biological contactors (RBCs), can form peaks of N2O emissions during or following a scouring or aeration event. N2O emissions from biofilm processes depend on the microbial composition, biofilm thickness, substrate concentrations and variability, and reactor type and operation. Given the complexity and difficulty in quantifying many of these factors, it may be difficult to accurately predict emissions for full-scale treatment plants. However, a better understanding of the mechanisms and the impacts of process configurations can help minimize N2O emission from biofilm processes for wastewater treatment.

Development of kinetics of wastewater treatment in the aerobic biofilm reactor

Development of kinetics of wastewater treatment in the aerobic biofilm reactor

Treatment of centrifugal mother liquid of polyvinyl chloride by internal circulation aerobic biofilm reactor: Lab to plant scale system

Centrifugal mother liquid (CML) is a typical poorly biodegradable organic wastewater generated during the production of polyvinyl chloride (PVC). A method of treating PVC centrifugal mother liquid by a high efficiency internal circulation aerobic biofilm reactor (four reactors lined up together) is presented in this study. The optimized technique was tested and evaluated in a lab and in a plant scale system. The effect of chemical oxygen demand (COD) removal efficiency was studied under different hydraulic retention time (HRT): 40, 20, 17, 14 and 12 h, respectively. Considering the efficiency and removal rate, the best HRT was 14 h. At HRT of 14 h, the average volume loading was 0.326 kg COD/(m3∙d). During the product shift from TL-1000 to TL-800 (two kinds of PVC products with different polymerization degree), the treatment efficiency of the system was not affected. Compared with the plant scale system, the results showed that in the lab setup the removal rates of centrifugal mother liquid of TL-1000 (total organic carbon (TOC) = 70–75 mg/L) and TL-800 (TOC = 20 mg/L) were 90%–95% and 80%–90%, respectively. While in the plant scale system, the removal rates of centrifugal mother liquid of TL-1000 and TL-800 were 80%–90% and 65%–80% (effluent TOC 7.5–12 mg/L), respectively. Biofilm formation by the microbial reproduction as well as intensive filamentous bacteria settlements were observed.