Survival of Yersinia pestis on Environmental Surfaces

The survival of two strains of Yersinia pestis (avirulent A1122 and virulent Harbin) on the surfaces of four materials was investigated. Viability was evaluated with epifluorescence microscopy by using the metabolic stain cyanoditolyl tetrazolium chloride and plate counts. Small numbers of cells suspended in phosphate buffer survived 2 to 4 h after visible drying on stainless steel, polyethylene, or glass and beyond 48 h on paper. Cells suspended in brain heart infusion broth (BHI) persisted more than 72 h on stainless steel, polyethylene, and glass. Small numbers of cells suspended in BHI were still viable at 120 h on paper. These data suggest that Y. pestis maintains viability for extended periods (last measured at 5 days) under controlled conditions.     

Effectiveness of disinfectants in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in a biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Effects of marine paints on microbial biofilm development on three materials

The development of biofilms of Pseudomonas aeruginosa PAO-1 was studied using modified Robbins devices. Biofilm development was measured using viable counts, acridine orange direct counts (AODC), and a colorimetric method for exopolysaccharide (EPS). Biofilms reached their maximum population 24–72 h after inoculation on coupons with no paint or on coupons coated with marine paint VC-18 without additives. Biofilms on stainless steel contained higher numbers of total cells and of viable cells than biofilms on fiberglass or aluminum. Coating the surfaces with marine paint VC-18 resulted in decreased numbers of cells on stainless steel but had little effect on numbers of cells on fiberglass or aluminum. Addition to the paint of Cu or tributyltin (TBT), the active components in two types of antifouling paints, inhibited the initial development of biofilms. However, by 72–96 h, most biofilms contained the same number of cells as surfaces without additives as shown by both viable counts and AODC. Biofilms that formed on surfaces coated with Cu- or TBT-containing paint did not synthesize more EPS, suggesting that P. aeruginosa PAO-1 does not respond to these compounds by synthesizing more EPS, which could bind the metal and protect the cells. Rather, these biofilms may contain Cu- or TBT-resistant cells. TBT-resistant cells made up 1–10% of the viable counts in biofilms on uncoated stainless steel, but in biofilms on stainless steel coated with marine paint containing TBT, TBT-resistant cells made up as much as 50% of the population. For non-coated stainless steel surfaces, Cu-resistant cells initially made up the majority of the population, but after 48 h they made up less than 1% of the population. On Cu-coated stainless steel, Cu-resistant cells predominated through 48 h, but after 48 h they comprised less than 10% of the population. These results suggest that the growth of TBT-resistant and Cu-resistant cells contributes to biofilms of P. aeruginosa PAO-1 at early stages of development but not at later stages. Received 16 December 1997/ Accepted in revised form 9 March 1998     

Effectiveness of Disinfectants in Killing Enterobacter sakazakii in Suspension, Dried on the Surface of Stainless Steel, and in a Biofilm

The effectiveness of 13 disinfectants used in hospitals, day-care centers, and food service kitchens in killing Enterobacter sakazakii in suspension, dried on the surface of stainless steel, and in biofilm was determined. E. sakazakii exhibited various levels of resistance to the disinfectants, depending on the composition of the disinfectants, amount and type of organic matrix surrounding cells, and exposure time. Populations of planktonic cells suspended in water (7.22 to 7.40 log CFU/ml) decreased to undetectable levels (<0.30 log CFU/ml) within 1 to 5 min upon treatment with disinfectants, while numbers of cells in reconstituted infant formula were reduced by only 0.02 to 3.69 log CFU/ml after the treatment for 10 min. The presence of infant formula also enhanced the resistance to the disinfectants of cells dried on the surface of stainless steel. The resistance of cells to disinfectants in 6-day-old and 12-day-old biofilms on the surface of stainless steel was not significantly different. The overall order of efficacy of disinfectants in killing E. sakazakii was planktonic cells > cells inoculated and dried on stainless steel > cells in biofilms on stainless steel. Findings show that disinfectants routinely used in hospital, day-care, and food service kitchen settings are ineffective in killing some cells of E. sakazakii embedded in organic matrices.     

Survival of human rhinovirus type 14 dried onto nonporous inanimate surfaces: effect of relative humidity and suspending medium

To study the survival of human rhinovirus 14 on environmental surfaces, each stainless steel disk (1 cm in diameter) was contaminated with 10 microL (about 10(5) plaque-forming units) of the virus suspended in either 1 chi tryptose phosphate broth (TPB), 5 mg/mL of bovine mucin in normal saline, or undiluted human nasal discharge. The inoculum was dried in a laminar flow cabinet for 1 h under ambient conditions. The disks were then placed in a glass chamber (20 +/- 1 degree C) with the relative humidity at either low (20 +/- 5%), medium (50 +/- 5%), or high (80 +/- 5%) level. At appropriate intervals, the disk to be tested was placed in 1 mL of tryptose phosphate broth and the eluate titrated in A-5 HeLa cells. When the virus was suspended in either tryptose phosphate broth, mucin, or the nasal discharge and subjected to initial drying, there was a 3.0 +/- 1.0, 82.0 +/- 6.7, and 89.0 +/- 3.0% loss in virus infectivity, respectively. The half-life of the TPB-suspended virus was about 14 h at the high relative humidity as compared with less than 2 h at the other two relative humidity levels. The half-lives for the mucin-suspended virus at the high, medium, and low relative humidity were 1.42, 0.55, and 0.24 h, respectively; the corresponding values for the nasal discharge suspended virus being 0.17, 0.25, and 0.09 h.     

The influence of milk and milk components on the attachment of bacteria to farm dairy equipment surfaces

Glass, rubber and stainless steel surfaces were exposed to various types of bacteria in the presence of milk and a number of milk components under both static and agitated incubation conditions. Numbers of bacteria attaching were enumerated by epifluorescence microscopy. Results were affected by the different bacterial types, the nature of the attachment surface and the substances in which the bacteria were suspended with a Moraxella -like species, stainless steel and lactose and non-casein protein solutions respectively resulting in greatest numbers of cells attaching. Agitation had no marked influence on attachment.     

Antimicrobial activity and effectiveness of a combination of sodium hypochlorite and hydrogen peroxide in killing and removing Pseudomonas aeruginosa biofilms from surfaces

AIMS: To evaluate both the antimicrobial activity and the effectiveness of a combination of sodium hypochlorite and hydrogen peroxide (Ox-B) for killing Pseudomonas aeruginosa ATCC 19142 cells and removing P. aeruginosa biofilms on aluminum or stainless steel surfaces. METHODS AND RESULTS: Pseudomonas aeruginosa biofilms were developed in tryptic soy broth containing vertically suspended aluminium or stainless steel plates. Biofilms were exposed to a mixed sodium hypochlorite and hydrogen peroxide solution as a sanitizer for 1, 5 and 20 min. The sanitizer was then neutralized, the cells dislodged from the test surfaces, and viable cells enumerated. Cell morphologies were determined using scanning (SEM) and transmission electron microscopy (TEM). Cell viability was determined by confocal scanning laser microscopy (CSLM). Biofilm removal was monitored by Fourier transform infrared (FTIR) spectrophotometry. Cell numbers were reduced by 5-log to 6-log after 1 min exposure and by 7-log after 5 min exposure to Ox-B. No viable cells were detected after a 20 min exposure. Treatment with equivalent concentrations of sodium hypochlorite reduced viable numbers by 3-log to 4-log after 1 min exposure and by 4-log to 6-log after 5 min, respectively. A 20 min exposure achieved a 7-log reduction. Hydrogen peroxide at test concentration treatments showed no effect. FTIR analysis of treated pseudomonad biofilms on aluminium or stainless steel plates showed either a significant reduction or complete removal of biofilm material after a 5 min exposure to the mixed sodium hypochlorite and hydrogen peroxide solution. SEM and TEM images revealed damage to cell wall and cell membranes. CONCLUSIONS: A combination of sodium hypochlorite and hydrogen peroxide effectively killed P. aeruginosa cells and removed biofilms from both stainless steel and aluminium surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of sodium hypochlorite and hydrogen peroxide can be used as an alternative disinfectant and/or biofilm remover of contaminated food processing equipment.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.     

AFM study of the colonisation of stainless steel by Aquabacterium commune

Aquabacterium commune, a member of the beta proteobacteria family that is a recently isolated, predominant member of various European drinking water distribution system biofilms, was selected as a test organism in this study. Attachment of A. commune cells onto two increasingly popular pipe materials, stainless steel EN1.4307 and medium density polyethylene (MDPE) was studied at 15 °C, 150 rpm, and a hydraulic retention time of 10.5 h in a rotating annular biofilm (RAB) reactor. Planktonic and sessile growth was monitored by spread plate technique. Atomic force microscopy (AFM) was used to obtain information about surface topography and biofilm formation pattern. Our study has shown that: (i) Steady-state conditions were reached after ca. 100 h for both materials; (ii) biofilm cell density on MDPE slides is four times greater than on stainless; (iii) the primary colonization of MDPE and stainless steel occurred at the edge of the slides; and (iv) no preferential attachment to stainless steel grain boundaries was observed. Stainless-steel manufacturers and suppliers, researchers, and companies working in the drinking-water sector will benefit from this paper. It is suggested that electropolishing of stainless-steel pipes for drinking water installations is not necessary to remove specific biofilm formation sites (i.e. grain boundaries). Furthermore, this paper provides, for the first time, some fundamental information for the continuous cultivation of the recently isolated drinking water microorganism, A. commune.     

Glass microspargers as effective frit spargers in single use bioreactors

For multiple-use bench scale and larger bioreactors, sintered stainless steel frit spargers are commonly used as microspargers. For bench-scale single-use bioreactors (SUBs), existing microspargers are sintered plastics, such as polyethylene. However, though plastics are readily sterilized by irradiation making them convenient for single use, these designs overlook surface energy properties of the materials of construction. For these sintered plastic spargers, forces at the water-air-surface interface cause bubble coalescence, leading to lower effective mass transfer, higher gas flow rates, and differing pCO₂ profiles in cell culture. Alternative materials of construction were evaluated based on contact angle information and bubble formation observations. Sintered glass was chosen over thermoplastic polymers for higher surface wettability as described in the glass/water contact angle, its history as a commonly sintered material, and availability at costs suitable for single use applications. Glass sintered spargers and traditional stainless steel frit spargers were compared by porosity, bubble size, and k_La studies. Mass transfer (k_La) and cell culture performance equal or greater than a standard 20 μm stainless steel microsparger mass transfer efficiency was achieved by a glass frit sparger, of international porosity standard “P40” according to ISO 4793-80, which corresponds to a range of 16–40 μm.     

Levan production by Zymomonas mobilis cells. Attached to plaited spheres

In this work, an immobilization method for polymer-levan production by a non-flocculating Z mobilis culture was developed. The extent of cell attachment to the stainless steel wire surface, culture growth and product synthesis were described. It was established that during short-term passive immobilization of non-flocculation Z mobilis cells on a stainless steel wire surface, sufficient amounts of biomass for proper levan and ethano fermentation could not be obtained. Adherence of cells was improved by pressing the paste-like biomass within stainless steel spheres knitted from wire with subsequent dehydration. Biomass fixed in metal spheres was used for repeated batch fermentation of levan. The activation period of cells within wire spheres (WS) was 48 h in duration. During this time, cell growth stabilized at production levels of ethanol and levan of Q_eth = 1.238 g/l × h and q_eth = 0.47 g/l × h; Q_eth = 0.526 g/l × h and q_eth = 0.20 g/l × h. Five stable fermentation cycles were realized using one wire sphere inoculum, and maintaining a stable ratio of 2.4 of biomass suspended in the medium to biomass fixed in the sphere. Using fixed Z mobilis biomass in the WS, the total amount of inoculum could be reduced for batch fermentation. Large plaited wire spheres with biomass may have potential in fermentation in viscous systems, including levan production.     

The use of graft copolymers to inhibit the adhesion of bacteria to solid surfaces

Twelve graft copolymers have been evaluated for their ability to prevent the adhesion of bacteria to substrata. The copolymers had polyethylene glycol (PEG) side-chains (‘teeth’) and a backbone that was either uncharged, acidic, basic or amphoteric. The copolymers were adsorbed onto glass, stainless steel and hydroxyapatite substrata, and 2-hpetri-dish adhesion experiments performed with bacteria isolated from marine (Pseudomonas sp. NCMB 2021), paper mill (S. marcescens NCIB 12211) and oral (S. mutans NCTC 10449) environments. The copolymers containing the most charged groups in the backbone had the most significant effect on bacterial adhesion levels, with anti-adhesive effects up to 99% achieved. An amphoteric copolymer (Compound 12) on glass, and acidic copolymer (Compound 11) on stainless steel and hydroxyapatite gave the most impressive anti-adhesive effects. These copolymers had non-specific bacterial anti-adhesive properties.It is proposed that the graft copolymers adsorbed onto hydrophilic surfaces via their charged backbone in such a way that the PEG side-chains were pointing out into the aqueous phase, and it was this orientation that was responsible for the observed anti-adhesive effect.     

Role of curli fimbriae in mediating the cells of enterohaemorrhagic Escherichia coli to attach to abiotic surfaces

AIMS: The objectives of this study were to evaluate the role of curli in assisting the cells of enterohaemorrhagic Escherichia coli (EHEC) in attaching to abiotic surfaces and to determine the influence of cell-surface contact time on the efficiency of the attachment. METHODS AND RESULTS: Three pairs of EHEC cultures, each with a curli-expressing and a noncurli-expressing variant (O111:H- 7-57C+ and O157:H7 5-9C-, O157:H7 5-11C+ and 5-11C-, as well as O103:H2 7-52C+ and 7-52C-), were allowed to interact with polystyrene, glass, stainless steel and rubber surfaces at 28 degrees C for 24 h (short-term attachment) or 7 days (long-term attachment). The quantities of the cells that attached to the surfaces were measured daily in the long-term attachment study, and in 4 h intervals in the short-term attachment study. Quantification of the cells that attached to the surfaces was accomplished with a crystal violet binding assay. The results of the long-term attachment study indicated that 7-57C+ attached to the polystyrene and glass surfaces more efficiently (P < 0.05) than did 5-9C-. The curli-expressing variant of 5-11 possessed a better ability to adhere to the polystyrene and glass surfaces than did its noncurli-expressing counterpart (P < 0.05). The differences in attachment between 7-52C+ and 7-52C- on polystyrene and stainless steel surfaces were statistically significant (P < 0.05). However, the attachment of the pair on the glass surfaces was statistically insignificant (P > 0.05). In addition, the two members of all three EHEC pairs attached equally well to rubber surfaces (P > 0.05). In the short-term attachment study, only the pair of 7-52 attached differently on glass and stainless steel surfaces (P < 0.05). CONCLUSIONS: These results suggest that curli could be an important cell surface component to mediate the attachment of some EHEC cells to certain abiotic surfaces. Cell-surface contact time could have a significant influence on EHEC attachment to abiotic surfaces. SIGNIFICANCE AND IMPACT OF THE STUDY: The study signifies a possible role of curli in assisting the cells of EHEC in attaching to food-contact surfaces. It underlines the importance of cleaning and sanitizing food-contact surfaces regularly and thoroughly, and of identifying chemical agents that can effectively remove the attached EHEC cells from these surfaces.     

A simple method for ultrastructural enzyme cytochemistry on small volumes and numbers of isolated cells

Summary A simple technique is described for ultrastructural enzyme cytochemistry using small volumes and numbers of single cells. Small aliquots (1–5 l) of single cells suspended in a glutaraldehyde fixative are incubated on poly-L-lysine-coated glass coverslips. Cells in contact with the coated coverslip become immobilized to that support. All changes of solutions are accomplished without repeated centrifugation.     

Comparison of adhesion of the food spoilage bacterium Shewanella putrefaciens to stainless steel and silver surfaces

AIMS: To compare the number of attached Shewanella putrefaciens on stainless steel with different silver surfaces, thus evaluating whether silver surfaces could contribute to a higher hygienic status in the food industry. METHODS AND RESULTS: Bacterial adhesion to three types of silver surface (new silver, tarnished silver and sulphide-treated silver) was compared with adhesion to stainless steel (AISI 316) using the Malthus indirect conductance method to estimate the number of cfu cm(-2). The number of attached bacteria on new silver surfaces was lower than on steel for samples taken after 24 h. However, this was not statistically significant (P > 0.05). The numbers of attached bacteria were consistently lower when tarnished silver surfaces were compared with stainless steel and some, but not all, experiments showed statistical significance (P < 0.05). Treating new silver with sulphide to reproduce a tarnished silver surface did not result in a similar lowering of adhering cells when compared with steel (P > 0.05). CONCLUSIONS: New or tarnished silver surfaces caused a slight reduction in numbers of attached bacteria; however, the difference was only sometimes statistically significant. SIGNIFICANCE AND IMPACT OF THE STUDY: The lack of reproducibility in differences in numbers adhering to the different surfaces and lack of statistical significance between numbers of adhered viable bacteria do not indicate that the tested silver surfaces can be used to improve hygienic characteristics of surfaces in the food industry.     

Influence of copper-alloying of austenitic stainless steel on multi-species biofilm development

AIMS: To investigate the bactericidal influence of copper-alloying of stainless steel on microbial colonization. METHODS AND RESULTS: Inhibition of bacterial adherence was investigated by monitoring (192 h) the development of a multi-species biofilm on Cu-alloyed (3.72 wt%) stainless steel in a natural surface water. During the first 120 h of exposure, lower numbers of viable bacteria in the water in contact with copper-containing steel relative to ordinary stainless steel were observed. Moreover, during the first 48 h of exposure, lower colony counts were found in the biofilm adhering to the Cu-alloyed steel. No lower colony or viable counts were found throughout the remainder of the experimental period. CONCLUSION: The presence of Cu in the steel matrix impedes the adhesion of micro-organisms during an initial period (48 h), while this bactericidal effect disappears after longer incubation periods. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of Cu-alloyed stainless steels for bactericidal purposes should be restricted to regularly-cleaned surfaces.     

Physiology of biofilms of thermophilic bacilli—potential consequences for cleaning

Thermophilic Bacillus species readily attached and grew on stainless steel surfaces, forming mature biofilms of >10^6.0 cells/cm² in 6 h on a surface inoculated with the bacteria. Clean stainless steel exposed only to pasteurized skim milk at 55 °C developed a mature biofilm of >10^6.0 cells/cm² within 18 h. When bacilli were inoculated onto the steel coupons, 18-h biofilms were 30 m thick. Biofilm growth followed a repeatable pattern, with a reduction in the numbers of bacteria on the surface occurring after 30 h, followed by a recovery. This reduction in numbers was associated with the production of a substance that inhibited the growth of the bacteria. Variations in the environment, including pH and molarity, affected the viability of the cells. Chemicals that attack the polysaccharide matrix of the biofilm were particularly effective in killing and removing cells from the biofilm, demonstrating the importance of polysaccharides in the persistence of these biofilms. Treatment of either the biofilm or a clean stainless steel surface with lysozyme killed biofilm cells and prevented the attachment of any bacteria exposed to the surface. This suggests that lysozyme may have potential as an alternative control method for biofilms of these bacteria.     

Use of a modified Robbins device to directly compare the adhesion of Staphylococcus epidermidis RP62A to surfaces

Staphylococcus epidermidis is a frequent cause of infection associated with the use of biomedical devices. Flow cell studies of the interaction between bacteria and surfaces do not generally allow direct comparison of different materials using the same bacterial suspension. The use of a modified Robbins Device (MRD) to compare the adhesion to different surfaces of Staph. epidermidis RP62A grown in continuous culture was investigated. Adhesion to glass was compared with siliconized glass, plasma-conditioned glass, titanium, stainless steel and Teflon. Attachment to siliconized glass was also compared with glass under differing ionic strength, and divalent cation concentrations. Both the differences in numbers adhering and changes in adhesion (slope) through the MRD were compared. There was a trend towards higher numbers adhering to the discs at the in-flow end of the MRD than at the outflow end, probably reflecting depletion of adherent bacteria in the interacting stream. Adhesion of Staph. epidermidis RP62A to siliconized glass and Teflon was reduced when compared to glass with increasing flow rates. Adhesion to stainless steel was not affected by flow rate and titanium gave a different slope of adhesion through the MRD when compared with glass, suggesting an interaction with different sub-populations within the interacting stream. Differences between siliconized glass and glass at flow rates of 300 ml h-1 were abolished by the addition of calcium or EDTA and reduced by the addition of magnesium. Increasing ionic strength reduced the statistical significance of the differences between glass and siliconized glass. Pre-conditioning of glass with pooled human plasma reduced adhesion compared with untreated glass and again gave a different slope to glass. The MRD linked to a chemostat can be used to compare directly bacterial adhesion to potential biomaterials. Variable depletion of the interacting stream should be taken into account in the interpretation of results. Divalent cation concentration, substrate properties and flow rate were important determinants of the comparative adhesion of Staph. epidermidis RP62A to surfaces.     

Changes in the strength of attachment of micro-organisms to surfaces following treatment with disinfectants and cleansing agents

Suspensions of Pseudomonas aeruginosa and Staphylococcus epidermidis , and biofilms established (16 h) on submerged glass and stainless steel (216 2B) coupons, were exposed to sodium hypochlorite (0·02% or 0·015% w/v), Dodigen (0·0015% w/v or 0·0006% w/v), sodium dodecylsulphate (6% w/v or 0·1% w/v) and Tween-80 (6% w/v) for 5 min at 20 °C. Survival was assessed by viable counts and blot succession. Biofilm bacteria were significantly less susceptible to these biocides than were planktonic cells, but their attachment to the surfaces was loosened by such treatments. Treatment with the non-ionic surfactant, Tween-80, however, strengthened the attachment of Staph. epidermidis to stainless steel. Such effects on attachment strength, which are species and surface dependent, have profound implications on post-treatment cleansing and possible re-contamination of product in clean-in-place (CIP) systems.     

The production of cellulase in a spouted bed fermentor using cells immobilized in biomass support particles

Continuous cellulase production by Trichoderma viride QM 9123, immobilized in 6 mm diameter, spherical, stainless steel biomass support particles, has been achieved using a medium containing glucose as the main carbon source. Experiments were carried out in a 10-L spouted bed fermentor. In this type of reactor-recycled broth is used to create a jet at the base of a bed of particles, causing the particles to spout and circulate. During the circulation, particles pass through a region of high shear near the jet inlet. This effectively prevents a buildup of excess biomass and thus enables steady-state conditions to be achieved during continuous operation. Continuous production of cellulase was achieved at significantly higher yield and productivity than in conventional systems. At a dilution rate of 0.15 h(-1) (nominal washout rate for freely suspended cells is 0.012 h(-1)), the yield of cellulase on glucose was 31% higher than that measured during batch operation, while the volumetric productivity (31.5 FPA U/L. h) was 53% greater than in the batch system. The specific cellulase productivity of the immobilized cells was more than 3 times that of freely suspended cells, showing that diffusional limitations can be beneficial. This offers significant opportunity for the further development of biomass support particles and associated bioreactors.