Efficient control of pathogenic bacteria, specifically Legionella pneumophila, is one of the main concerns when operating industrial cooling towers. Common practices to limit proliferation involves use of disinfectants, leading to formation of disinfection by-product and increase in water corrosiveness. A disinfectant-free Legionella control method would make the industry more environmentally friendly. A pilot-scale cooling tower (1 m3/h) operated with demineralized water was used to investigate the potential of high-pH conditioning as a disinfectant-free alternative for control of L. pneumophila and other pathogens. One control experiment was performed under standard full-scale operation involving sodium hypochlorite dosage. Thereafter 3 alkaline pHs of the cooling water were tested: 9.0, 9.4 and 9.6. The tests lasted between 25 and 35 days. The cooling water from the basins were analysed for total cell count by flow cytometry, L. pneumophila concentration by plate count and occasional qPCR analyses targeting the mip-gene, bacterial and eukaryotic community analyses with 16S and 18S rRNA gene amplicon sequencing, relative abundance of eukaryotic to prokaryotic DNA by qPCR of the 16S and 18S rRNA gene. The L. pneumophila analyses showed considerable growth at pH 9.0 and pH 9.4 but was maintained below detection limit (< 100 CFU/L) at pH 9.6 without disinfection. Interestingly, the results correlated with the overall abundance of protozoa in the water samples but not directly with the relative abundance of specific reported protozoan hosts of Legionella. The pathogenicity based on 16S rRNA gene amplicon sequencing of the cooling water DNA decreased with increasing pH with a strong decline between pH 9.0 and pH 9.4, from 7.1% to 1.6% of relative abundance of pathogenic genera respectively. A strong shift in microbiome was observed between each tested pH and reproducibility of the experiment at pH 9.6 was confirmed with a duplicate test lasting 80 days. High-pH conditioning ≥ 9.6 is therefore considered as an efficient disinfectant-free cooling tower operation for control of pathogenicity, including L. pneumophila.

Temperature change over the length of heat exchangers might be an important factor affect-ing biofouling. This research aimed at assessing the impact of temperature on biofilm accumulation and composition with respect to bacterial community and extracellular polymeric substances. Two identical laboratory-scale plate heat exchanger modules were developed and tested. Tap water sup-plemented with nutrients was fed to the two modules to enhance biofilm formation. One “reference” module was kept at 20.0 ± 1.4^◦ C and one “heated” module was operated with a counter-flow hot water stream resulting in a bulk water gradient from 20 to 27^◦ C. Biofilms were grown during 40 days, sampled, and characterized using 16S rRNA gene amplicon sequencing, EPS extraction, FTIR, protein and polysaccharide quantifications. The experiments were performed in consecutive triplicate. Monitoring of heat transfer resistance in the heated module displayed a replicable biofilm growth profile. The module was shown suitable to study the impact of temperature on biofouling formation. Biofilm analyses revealed: (i) comparable amounts of biofilms and EPS yield in the reference and heated modules, (ii) a significantly different protein to polysaccharide ratio in the EPS of the reference (5.4 ± 1.0%) and heated modules (7.8 ± 2.1%), caused by a relatively lower extracellular sugar production at elevated temperatures, and (iii) a strong shift in bacterial community composition with increasing temperature. The outcomes of the study, therefore, suggest that heat induces a change in biofilm bacterial community members and EPS composition, which should be taken into consideration when investigating heat exchanger biofouling and cleaning strategies. Research potential and optimization of the heat exchanger modules are discussed.

Understanding the bacterial dynamics in cooling towers is imperative for the assessment of disinfection efficiency and management of microbial risks linked to aerosol formation. The objective of this study was to evaluate the impact of feed water on the cooling water bacterial microbiome and investigate the survival ability of its members when exposed to continuous chlorine disinfection. Water from an industrial cooling water system (2600 m³/h) was collected over a 5-month period at 3 locations along the feed water line and 3 locations in the cooling tower. ATP measurements suggested that the average ATP-per-cell in the cooling tower evolved independently from the average ATP-per-cell in the feed water. Flow cytometry and 16S rRNA gene amplicon sequencing were then combined to quantify the bacterial dynamics in the whole system. A mass balance based equation was established to determine net growth and net decay of the cooling tower bacterial communities in order to evaluate the impact of continuous chlorination (0.35–0.41 mg Cl₂/L residual chlorine). The results indicated that cooling tower main community members were determined by the input feed water microbiome and the bacterial community structure was further shaped by varying decay rates of the microorganisms. Notably, the order Obscuribacterales showed to be growing in the cooling tower in the presence of residual chlorine up to 0.4 mg Cl₂/L, with a recurrent net growth of 260 ± 95%, taking into account the impact of the concentration factor. This conclusion was only possible thanks to the systematic analysis described in this paper and generates discussion about the resistance of Obscuribacterales to residual chlorine. The described mass balance approach provides a high level of understanding on bacterial dynamics and should be considered for future characterization studies of cooling towers in which accurate investigation of microbiome changes is essential.

Sialic acids in the structural matrix of biofilms developing in engineered water systems constitute a potential target in the battle against biofouling. This report focuses specifically on the presence of sialic acids as part of the extracellular polymeric substances (EPS) of biofilms forming in cooling towers and the potential effect of nutrient starvation on sialic acid presence and abundance. Two cooling water compositions were compared in parallel pilot-scale cooling towers, one poor in nutrients and one enriched in nutrients. Fresh deposits from the two cooling towers were collected after a five-week operation period. EPS extractions and analyses by Fourier transform infrared spectroscopy (FTIR) and high-resolution mass spectrometry (MS), along with 16S rRNA gene amplicon sequencing were performed. The results of MS analyses showed the presence of pseudaminic/legionaminic acids (Pse/Leg) and 2-keto-3-deoxy-d-glycero-d-galacto-nononic acid (KDN) in both biofilm EPS samples. FTIR measurements showed the characteristic vibration of sialic acid-like compounds ν(C=O)OH in the nutrient poor sample exclusively. Our findings, combined with other recent studies, suggest that bacterial sialic acids are common compounds in environmental biofilms. Additionally, the conservation of sialic acid production pathways under nutrient starvation highlights their importance as constituents of the EPS. Further in-depth studies are necessary to understand the role of sialic acids in the structural cohesion and protection of environmental biofilm layer.

Phosphate limitation has been suggested as a preventive method against biofilms. P-limited feed water was studied as a preventive strategy against biofouling in cooling towers (CTs). Three pilot-scale open recirculating CTs were operated in parallel for five weeks. RO permeate was fed to the CTs (1) without supplementation (reference), (2) with supplementation by biodegradable carbon (P-limited) and (3) with supplementation of all nutrients (non-P-limited). The P-limited water contained ≤10 µg PO₄ l⁻¹. Investigating the CT-basins and coupons showed that P-limited water (1) did not prevent biofilm formation and (2) resulted in a higher volume of organic matter per unit of active biomass compared with the other CTs. Exposure to external conditions and cycle of concentration were likely factors that allowed a P concentration sufficient to cause extensive biofouling despite being the limiting compound. In conclusion, phosphate limitation in cooling water is not a suitable strategy for CT biofouling control.

Chemical cleaning is routinely performed in reverse osmosis (RO) plants for the regeneration of RO membranes that suffer from biofouling problems. The potential of urea as a chaotropic agent to enhance the solubilization of biofilm proteins has been reported briefly in the literature. In this paper the efficiency of urea cleaning for RO membrane systems has been compared to conventionally applied acid/alkali treatment. Preliminary assessment confirmed that urea did not damage the RO polyamide membranes and that the membrane cleaning efficiency increased with increasing concentrations of urea and temperature. Accelerated biofilm formation was carried out in membrane fouling simulators which were subsequently cleaned with (i) 0.01M sodium hydroxide (NaOH) and 0.1M hydrochloric acid (HCl) (typically applied in industry), (ii) urea (CO(NH ₂ ) ₂ ) and hydrochloric acid, or (iii) urea only (1340 g/L _water ). The pressure drop over the flow channel was used to evaluate the efficiency of the applied chemical cleanings. Biomass removal was evaluated by measuring chemical oxygen demand (COD), adenosine triphosphate (ATP), protein, and carbohydrate content from the membrane and spacer surfaces after cleaning. In addition to protein and carbohydrate quantification of the extracellular polymeric substances (EPS), fluorescence excitation−emission matrix (FEEM) spectroscopy was used to distinguish the difference in organic matter of the remaining biomass to assess biofilm solubilization efficacy of the different cleaning agents. Results indicated that two-stage CO(NH ₂ ) ₂ /HCl cleaning was as effective as cleaning with NaOH/HCl in terms of restoring the feed channel pressure drop (>70% pressure drop decrease). One-stage cleaning with urea only was not as effective indicating the importance of the second-stage low pH acid cleaning in weakening the biofilm matrix. All three chemical cleaning protocols were equally effective in reducing the concentration of predominant EPS components protein and carbohydrate (>50% reduction in concentrations). However, urea-based cleaning strategies were more effective in solubilizing protein-like matter and tyrosine-containing proteins. Furthermore, ATP measurements showed that biomass inactivation was up to two-fold greater after treatment with urea-based chemical cleanings compared to the conventional acid/alkali treatment. The applicability of urea as an alternative, economical, eco-friendly and effective chemical cleaning agent for the control of biological fouling was successfully demonstrated.

Biocides may be used to control biofouling in spiral-wound reverse osmosis (RO) and nanofiltration (NF) systems. The objective of this study was to investigate the effect of biocide 2,2-dibromo-3-ni-trilopropionamide (DBNPA) dosage on biofouling control. Preventive biofouling control was studied applying a continuous dosage of substrate (0.5 mg/L) and DBNPA (1 mg/L). Curative biofouling control was studied on pre-grown biofilms, once again applying a continuous dosage of substrate (0.5 mg acetate C/L) and DBNPA (1 and 20 mg/L). Biofouling studies were performed in membrane fouling simulators (MFSs) supplied with biodegradable substrate and DBNPA. The pressure drop was monitored in time and at the end of the study, the accumulated biomass in MFS was quantified by adenosine triphosphate (ATP) and total organic carbon (TOC) analysis. Continuous dosage of DBNPA (1 mg/L) prevented pressure drop increase and biofilm accumulation in the MFSs during a run time of 7 d, showing that biofouling can be managed by preventive DBNPA dosage. For biofouled systems, continuous dosage of DBNPA (1 and 20 mg/L) inactivated the accumulated biomass but did not restore the original pressure drop and did not remove the accumulated inactive cells and extracellular polymeric substances (EPS), indicating DBNPA dosage is not suitable for curative biofouling control.