Feed substrates influence biofilm formation on reverse osmosis membranes and their cleaning efficiency

The dairy industry is increasingly using reverse osmosis (RO) membranes for concentration of various fluid feed materials such as whey and ultrafiltration (UF) permeate. This study compared the effect of UF permeate and whey on membrane biofilm formation. A Bacillus sp., previously isolated in our laboratory from a cleaning-resistant membrane biofilm, was used to develop 48-h-old static biofilms on RO membrane pieces, using the different feed substrates (UF permeate, whey, and an alternating whey/UF feed). Biofilms were analyzed for viable counts by the swab technique, and we used scanning electron and atomic force microscopy for microstructure imaging. The membrane cleaning process included 6 sequential steps. We observed differences in the resistance pattern of the 3 types of biofilms to the typical cleaning process. The mean pretreatment counts of the 48-h UF permeate biofilms were 5.39 log cfu/cm², much higher than the whey biofilm counts of 3.44 log, and alternating whey/UF biofilm counts of 4.54 log. After a 6-step cleaning cycle, we found 2.54 log survivors of the Bacillus isolate on UF biofilms, whereas only 1.82 log survivors were found in whey biofilm, and 2.14 log survivors on whey/UF permeate biofilms. In conclusion, the UF permeate biofilms was more resistant to the biofilm cleaning process compared with the whey or whey/UF permeate biofilms. Scanning electron micrographs showed different microstructures of biofilms based on the type of feed. For UF permeate and whey/UF permeate biofilms, bacilli were present in multilayers of cells in aggregates or irregular clusters with foulant layers. In contrast, those in whey biofilms were in monolayers, with a smoother, flatter appearance. Atomic force microscopy analysis indicated that UF permeate biofilms had the greatest surface roughness among the biofilms, reflecting intensified bacterial colonization. The biofilm micro- and nanostructure variations for the 2 feed substrates and their combination may have resulted in differences in their resistance to the cleaning process.     

Biofilm formation characteristics of bacterial isolates retrieved from a reverse osmosis membrane

High-quality water purification systems using reverse osmosis (RO) membrane separation have faced a major challenge related to biofilm formation on the membrane surface, or biofouling. To understand this issue, the biofilm formation characteristics of four bacterial isolates previously retrieved from an RO membrane treating potable water were investigated. Biofilm formation of all four isolates occurred to different extents in microtiter plates and could be related to one or more cell properties (hydrophobicity, surface charge, and motility). For Dermacoccus sp. strain RO12 and Microbacterium sp. strain RO18, bacterial adhesion was facilitated by cell surface hydrophobicity, and for Rhodopseudomonas sp. strain RO3, adhesion was assisted by its low surface charge. Sphingomonas sp. strain RO2 possessed both twitching and swarming motilities, which could be important in mediating surface colonization. Further, strains RO2, RO3, and RO12 did not exhibit swimming motility, suggesting that they could be transported to RO membrane surfaces by other mechanisms such as convective permeate flow. The biofilm formation of RO2 was further tested on different RO membranes made of cellulose acetate, polyamide, and thin film composite in continuous flow cell systems. The resultant RO2 biofilms were independent of membrane surface properties and this was probably related to the ex-opolysaccharides secreted bythe biofilm cells. These results suggested that RO2 could colonize RO membranes effectively and could be a potential fouling organism in RO membranes for freshwater purification.     

Studying the role of common membrane surface functionalities on adsorption and cleaning of organic foulants using QCM-D

Adsorption of organic foulants on nanofiltration (NF) and reverse osmosis (RO) membrane surfaces strongly affects subsequent fouling behavior by modifying the membrane surface. In this study, impact on organic foulant adsorption of specific chemistries including those in commercial thin-film composite membranes was investigated using self-assembled monolayers with seven different ending chemical functionalities (-CH(3), -O-phenyl, -NH(2), ethylene-glycol, -COOH, -CONH(2), and -OH). Adsorption and cleaning of protein (bovine serum albumin) and polysaccharide (sodium alginate) model foulants in two solution conditions were measured using quartz crystal microbalance with dissipation monitoring, and were found to strongly depend on surface functionality. Alginate adsorption correlated with surface hydrophobicity as measured by water contact angle in air; however, adsorption of BSA on hydrophilic -COOH, -NH(2), and -CONH(2) surfaces was high and dominated by hydrogen bond formation and electrostatic attraction. Adsorption of both BSA and alginate was the fastest on -COOH, and adsorption on -NH(2) and -CONH(2) was difficult to remove by surfactant cleaning. BSA adsorption kinetics was shown to be markedly faster than that of alginate, suggesting its importance in the formation of the conditioning layer. Surface modification to render -OH or ethylene-glycol functionalities are expected to reduce membrane fouling.     

Use of reverse osmosis membranes to remove perfluorooctane sulfonate (PFOS) from semiconductor wastewater

Perfluorooctane sulfonate (PFOS) and related substances are persistent, bioaccumulative, and toxic, and thus of substantial environmental concern. PFOS is an essential photolithographic chemical in the semiconductor industry with no substitutes yet identified. The industry seeks effective treatment technologies. The feasibility of using reverse osmosis (RO) membranes for treating semiconductor wastewater containing PFOS has been investigated. Commercial RO membranes were characterized in terms of permeability, salt rejection, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and membrane surface zeta potential (streaming potential measurements). Filtration tests were performed to determine the membrane flux and PFOS rejection. Over a wide range of feed concentrations (0.5 - 1500 ppm), the RO membranes generally rejected 99% or more of the PFOS. Rejection was better for tighter membranes, but was not affected by membrane zeta potential. Flux decreased with increasing PFOS concentration. While the flux reduction was severe for a loose RO membrane probably due to its higher initial flux, very stable flux was maintained for tighter membranes. At a very high feed concentration (about 500 ppm), all the membranes exhibited an identical stable flux. Isopropyl alcohol, present in some semiconductor wastewaters, had a detrimental effect on membrane flux. Where present it needs to be removed from the wastewater prior to using RO membranes.     

Development and Control of Bacterial Biofilms on Dairy Processing Membranes

Membrane fouling is a major operational problem that leads to reduced membrane performance and premature replacement of membranes. Bacterial biofilms developed on reverse osmosis membranes can cause severe flux declines during whey processing. Various types of biological, physical, and chemical factors regulate the formation of biofilms. Extracellular polymeric substances produced by constitutive microflora provide an effective barrier for the embedded cells. Cultural and microscopic techniques also revealed the presence of biofilms with attached bacterial cells on membrane surfaces. Presence of biofilms, despite regular cleaning processes, reflects ineffectiveness of cleaning agents. Cleaning efficiency depends upon factors such as pH of the cleaning agent, temperature, pressure, cleaning agent dose, optimum cleaning time, and cross‐flow velocity during cleaning. Among different cleaning agents, surfactants help to prevent bacterial attachment to surfaces by reducing the surface tension of water and interfacial tension between the layers. Enzymes mixed with surfactants and chelating agents can be used to penetrate the biofilm matrix formed by microbes. Recent studies have shown the role of quorum‐sensing‐based cell‐to‐cell signaling, which provides communication within bacterial cells to form a mature biofilm, and also the role of applying quorum inhibitors to prevent biofilm formation. Major cleaning applications are also summarized in Table 1 .     

Performance of forward (direct) osmosis process: membrane structure and transport phenomenon

The performance of a forward (direct) osmosis (FO) process was investigated using a laboratory-scale unit to elucidate the effect of membrane structure and orientation on waterflux. Two types of RO membrane and a FO membrane were tested using ammonium bicarbonate, glucose, and fructose as the draw solution to extract water from a saline feed solution. The FO membrane was able to achieve higher water flux than the RO membranes under the same experimental conditions while maintaining high salt rejection of greater than 97%. Increasing operating temperature increased the water flux in FO process. To investigate the effect of membrane orientation on water flux, the FO membrane was tested normally (dense selective layer facing draw solution) and reversely (dense selective layer facing feed solution). Explanations on transport phenomenon in FO process were proposed which explain the observation that the FO membrane, when used in the normal orientation, performed better due to lesser internal concentration polarization. This study suggests that an ideal FO membrane should consist of a thin dense selective layer without any loose fabric support layer.     

Swelling and morphology of the skin layer of polyamide composite membranes: an atomic force microscopy study

The paper introduces a new methodology for studying polyamide composite membranes for reverse osmosis (RO) and nanofiltration (NF) in liquid environments. The methodology is based on atomic force microscopy of the active layer, which had been separated from the support and placed on a solid substrate. The approach was employed to determine the thickness, interfacial morphology, and dimensional changes in solution (swelling) of polyamide films. The face (active) and back (facing the support) surfaces of the RO films appeared morphologically similar, in agreement with the recently proposed model of skin formation. Measured thickness and swelling data in conjunction with the intrinsic permeability of the membranes suggest that the selective barrier in RO membrane constitutes only a fraction of the polyamide skin, whereas NF membranes behave as nearly uniform films. For NF membranes, there was reasonable correlation between the changes in the swelling and in the permeability of the membrane and the salinity and pH of the feed.     

Spore formation in Bacillus subtilis biofilms

Spore formation by a Bacillus strain (Bacillus subtilis SpoIVFB-GFP) engineered with a green fluorescent protein (GFP) fused to a polytopic membrane protein (SpoIVF) that fluoresces during sporulation was observed. Biofilms of B. subtilis SpoIVFB-GFP containing ca. 8 log CFU/ml vegetative cells and spores below the lower detection limit (i.e., <1 log CFU/ ml) were allowed to develop on glass wool (37 degrees C). These biofilms were subsequently exposed to nutrient limitation to stimulate spore formation, which was monitored for fluorescence by confocal scanning laser microscopy. Sporulation in corresponding planktonic cells was also monitored for comparative purposes. Planktonic B. subtilis SpoIVFB-GFP cells began fluorescing after 5 h, while B. subtilis SpoIVFB-GFP biofilm cells began fluorescing after 30 h. Results suggested that an existing biofilm of vegetative B. subtilis cells may be stimulated to form spores when exposed to conditions of nutrient limitation. From a practical point of view, it may be suggested that a window of time does exist before sporulation occurs in attached Bacillus biofilms highlighting the need for shorter operating runs between cleaning and sanitation of food-processing equipment surfaces.     

Role of soluble microbial products (SMP) in membrane fouling and flux decline

Soluble microbial products (SMP), a significant component of effluent organic matter (EfOM), play an important role in membrane fouling and flux decline in wastewater reclamation/reuse applications. The SMP compounds of a microbial origin are derived during biological processes of wastewater treatment. They exhibit the characteristics of hydrophilic organic colloids and macromolecules. These high molecular weight compounds play an important role in creating high resistance of the membrane, leading to a reduction of permeate flux. The SMP fouling of RO, NF, and tight UF membranes is associated with formation of a cake/gel layer due to size (steric) exclusion. FTIR spectra of SMP- and EfOM-fouled membranes exhibited foulants' composition, consisting of polysaccharides, proteins, and/or aminosugar-like compounds. This finding reveals the important role of the SMP components as factors in membrane fouling and flux decline associated with EfOM source waters. Solids retention time (SRT) affects the characteristics and amounts of SMP, however, SRT did not affect flux decline trends of RO and NF membranes.     

Effect of fractal flocculation behavior on fouling layer resistance during apple juice microfiltration

The colloidal material in juice obtained from Red Delicious apples held in cold storage for over 6 months was observed to spontaneously aggregate to form ordered flocculation patterns. These flocculation patterns could be altered by heat treatment and by gelatin or antioxidant addition. The specific resistance of the fouling layer produced by each juice during microfiltration could be qualitatively predicted by examining the structure of macroscopic flocculation patterns. Environmental scanning electron microscopy (ESEM) photos of the various hydrated fouling layers indicated that loose flocs appeared to compress and rearrange on the membrane surface, producing smooth, low porosity secondary membranes. Densely packed cross-linked aggregate networks also formed low porosity structures, once again creating a fouling layer with high resistance. The lowest resistance fouling layers were produced when the flocs were dense enough to resist compression but porous enough to provide pathways for permeate flow. Field emission scanning electron microscopy (FESEM) and ESEM images indicated that, in many cases, the spacial distribution of solids in the microscopic aggregates were reminiscent of those in the macroscopic flocs. Such scaling relationships are typical of fractal systems.     

Organic fouling and chemical cleaning of nanofiltration membranes: measurements and mechanisms

Fouling and subsequent chemical cleaning of nanofiltration (NF) membranes used in water quality control applications are often inevitable. To unravel the mechanisms of organic fouling and chemical cleaning, it is critical to understand the foulant-membrane, foulant-foulant, and foulant-cleaning agent interactions at the molecular level. In this study, the adhesion forces between the foulant and the membrane surface and between the bulk foulant and the fouling layer were determined by atomic force microscopy (AFM). A carboxylate modified AFM colloid probe was used as a surrogate for humic acid, the major organic foulant in natural waters. The interfacial force data were combined with the NF membrane water flux measurements to elucidate the mechanisms of organic fouling and chemical cleaning. A remarkable correlation was obtained between the measured adhesion forces and the fouling and cleaning behavior of the membrane under various solution chemistries. The AFM measurements further confirmed that divalent calcium ions greatly enhance natural organic matter fouling by complexation and subsequent formation of intermolecular bridges among organic foulant molecules. Efficient chemical cleaning was achieved only when the calcium ion bridging was eliminated as a result of the interaction between the chemical cleaning agent and the fouling layer. The cleaning efficiency was highly dependent on solution pH and the concentration of the chemical cleaning agent.     

Formation of biofilms by Listeria monocytogenes under various growth conditions

Eight strains of Listeria monocytogenes (7644, 19112, 15313, Scott A, LCDC, 10403S, SLCC, and 1370) produce biofilms when grown on polyvinyl chloride microtiter well plates. The growth medium (tryptic soy broth [TSB] or modified Welshimer's broth [MWB] at 32 degrees C) influenced the amount of biofilm formed; maximum biofilms were formed in MWB by six strains and in TSB by the remaining two strains. This result suggests that the growth medium is critical in development of L. monocytogenes biofilm. This organism also produced biofilms on stainless steel chips. Biofilm formation on these chips was observed following growth in TSB at 4, 20, and 37 degrees C. After 20 h of incubation at 20 or 37 degrees C, the cell density was approximately 10(6) CFU per chip, and after 4 days incubation at 4 degrees C, the cell density was 10(5) CFU per chip. L. monocytogenes strain Scott A biofilm formation on stainless steel chips was visualized using scanning electron microscopy, which revealed dense aggregates of cells held together by meshlike webbing.     

Modes of natural organic matter fouling during ultrafiltration

The fouling of ultrafiltration membranes by natural organic matter (NOM), isolated from a potable surface water source, was studied with an emphasis on elucidating fouling modes and the role of aggregates. NOM size was related to membrane pore sizes using parallel membrane fractionation and size exclusion chromatography, such analyses confirmed the predominance of low MW species and identified the presence of aggregates in concentrated NOM solutions. Cake formation was the dominant mode of fouling by the unfiltered feed, which contained aggregates. This was identified by a constant rate of increase in membrane resistance with permeate throughput and was independent of pore size over a 10-1000 kDa molecular weight cutoff (MWCO) range. Prefiltration (to remove aggregates) and dilution (to reduce aggregate concentration) reduced the rate of increase in membrane resistance for the low MWCO membranes but did not change the fouling mode. In contrast, such pretreatment prevented cake formation on the larger MWCO membranes and shifted the mode of fouling to pore blockage. The date lend support for the idea that an initial fouling layer of large aggregates can catalyze the fouling by lower MW species. The fouling layer could be removed from the large MWCO membranes by backwashing, but the lower MWCO membranes exhibited some irreversible fouling, suggesting that low MW species penetrated into the pore structure. A combined pore blockage-cake formation model described the data well and provided insight into how fouling modes evolve during filtration.     

Beneficial biofilm formation by industrial bacteria Bacillus subtilis and related species

Biofilms are densely packed multicellular communities of microorganisms attached to a surface or interface. Bacteria seem to initiate biofilm formation in response to specific environmental cues, such as nutrient and oxygen availability. Biofilms undergo dynamic changes during their transition from free-living organisms to sessile biofilm cells, including the specific production of secondary metabolites and a significant increase in the resistivity to biological, chemical, and physical assaults. Bacillus subtilis is an industrially important bacterium exhibiting developmental stages. It forms rough biofilms at the air-liquid interface rather than on the surface of a solid phase in a liquid, due to the aerotaxis of the cells. Biofilm formation by B. subtilis and related species permits the control of infection caused by plant pathogens, the reduction of mild steel corrosion, and the exploration of novel compounds. Although it is obviously important to control harmful biofilm formation, the exploitation of beneficial biofilms formed by such industrial bacteria may lead to a new biotechnology.     

Formation of biofilm by Staphylococcus xylosus

The ability of 12 Staphylococcus xylosus strains to form biofilm was determined through the study of different criteria. Eleven out of the 12 strains were able to form biofilm, 10 preferentially on hydrophilic support (glass) and one, S. xylosus C2a, on both hydrophilic and hydrophobic (polystyrene) supports. The determination of bacterial surface properties showed that all strains were negatively charged with five strains moderately hydrophobic and seven hydrophilic. The bap and icaA genes, important for biofilm formation of some staphylococci, were searched. All strains were bap positive but icaA negative. Furthermore, S. xylosus strain C2a was studied on two supports widely used in the food industry, polytetrafluoroethylene (PTFE, hydrophobic) and stainless steel (hydrophilic) and appeared to adhere preferentially on stainless steel. Addition of 20 g/l of NaCl to Tryptic Soy Broth medium (TSB) did not improve significantly its adhesion but enhanced both bacterial growth and cell survival, which were optimum in this medium. Environmental scanning electron microscopy showed that S. xylosus C2a colonized the surface of stainless steel chips with intercellular spaces. The strain formed cell aggregates embedded in an amorphous polysaccharidic matrix. Indeed, synthesis of polysaccharides increased during growth on stainless steel chips in TSB.     

In situ localization of beta-glucans in the cell wall of Schizosaccharomyces pombe

The chemical composition of the cell wall of Sz. pombe is known as beta-1,3-glucan, beta-1,6-glucan, alpha-1,3-glucan and alpha-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with alpha-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of beta-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly beta-1,6-branched beta-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the alpha-galactomannan layer, suggesting that some of the radial filaments contain beta-1,6-branched beta-1,3-glucan. beta-1,6-glucan is preferentially located underneath the alpha-galactomannan layer. Linear beta-1,3-glucan is exclusively located in the primary septum of dividing cells. beta-1,6-glucan only labels the secondary septum and does not co-localize with linear beta-1,3-glucan, while beta-1,6-branched beta-1,3-glucan is present in both septa. Linear beta-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear beta-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found beta-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that beta-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.     

Tailoring the structure of thin film nanocomposite membranes to achieve seawater RO membrane performance

Herein we report on the formation and characterization of pure polyamide thin film composite (TFC) and zeolite-polyamide thin film nanocomposite (TFN) reverse osmosis (RO) membranes. Four different physical-chemical post-treatment combinations were applied after the interfacial polymerization reaction to change the molecular structure of polyamide and zeolite-polyamide thin films. Both TFC and TFN hand-cast membranes were more permeable, hydrophilic, and rough than a commercial seawater RO membrane. Salt rejection by TFN membranes was consistently below that of hand-cast TFC membranes; however, two TFN membranes exhibited 32 g/L NaCl rejections above 99.4%, which was better than the commercial membrane under the test conditions employed. The nearly defect-free TFN films that produced such high rejections were achieved only with wet curing, regardless of other post-treatments. Polyamide films formed in the presence of zeolite nanoparticles were less cross-linked than similarly cast pure polyamide films. At the very low nanoparticle loadings evaluated, differences between pure polyamide and zeolite-polyamide membrane water and salt permeability correlated weakly with extent of cross-linking of the polyamide film, which suggests that defects and molecular-sieving largely govern transport through zeolite-polyamide thin film nanocomposite membranes.     

Current and Recent Advanced Strategies for Combating Biofilms

Biofilms are matrix‐enclosed microbial aggregates that adhere to a biological or nonbiological surface. Biofilm formation is a significant problem in the medical, food, and marine industries and can lead to substantial economic and health problems. The complex microbial community of a biofilm is highly resistant to antibiotics and sanitizers and confers persistent survival that is a challenge to overcome. There are several conventional approaches to combating biofilms, physical and/or mechanical removal, chemical removal, and the use of antimicrobials, sanitizers, or disinfectants to kill biofilm organisms. However, biofilms are highly resistant to these approaches as opposed to planktonic cells. Thus, novel approaches other than the conventional methods are urgently needed. In this review, we discuss current and new advanced antibiofilm strategies that are superior to the conventional method in terms of addressing the biofilm problem for the improvement of healthcare, food safety, and in industrial processes.     

Membrane fouling during filtration of milk––a microstructural study

The surface morphology and internal microstructure of a membrane are the result of membrane manufacturing processes and subsequent use during fluid processing in industry. Both these structural factors have a great effect on fouling and filtration performance.

In this study, scanning electron microscopy (SEM), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy were used to comprehensively characterise the surface of unused microfiltration and ultrafiltration membranes, the fouled layer on the surface of membranes used for milk filtration and the internal fouling within the used membranes.

A simple and effective sample preparation method was developed to study the internal membrane structure using high-resolution field emission SEM with low-accelerating voltage. Various methods of structural characterisation were compared and the results showed that for flat sheet membranes AFM is an appropriate and convenient technique for examining the surface topography of membranes. In contrast SEM is a very appropriate technique for examining the cross-sectional and internal structure of a membrane, either unused or fouled.

Using these complimentary techniques it has been shown that internal fouling, during filtration of skim milk, proceeds by protein–polymer and protein–protein interactions. A gel layer forms on the surface of the membrane and has been imaged using SEM. This layer is slightly compressible and densifies as it grows. Fouling initiation commences after a very short filtration time.     

培养条件对奇异变形杆菌生物膜生长的影响

采用改良微孔板培养,结晶紫染色、平板计数以及电镜扫描方法研究不同初始菌浓度、培养环境(温度、气体环境)、培养基成分(pH、氯化钠浓度、碳源种类和浓度),培养基用量及接触材料对奇异变形杆菌(Proteus mirabilis,P. mirabilis)生物膜生长的影响。经过24 h培养,初始菌浓度10 CFU/cm2时,生物膜形成量较低,初始菌浓度102~108 CFU/cm2时,生物膜形成量明显;37 ℃有氧条件适宜P. mirabilis生物膜形成;培养基组成(2.0% NaCl,1.0%麦芽糖,pH8.0)和加倍用量对P. mirabilis生物膜形成具有明显的促进作用;粗糙的木制表面最易使P. mirabilis粘附并形成生物膜,硅胶次之,聚丙烯塑料和盖玻片表面比较光滑,菌体不易粘附,生物膜难以形成;不同接触材料形成的P. mirabilis生物膜结构和形态不同,硅胶上生物膜为蘑菇状,盖玻片上生物膜成扁平状。本文研究结果可为食品生产加工过程中P. mirabilis污染防控提供理论依据和技术支撑。