Efficacy of a typical clean-in-place protocol against in vitro membrane biofilms

This study evaluates the effectiveness of a typical clean-in-place (CIP) protocol against in vitro biofilms on whey reverse osmosis (RO) membranes developed under static condition. Bacterial isolates obtained from RO membrane biofilms were used to develop single and multispecies biofilms under laboratory conditions. A typical commercial CIP protocol was tested against the 24-h-old biofilms, and included 6 sequential treatment steps based on alkali, surfactant, acid, enzyme, a second surfactant, and a sanitizer treatment step. Experiments were conducted in 4 replicates and the data were statistically analyzed. The results revealed a variation in the resistance of mixed-species biofilms against the individual steps in the sequential CIP protocol. The overall 6 steps protocol, although resulted in a greater reduction, also resulted in the detection of survivors even after the final sanitizer step, reflect the ineffectiveness of the CIP protocol for complete removal of biofilms. Posttreatment counts of 0.71 log after the sequential CIP of mixed-species biofilm revealed the resistance of biofilm constitutive microbiota. Mixed-species biofilms, constituting different genera including Bacillus, Staphylococcus, and Streptococcus, were observed to be more resistant than most of the single-species biofilms. However, among the single-species biofilms, significantly different resistance pattern was observed for Bacillus isolates compared with the other bacterial isolates. All 5 isolates of Bacillus were found resistant with survivor counts of more than 1.0 log against the sequential CIP protocol tested. Thus, it can be concluded that the tested CIP protocol had a limited effectiveness to clean membrane biofilms formed on the whey RO membranes.     

Ultrafiltration/Reverse Osmosis Concentration of Lobster Extract

A membrane concentration system consisting of tubular polysulphone ultrafiltration (UF) and polyamide reverse osmosis (RO) was evaluated for concentrating key water soluble flavor compounds from lobster extracts. Major flavor-giving compounds in the extract were glutamic acid, glycine, arginine, uridine 5′-monophosphate (UMP), succninic acid and glucose. Factors affecting performance of the UF/RO systems, such as flow rate, feed solid level, temperature and pressure, on permeate flux and solids rejection were measured. The optimum UF conditions were 1.5% feed solid level, 15 L/min feed flow rate, 50°C feed temperature and 1 MPa log mean transmembrane pressure. The RO system retained all dissolved flavor components and its ideal operating conditions were 40°C, 2.8 MPa log mean transmembrane pressure and a flow rate of 15 L/min.     

The effects of biofilms formed on whey reverse osmosis membranes on the microbial quality of the concentrated product

The study investigated the development of bacterial biofilms on spiral wound reverse osmosis (RO) whey concentration membranes and their influence on the microbial quality and safety of concentrated whey (retentate). Used RO membranes, obtained from a commercial whey processing plant, were evaluated at intervals of 2 months for a total duration of 14 months using standard techniques. Results confirmed the presence of multi‐species bacterial biofilms on whey RO membranes. Considerable variations were noticed in the distribution pattern of biofilms constitutive microflora as the membranes aged. A greater increase in retentate counts as compared to feed suggested the possibilities of cross‐contamination from the membrane biofilms.     

Short communication: A competitive exclusion study reveals the emergence of Bacillus subtilis as a predominant constitutive microorganism of a whey reverse osmosis membrane biofilm matrix

Microbial attachment and colonization on separation membranes lead to biofilm formation. Some isolates within the biofilm microflora acquire greater resistance to the chemical cleaning protocols on prolonged use of membranes. It is thus likely that the constitutive microflora might compete with each other and result in certain species emerging as predominant, especially within older biofilms. To understand the microbial interactions within biofilms, the emergence of predominance was studied in the current investigation. An 18-mo-old reverse osmosis membrane was procured from a whey processing plant. The membrane pieces (2.54 × 2.54 cm²) were neutralized by dipping in Letheen broth. The resuscitation step was done in tryptic soy broth (TSB) at 37°C, followed by plating on tryptic soy agar (TSA) to recover the constitutive microflora. Distinct colonies of isolates were further identified using MALDI-TOF as Bacillus licheniformis, Exiguobacterium aurantiacum, Acinetobacter radioresistens, Bacillus subtilis (rpoB sequencing), and 1 unidentified species each of Exiguobacterium and Bacillus. Further, the competitive exclusion study helped establish the emergence of predominance using a co-culturing technique. Fifteen combinations (of 2 isolates each) were prepared from the isolates. Pure cultures of the respective isolates were spiked in a ratio of 1:1 in TSB and incubated at 37°C for 24 h, followed by plating on TSA. The enumerated colonies were distinguished based on colony morphology, Gram staining, and MALDI-TOF to identify the type of the isolate. Plate counts of B. subtilis emerged as predominant with mean log counts of 7.22 ± 0.22 cfu/mL. The predominance of B. subtilis was also validated using the process of natural selection in a multispecies growth environment. In this instance, the TSB culture with overnight-incubated membrane piece (with mixed-species biofilm) at 37°C for 12 h was inoculated in fresh TSB and incubated for the second cycle. Overall, 5 such sequential broth-culture incubation cycles were carried out, followed by pour plating on TSA plates, at the end of each cycle. The isolates obtained were identified using a similar methodology as mentioned above. The fifth subsequent transfer depicted the presence of only 1 B. subtilis isolate on plating, thereby validating its predominance under the conditions of the experiment.     

Permeate Flux During Ultrafiltration of Whey: Influence of Milk Coagulant Used for Cheese Manufacture

A pilot-scale plate and frame UF system was used to fractionate Cheddar cheese whey and study the effects of different commercial milk coagulants on permeate flux. Coagulants used in this study were calf rennet, Mucor pusillus protease, and Mucor miebei protease. Whey UF performance studies were conducted at a commercial Cheddar cheese plant and at Cornell under controlled conditions. Ultrafiltration was done in a continuous mode and initial concentration factor was set at 2× to simulate the first stage of a multistage whey UF system.Permeate flux decline was rapid in the first 30 min of UF for all wheys studied. More important, the type of milk coagulant used in cheese making had a profound effect on permeate flux during whey UF. No differences in the gross composition of the various wheys were correlated with differences in permeate flux. The highest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using coagulant derived from Mucor pusillus. Lowest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using calf rennet. Whey from cheese manufactured using Mucor miebei coagulant had flux performance intermediate to Mucor pusillus and calf rennet. The impact of milk coagulants on whey UF process efficiency should be considered by cheese makers.     

Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol

This experiment evaluates the effectiveness of individual steps of a clean-in-place protocol against the biofilm constitutive microflora isolated from the biofilms developed on whey reverse-osmosis membranes, aged 2 to 14 mo, under industrial processing conditions. The isolates used for the in vitro resistance studies included species of Bacillus, Enterococcus, Streptococcus, Staphylococcus, Micrococcus, Aeromonas, Corynebacterium, Pseudomonas, Klebsiella, and Escherichia. The 6 cleaning steps (alkali, surfactant, acid, enzyme, a second surfactant, and sanitizer treatment) revealed resistance of isolates in both planktonic and biofilm-embedded cell states. The most effective step was the acid treatment, which resulted in 4.54 to 7.90 and 2.09 to 5.02 log reductions of the planktonic and biofilm-embedded cells, respectively. Although the sanitizer step causing a reduction of 4.91 to 8.33 log in the case of planktonic cells, it was less effective against the biofilm-embedded cells, resulting in a reduction of 0.59 to 1.64 log. Bacillus spp. showed the highest resistance in both planktonic, as well as embedded cell states.     

Bacteriological Aspects of Microfiltration of Cheese Whey,

Microfiltration of cheese whey using 1.2-μm pore size membranes reduced bacterial counts by one to three times. Increased fat concentration in the feed stream governed the decrease in bacterial counts. Fat was trapped onto the membrane proportionally to its amount in the feed stream, and thus, formed a barrier to microorganism penetration into the permeate.     

PROCESSING WHEY-TYPE BY-PRODUCT LIQUIDS FROM COTTONSEED PROTEIN ISOLATION WITH ULTRAFILTRATION AND REVERSE OSMOSIS MEMBRANES

Cottonseed wheys resulting from protein isolation from cottonseed flour were processed by semi-permeable ultrafiltration (UF) and reverse osmosis (RO) membranes. The UF membrane fractionated the soluble whey constituents by retaining protein and passing through salts, carbohydrates and other non-protein components along with most of the water. The UF membrane effluent was then processed through an RO membrane to recover a secondary product containing the whey materials not retained in the protein product from the UF membrane. The feasibility of recycling effluent from the RO membrane for reuse in subsequent protein extractions was demonstrated. Thus, the threat of water pollution from effluent disposal could be eliminated completely and process water requirements drastically reduced. Spray-dried UF protein concentrates were tested for utilization in protein fortification of breads and noncarbonated beverages and as whipping products. They exhibited commercial potential for use in these food applications. The economics of processing the whey-type liquids by the membrane process under investigation were analyzed. Membrane processing of wheys by each of two alternative whey processing systems proved to be economically attractive.     

Effective control of microbial populations in polysulfone ultrafiltration membrane systems

Sanitizers currently used in the food industry are not efficient in destroying bacterial populations in polysulfone UF membrane systems. A new sanitizer composition that releases chlorous acid and chlorine dioxide from sodium chlorite at pH 2.7 was evaluated. Polysulfone UF membranes were soiled for 2.5 h by circulating and concentrating Cheddar cheese whey and skim milk. A cleaning regimen was established whereby acid and caustic cleaning solutions were circulated to clean the UF membrane system. Restoring permeate flux to initial values did not indicate that the system was effectively cleaned. The UF system was sanitized by recycling sanitizer solutions. Stainless steel and membrane surfaces were examined by swabbing to determine bacterial populations and sections of membranes were removed for examination using a scanning electron microscope. The new sanitizer appeared to control microbial populations effectively in UF membrane systems.     

Microscopic observation of multispecies biofilm of various structures on whey concentration membranes

The objective of this study was to evaluate biofilm formation on polyamide reverse osmosis (RO) whey concentration membranes. Biofilms were observed with scanning electron and fluorescence microscopy. For scanning electron microscopy, pieces of 6-, 12-, and 14-mo-old membranes were allowed to air dry at room temperature (22°C) for 24 h followed by sputter coating with a 5-nm layer of gold and microscopic observations. Scanning electron microscopy images revealed that the hydrophilic layer, used to prevent membrane plugging, was not evenly distributed on the surface. Although this hydrophilic layer seemed to prevent the attachment of proteins, it supported biofilm formation. Three different structures of multispecies biofilm were observed on the retentate side of the membrane: 1) a mono layer, 2) a 3-dimensional structure of a dense matrix of extracellular polymeric substances where different types of bacterial cells were embedded, and 3) cell aggregates. In some of the biofilms, a smooth layer (shell) covered cell aggregates. In the 6-mo-old membranes, part of the shell layer was broken off. Biofilms as observed on the RO membrane were described as having a hill-and-valley type of structure, with hills showing a mushroom-like appearance and valleys comprising dense matrices of extracellular polymers with embedded bacterial cells. Fluorescence microscopy showed live cells on the surface of the biofilm. It is concluded that both cells in the deep layers of biofilm and surface cells may resist cleaning and sanitation. The extent of biofilm formation and the presence of live cells on RO membranes after regular clean in place cycles indicate the need for a more effective cleaning regimen customized for dairy separation systems.     

Evaluation of efficacy of four commercial enzyme-based cleaners of ultrafiltration systems

Use of UF and RO in the dairy industry is rapidly expanding. Because the dairy industry demands high levels of cleanliness, this new technology requires close evaluation to assure adherence to these standards. Efficacy of four commercial enzyme-based cleaners (pH 7.0 to 8.4) in UF systems was determined by microbiological evaluation and permeate flux restoration. The UF system containing two polysulfone UF membranes in parallel, was soiled by recycling 380 L of sweet whey (40 degrees C) for 2.0 h followed by concentrating whey for .5 h. The cleaning cycle consisted of acid cleaner (.5 h, 40 degrees C), followed by enzyme cleaner (10.0 h, 40 degrees C), and rinsing (2.0 h, 40 degrees C). A chlorine sanitizer was circulated (5 min, 40 degrees C) and the unit containing sanitizing solution left idle overnight. Flux was determined and swabs and rinse water samples were taken immediately after soiling, after cleaning, and the next morning to check sanitizing. The four enzyme-based cleaners were unsatisfactory when microbiological criteria were considered. Loss of sanitizer strength and problems with yeast and especially mold growth over time also indicated lack of effective cleaning. Flux, however, was restored easily and did not correlate with efficacy of cleaning based on numbers of microorganisms remaining.     

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.     

Ultrafiltration and Reverse Osmosis of the Waste Water from Sweet Potato Starch Process

The primary waste water discharged from pilot plant scale sweet potato starch manufacturing was processed by ultrafiltration (UF). The UF permeate was then concentrated by reverse osmosis (RO). Growth of microorganisms in waste water would reduce the flux of UF. When the feed velocity of UF was higher than 2.5 m/sec, its positive effect on permeation rate was no longer existent. Relationships between transmembrane pressure and permeate flux were linear at all tested concentrations. UF filtered protein and calcium reduced two-thirds of the biochemical oxygen demand (BOD) and half the chemical oxygen demand (COD) at weight concentration ratio (WCR) of 5. With RO the rest of the components were recovered and BOD and COD were reduced more than 99% and 98%, respectively, at a WCR of 6.     

Buffering curves of ideal whey fractions obtained from a cascade membrane separation process

Skimmed milk was fractionated via a cascade system: Graphic plotting of microfiltration (MF); ultrafiltration (UF); nanofiltration (NF); and reverse osmosis (RO). The buffering curves of each fraction were studied over the pH range 4–7. Depending on their composition, the individual permeate streams showed different buffering capacity values and pH ranges where the buffering occurred. The concentration of active buffering substances in the permeates decreased (in mmol/L) from ~26.6 (MF) to ~17.4 (UF) to 1.39 (NF) to 0.07 (RO). Contributions to the total buffering capacity for MF permeate, which represents the serum phase of milk, were ~37% from whey proteins and ~63% from milk salts (especially citrates, phosphates and carbonates) including lactose and water.     

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 .     

Mushroom Blanch Water Concentration by Membrane Processes

Mushroom blanch water was concentrated by ultrafiltration (UF) and reverse osmosis (RO). UF prefiltration was essential in preventing severe fouling during the RO process. When the UF blanch water permeate was processed by RO, linear relationships between pressure and flux were observed at all concentrations tested. The blanch water was concentrated by UF/RO from 2% to 13% total solids at 60°C and 120 KPa/5000 KPa operating pressures with flux higher than 15 L/m² hr. Maximum concentration obtained was approximately 20% total solids with 90% recovery of the nonvolatiles. Recoveries of some major volatiles were above 50%. Panelists could not differentiate the original from the reconstituted blanch waters in sensory evaluations.     

Determination of the efficiency of removal of whey protein from sweet whey with ceramic microfiltration membranes

Our research objective was to measure percent removal of whey protein from separated sweet whey using 0.1-µm uniform transmembrane pressure ceramic microfiltration (MF) membranes in a sequential batch 3-stage, 3× process at 50°C. Cheddar cheese whey was centrifugally separated to remove fat at 72°C and pasteurized (72°C for 15 s), cooled to 4°C, and held overnight. Separated whey (375 kg) was heated to 50°C with a plate heat exchanger and microfiltered using a pilot-scale ceramic 0.1-µm uniform transmembrane pressure MF system in bleed-and-feed mode at 50°C in a sequential batch 3-stage (2 diafiltration stages) process to produce a 3× MF retentate and MF permeate. Feed, retentate, and permeate samples were analyzed for total nitrogen, noncasein nitrogen, and nonprotein nitrogen using the Kjeldahl method. Sodium dodecyl sulfate-PAGE analysis was also performed on the whey feeds, retentates, and permeates from each stage. A flux of 54 kg/m² per hour was achieved with 0.1-µm ceramic uniform transmembrane pressure microfiltration membranes at 50°C. About 85% of the total nitrogen in the whey feed passed though the membrane into the permeate. No passage of lactoferrin from the sweet whey feed of the MF into the MF permeate was detected. There was some passage of IgG, bovine serum albumen, glycomacropeptide, and casein proteolysis products into the permeate. β-Lactoglobulin was in higher concentration in the retentate than the permeate, indicating that it was partially blocked from passage through the ceramic MF membrane.     

Purification of Steviosides by Membrane and Ion Exchange Processes

An ultrafiltration (UF) process removed more than 96% of the pigments and recovered 45% of steviosides non-nutritive sweetener from an extract of leaves. UF retentate was further processed by diafiltration (DF), and the permeate was concentrated by reverse osmosis (RO). The membrane processes (UF, DF, RO) achieved an overall steviosides recovery of more than 90% with product purity 46%. Final purification was conducted by two consecutive mixed bed ion exchange processes. The ion exchangers improved purity of the final product to 90%.     

超滤法澄清西番莲果汁的研究

以寻求提高西番莲原汁超滤时的渗透通量为目的，用国产芳香聚酰胺膜平板超滤器进行试验，对超滤操作条件，果汁预处理，膜的清洗进行研究。结果表明，西番莲原汁超滤时渗透通量最高的操作压力为0．15MPa，最佳进料速度为22mL/s，操作温度为室温；原汁经海藻酸钠—碳酸钠澄清剂预处理后可以明显增大渗透通量；超滤处理后。原汁的西番莲固有滋味和品质得到较好保留。采用超滤澄清法生产高质量的西番莲原汁是可行的。     

Ultrafiltration and Reverse Osmosis Recovery of Limonene from Citrus Processing Waste Streams

Commercial ultrafiltration (UF) and reverse osmosis (RO) membranes were used to concentrate the terpene, limonene, present in cold pressed oil centrifuge effluent and molasses evaporator condensate. UF membrane rejections were 78–97% for mixtures with initial limonene concentrations from 0.04–0.6%v/v. RO membrane rejection of limonene ranged from 87–99% for feed streams containing 0.06–0.23% limonene. Initial membrane flux rates for centrifuge effluents were in the range 10–100 kg/m²/hr. Evaporator condensate fluxes were higher, 25–400, while pure water rates ranged from 25 (RO) to 1000 kg/m²/hr (UF). Contact with limonene adversely affected membrane flux rates in decreasing order of severity: polysulfone > cellulose acetate > teflon-type.