Biofilm development on new and cleaned membrane surfaces

This thesis presents a comprehensive research report on microbiological aspects of biofouling occurrence in full-scale reverse osmosis (RO) systems. Biofouling is a process in which microorganisms attach to membranes and develop into a thick film that can choke the entire RO system. Management of this problem requires basic understanding of the mechanism of this phenomenon. The basic questions of this PhD research project therefore addressed the origin, succession and spatiotemporal development of biofilms in full-scale RO systems, in particular in relation to operational aspects of RO systems. The multifaceted research strategy involving acquisitions of representative samples and use of many molecular and microscopic analysis techniques in parallel was employed. The investigation showed that biofilms are able to grow on any surface in a full-scale RO plant. This gives local niches for detachment of biomass, either as single cells or cell clumps, and results in a spreading of bacteria to the further stages of the plant. In the RO membrane modules, the enriched bacteria might more easily colonise the surfaces since they will be better adapted to growth in the system than bacteria present in the feed water. Initially, the single cell colonizers (sphingomonads) form a number of flat and abundantly EPS-embedded cell monolayers over the entire membrane surface. The clumps-associated pioneers (mainly Beta- and Gammaproteobacteria) appear to be trapped mainly in the first part of the module, most likely due to a filtering action of the spacer. In time, these bacteria develop in pillar-like structures and slowly spread throughout the whole membrane module on top of the established sphingomonads biofilm. The secondary colonisers (bacteria and eukaryotes) occur in the resulting biofilm formations. Although composition of the biofilm microbial community undergoes a succession in time, the architecture of an established biofilm appears to be rather stable. Conventional treatment of RO membrane modules with chemicals did not lead to cleaning: the sphingomonads cells can be detected under the collapsed but obviously not removed biofilm EPS matrix. After cleaning, the biofouling layer seemed to grow faster (within 6 days) than a fresh biofilm (16 days). To conclude, biofouling is a complex phenomenon with two appearances: a fouling layer on the membrane limiting the water flux and a fouling layer on the spacer limiting the water flow through the spacer channel and resulting in an increased pressure drop. It became clear that cleaning strategies should focus more on the removal of accumulated biomass and not only on the killing of cells. Moreover, the basal Sphingomonas layer requires further research to appropriately control biofouling in RO systems. It might also be possible to design the RO - membrane module in a different manner, leading to a different biofilm morphology which gives less rise to operational problems

Survival, biofilm formation, and growth potential of environmental and enteric escherichia coli strains in drinking water microcosms

E. coli is the most commonly used indicator for faecal contamination in a drinking water distribution system (WDS). The assumption is that E. coli are of enteric origin and cannot persist for long outside their host, therefore acting as indicators of recent contamination events. This study investigates the fate of E. coli in drinking water; specifically addressing survival, biofilm formation under shear stress, and regrowth in a series of laboratory-controlled experiments. We show the extended persistence of three E. coli strains (two enteric and one soil isolate) in sterile and non-sterile drinking water microcosms, at 8 and 17°C, with T90 values ranging from 17.4 ± 1.8 to 149 ± 67.7 days, using standard plate counts and a series of (RT)-Q-PCR assays targeting 16S rRNA, tuf, uidA, and rodA genes and transcripts. Furthermore, each strain was capable of attaching to a surface and replicating to form biofilm in the presence of nutrients under a range of shear stress values (0.6, 2.0, and 4.4 dyn cm-2; BioFlux, Fluxion); however, cell numbers did not increase when drinking water was flowed over (t-test; p > 0.05). Finally, E. coli regrowth within drinking water microcosms containing PE-100 pipe-wall material was not observed in the biofilm or water phase using a combination of culturing and Q-PCR methods for E. coli. The results of this work highlight that when E. coli enters drinking water it has the potential to survive and attach to surfaces but that regrowth within drinking water or biofilm is unlikely

The microbial ecology of a Mediterranean chlorinated drinking water distribution systems in the city of Valencia (Spain)

Drinking water distribution systems host extensive microbiomes with diverse biofilm communities regardless of treatment, disinfection, or operational practices. In Mediterranean countries higher temperatures can accelerate reactions and microbial growth that may increase aesthetic water quality issues, particularly where material deposits can develop as a result of net zero flows within looped urban networks. This study investigated the use of flow and turbidity monitoring to hydraulically manage mobilisation of pipe wall biofilms and associated material from the Mediterranean city of Valencia (Spain). Pipe sections of different properties were subjected to controlled incremental flushing with monitoring and sample collection for physico-chemical and DNA analysis with Illumina sequencing of bacterial and fungal communities. A core microbial community was detected throughout the network with microorganisms like Pseudomonas, Aspergillus or Alternaria increasing during flushing, indicating greater abundance in underlying and more consolidated material layers. Bacterial and fungal communities were found to be highly correlated, with bacteria more diverse and dynamic during flushing whilst fungi were more dominant and less variable between sampling sites. Results highlight that water quality management can be achieved through hydraulic strategies yet understanding community dynamics, including the fungal component, will be key to maintaining safe and ultimately beneficial microbiomes in drinking water distribution systems

A novel method for viability enumeration for single-droplet drying of Lactobacillus plantarum WCFS1

Survival of probiotic bacteria during drying is not trivial. Survival percentages are very specific for each probiotic strain and can be improved by careful selection of drying conditions and proper drying carrier formulation. An experimental approach is presented, comprising a single-droplet drying method and a subsequent novel screening methodology, to assess the microbial viability within single particles. The drying method involves the drying of a single droplet deposited on a flat, hydrophobic surface under well-defined drying conditions and carrier formulations. Semidried or dried particles were subjected to rehydration, fluorescence staining, and live/dead enumeration using fluorescence microscopy. The novel screening methodology provided accurate survival percentages in line with conventional plating enumeration and was evaluated in single-droplet drying experiments with Lactobacillus plantarum WCFS1 as a model probiotic strain. Parameters such as bulk air temperatures and the carrier matrices (glucose, trehalose, and maltodextrin DE 6) were varied. Following the experimental approach, the influence on the viability as a function of the drying history could be monitored. Finally, the applicability of the novel viability assessment was demonstrated for samples obtained from drying experiments at a larger scale

Effect of conventional chemical treatment on the microbial

The impact of conventional chemical treatment on initiation and spatiotemporal development of biofilms on reverse osmosis (RO) membranes was investigated in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The flow cells got the same feed (extensively pre-treated fresh surface water) and operational conditions (temperature, pressure and membrane flux) as the full-scale installation. With regular intervals both the full-scale RO membrane modules and the flow cells were cleaned using conventional chemical treatment. For comparison some flow cells were not cleaned. Sampling was done at different time periods of flow cell operation (i.e., 1, 5, 10 and 17 days and 1, 3, 6 and 12 months). The combination of molecular (FISH, DGGE, clone libraries and sequencing) and microscopic (field emission scanning electron, epifluorescence and confocal laser scanning microscopy) techniques made it possible to thoroughly analyze the abundance, composition and 3D architecture of the emerged microbial layers. The results suggest that chemical treatment facilitates initiation and subsequent maturation of biofilm structures on the RO membrane and feed-side spacer surfaces. Biofouling control might be possible only if the cleaning procedures are adapted to effectively remove the (dead) biomass from the RO modules after chemical treatment.

Effect of conventional chemical treatment on the microbial population in a biofouling layer of reverse osmosis systems

The impact of conventional chemical treatment on initiation and spatiotemporal development of biofilms on reverse osmosis (RO) membranes was investigated in situ using flow cells placed in parallel with the RO system of a full-scale water treatment plant. The flow cells got the same feed (extensively pre-treated fresh surface water) and operational conditions (temperature, pressure and membrane flux) as the full-scale installation. With regular intervals both the full-scale RO membrane modules and the flow cells were cleaned using conventional chemical treatment. For comparison some flow cells were not cleaned. Sampling was done at different time periods of flow cell operation (i.e., 1, 5, 10 and 17 days and 1, 3, 6 and 12 months). The combination of molecular (FISH, DGGE, clone libraries and sequencing) and microscopic (field emission scanning electron, epifluorescence and confocal laser scanning microscopy) techniques made it possible to thoroughly analyze the abundance, composition and 3D architecture of the emerged microbial layers. The results suggest that chemical treatment facilitates initiation and subsequent maturation of biofilm structures on the RO membrane and feed-side spacer surfaces. Biofouling control might be possible only if the cleaning procedures are adapted to effectively remove the (dead) biomass from the RO modules after chemical treatmen

Investigation of microbial communities on reverse osmosis membranes used for process water production

In the present study, the diversity and the phylogenetic affiliation of bacteria in a biofouling layer on reverse osmosis (RO) membranes were determined. Fresh surface water was used as a feed in a membrane-based water purification process. Total DNA was extracted from attached cells from feed spacer, RO membrane and product spacer. Universal primers were used to amplify the bacterial 16S rRNA genes. The biofilm community was analysed by 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) and the phylogenetic affiliation was determined by sequence analyses of individual 16S rDNA clones. Using this approach, we found that five distinct bacterial genotypes (Sphingomonas, Beta proteobacterium, Flavobacterium, Nitrosomonas and Sphingobacterium) were dominant genera on surfaces of fouled RO membranes. Moreover, the finding that all five ¿key players¿ could be recovered from the cartridge filters of this RO system, which cartridge filters are positioned before the RO membrane, together with literature information where these bacteria are normally encountered, suggests that these microorganisms originate from the feed water rather than from the RO system itself, and represent the fresh water bacteria present in the feed water, despite the fact that the feed water passes an ultrafiltration (UF) membrane (pore size approximately 40 nm), which is able to remove microorganisms to a large extent

Investigation of microbial communities on reverse osmosis membranes used for process water production

In the present study, the diversity and the phylogenetic affiliation of bacteria in a biofouling layer on reverse osmosis (RO) membranes were determined. Fresh surface water was used as a feed in a membrane-based water purification process. Total DNA was extracted from attached cells from feed spacer, RO membrane and product spacer. Universal primers were used to amplify the bacterial 16S rRNA genes. The biofilm community was analysed by 16S rRNA-gene-targeted denaturing gradient gel electrophoresis (DGGE) and the phylogenetic affiliation was determined by sequence analyses of individual 16S rDNA clones. Using this approach, we found that five distinct bacterial genotypes (Sphingomonas, Beta proteobacterium, Flavobacterium, Nitrosomonas and Sphingobacterium) were dominant genera on surfaces of fouled RO membranes. Moreover, the finding that all five ¿key players¿ could be recovered from the cartridge filters of this RO system, which cartridge filters are positioned before the RO membrane, together with literature information where these bacteria are normally encountered, suggests that these microorganisms originate from the feed water rather than from the RO system itself, and represent the fresh water bacteria present in the feed water, despite the fact that the feed water passes an ultrafiltration (UF) membrane (pore size approximately 40 nm), which is able to remove microorganisms to a large extent

Dehydration and thermal inactivation of Lactobacillus plantarum WCFS1: Comparing single droplet drying to spray and freeze drying

We demonstrated that viability loss during single droplet drying can be explained by the sum of dehydration and thermal inactivation. For Lactobacillus plantarum WCFS1, dehydration inactivation predominantly occurred at drying temperatures below 45 °C and only depended on the moisture content. Above 45 °C the inactivation was due to a combination of dehydration and thermal inactivation, which depended on the moisture content, temperature, and drying time. A Weibull model was successfully applied to describe the thermal and dehydration inactivation and enabled the prediction of residual viability of L. plantarum WCFS1 after single droplet drying. Subsequently, the model was evaluated to predict the viability loss during laboratory scale spray drying, showing a remarkable agreement if assumed that only thermal inactivation occurred. This indicated that very high drying rates in laboratory scale spray drying could induce instant fixation of the cell suspensions in a vitrified matrix and thereby preventing dehydration inactivation. Finally, the influence of drying rate on remaining viability was evaluated by comparing single droplet drying, freeze drying and laboratory scale spray drying of the same bacterial suspension. It was shown that slow drying leads to large dehydration inactivation, which diminished in fast drying processes such as laboratory scale spray drying where thermal inactivation appears to be the predominant mechanism of inactivation